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    • 专利摘要:
    The present invention is concernd with a) isolation and purification of the soluble IgE binding factor (Fc epsilon R) secreted or shed by lymphoblastoid cells, b) partial sequencing the obtained Fc epsilon R-receptor, isolating the thus obtained fragments and determination of the sequences of the thus obtained fragments, c) preparation of an oligonucleotide encoding to one of the said partial sequences, d) isolating and identifying expression vehicles containing the gene coding for Fc epsilon -receptor, comprising the steps of: synthesizing cDNA from a RNA matrix derived from lymphoblastoid cells producing Fc epsilon -receptor mRNA, incorporating said synthesized cDNA in expression vehicles to form an expression vehicle bank, hydribizing said incorporated cDNA to identify those expression vehicles which contain a gene coding for Fc epsilon -receptor, with a labeled probe comprising an oligonucleotide common to the gene for Fc epsilon --receptor, replication and expression of the thus obtained gene, and sequencing the thus obtained gene, e) determination of the gene coding for the active human low-affinity Fc epsilon -receptor utilising isolated cDNA sequence obtained from the vehicles from operation d), f) expression of the Fc epsilon R cDNA in Cos 7 cells and g) preparing an expression vector containing the DNA sequences coding for a soluble fragment of the Fc epsilon -receptor and expressing said soluble fragments in a microorganism or in mammalian cells. The water-soluble fragment of the Fc epsilon -receptor is suitable for treatment of allergic reactions.
    公开号:SU1727533A3
    申请号:SU874203195
    申请日:1987-08-20
    公开日:1992-04-15
    发明作者:Кишимото Тадамитсу；Суемура Масаки；Кикутани Хитоши；Барсумиан Эдвард
    申请人:Тадамитсу Кишимото (Фирма)；
    IPC主号:
    专利说明:

    The invention relates to biotechnology and medicine, in particular to methods for producing polypeptides suitable for controlling local and systemic allergic reactions.
    The method includes the following operations.
    1. Isolation of the water-soluble part of IgE binding factoro (Fcj R) from lymphoblastoid cells.
    2. Analysis of the sequences of the water-soluble part of the human receptor of small affinity of the RS by hydrolysis, the selection of the resulting fragments and the determination of their sequences.:
    3. Preparation of two hybridization samples.
    Sample 1: obtaining a transforming FcЈ + L cell with additional DNA (hereinafter referred to as dDNK) using multistage cyclic extraction of RNA encoding a polypeptide (hereinafter mRNA).
    Sample 2: obtaining oligonucleotides of the formula
    tdl
    3 - TTC ACCTAGTTGAAAc GT-5 A
    encoding the amino acid sequence of the formula
    Lys-Trp-lfe-Asn-Phe-G fn.
    x | Yu v |
    cl
    CJ
    with

    ICJ
    4. Isolation of genes encoding human affinity FcЈ receptor from the RNA matrix of lymphoblastoid cells producing the Fc receptor mRNA, synthesis of DNA, followed by identification of expression carriers containing the gene encoding the Fc receptor, using two labeled samples containing Fc специфи R specific to cells + L DNA
    and an oligonucleotide that is common with the FcЈ receptor gene of small affinity, and replication of the FcЈ receptor gene thus obtained.
    5. Expression of DNA Fc R.
    6. Determination of the gene encoding the human Fcg receptor using DNA obtained from the carriers according to paragraph 5.
    7. Expression of mRNA.
    8. Preparation of a vector containing DNA sequences encoding a water-soluble fragment of the FC- receptor. , and its 0-glycosylated derivatives, as well as the expression of this soluble fragment in microorganisms or mammalian cells.
    The operations described above are carried out as follows.
    1. Isolation and purification of the water-soluble part of Fcf R.
    Human lymphoblastoid cells B, for example, RPM1-8866 cells, are isolated on the surface of Fc. R with a molecular mass of approximately 46 cd and give a fragment with a molecular mass of approximately 25 cd to the culture supernatant. As a result of the experiment using an enzyme-linked immunosorbent, two monoclonal antibodies were isolated (hereinafter referred to as the method enim). The Fc R activity released by the lymphoblastoid cells RPM1 8866 is higher in the concentrated culture supernatant than in the surface receptor-solubilized PN-40 membrane receptors, although the same number of cells, for example, 105 are used to obtain FcR / ml. In addition, after chromatography of purified supernatants on a polyacrylate gel using sodium dodecyl sulfate and elution of an Fc R activity gel, activity was detected in areas 45-46 cd and 24-25 cd. At the same time, the concentrated supernatants have a higher activity content in the region of 25 cd. Therefore, serum-free supernatant culture fluids are used as a receptor source.
    A purification of FCfR with a molecular weight of about 25 cd is carried out in columns containing 10 mg / ml of purified monoclonal antibody bound to sepharose beads. As a result of successive adsorption of culture fluid on bovine serum albumin-bound sepharose and on sepharose associated with murine immunoglobulin, 70% of the initial activity is obtained (without improvement in specific activity). After pre-adsorption, the receptor material is allowed to bind to the sepharose for 4-16 hours, the total activity of the elution product with acetic acid decreases, but the specific activity rises to 83 u / µg and the purity increases 190 times. As a result of further purification of the receptor by chromatography on a C-4 column with reversed phase and using a linear gradient of 0-65% acetonitrile and 0.1% trifluoroacetic acid, the specific activity increases to 1630 U / µg and the purity of the receptor is increased 3710 times. However, the recovery rate is only 33% of the original material. The surfactant solubilized membrane Fee R is also subjected to purification, but the specific activity thus obtained is much less than with the use of the culture supernatant. The recovery rate depends on the buffer used for the elution, i.e. by elution with 2.5% acetic acid followed by rapid neutralization, the receptor yield can be significantly increased.
    Purification of Fc R using monoclonal antibodies in the solid phase is not sufficient to obtain a pure receptor. Therefore, after elution, Fc R is subjected to further purification using reverse phase liquid chromatography. In this case, the freshly eluted material is fed onto a C-4 column and subjected to chromatography using a linear gradient of 0-65% acetonitrile. Fc R is eluted with acetonitrile at a concentration of 44-45%.
    The FcЈ R activity, obtained by liquid chromatography on a C-4 column, shows one band corresponding to a material with a molecular weight of 25 cd, which exhibits very high activity when analyzed by the method of Enim. It should be noted that the 25 kd band corresponds to a smaller Fc, R species, which is detected by the same monoclonal antibodies after surface iodination and Fcw R immunodeposition using the same antibodies. The 46 and 25 cd portions are assumed to be antigenically identical, the latter being the water-soluble portion of Pe, - R,
    2. Analysis of partial sequences of the water-soluble part.
    The sequence of amino acid residues is determined using trypsin or isyl endopeptidase. When processing with trypsin, 11 fragments are obtained, and during processing with lysilendopeptidase, 12 fragments. The following fragments were obtained:
    sh
    Met-Glu-Leu-Gln-Val-Ser-Ser-Gly-Phe-Val- (N-KOw eeoU) j
    Gly-Glu-Phe-Ile-Trp-Val-Asp-Gly-Ser-His-Val-Asp-Tyr-Ser-Asn-Trp-Ala-Pro-Gly-Glu-Pro-Thr-
    Lys-His-Ala-Ser-His-Thr-Gly-Ser-Trp-Ile-Gly-Leu-Arg-Asn-Leu-Y / G / L / LU-LtiS-20
    and Lys - 7rp -jЈ & .jfn - pffg. # Јf 3. Obtaining hybridization samples.
    Sample 1 (ddna specific for L-cells positive for Fc R).
    L cells lacking thymidine kinase (hereinafter TK), for example, LtK cells, are transfected with high molecular DNA from RPM1-8866 cells and TK. Herpes simplex virus genome. After selection of HAT TK, positive transformants are treated with biotinylated anti-FCf R antibody (8-30) and FITC-avidin and classified. The result is two transformed cell L lines. L-V-8-30 and L-VI-8-30.
    RNA is obtained from L-V-8-30 cells using guanidine isothiocyanate and cesium chloride. DNAs supplemented to mRNA from LV-8-30 cells are synthesized by reverse transcriptase on oligo / t / primer. DNA by introducing labeled alpha-P-deoxy-CTP is hybridized with a 10-fold excess of poly / A / + -RNA, derived from LtK cells. The reaction mixture was applied to a column with oxyapatite. Unbound single-stranded DNA is subjected to re-hybridization with poly / A / + - RNA obtained from LtK cells. The first strand of dna, specific for transformants of L cells positive for Fc R, is used as a probe for the detection of a gene for Fc. R in the lambda dt-yu library.
    Sample 2: synthesis of a mixture of oligonucleotides of the formula
    five
    0
    five
    0
    five
    0
    five
    3- TTSTASS TAGTTGAAAGAGT-5 A
    This digonucleotide is used as a probe to detect a gene for an Fc receptor.
    4. Isolation and identification of media containing the gene for human affinity Fc R coding.
    mRNA was isolated from RPM1-8866 cells by centrifuging in a gradient of cesium chloride, extraction with phenol and precipitation with ethanol. Poly (A) -RNA is isolated by chromatography on oligo / oT / cellulose.
    Poly / A / + - RNA is used as a template for the production of dna through reverse transcriptase and oligo / oT / primer. After RN is treated with Nase H, a second DNA strand is synthesized using DNA polymerase 1. DNA is modified using T4 DNA polymerase so as to remove the residue of 3-spikes from the first DNA strand and are ligated into an EcoRI linker. dna 1 length more than 1000 p, o. fractionation and excess linkers are removed by column chromatography. DNA is cloned into lambda-d110 phage DNA at the EcoR1 site and transformed into Escherichla coli strain 0600 hfe.
    By colony hybridization, those that are specific for Fc специфиR are identified. Using the above samples.
    Twenty-three of approximately ZOOEP clones that hybridize with both are selected. samples.
    5. Expression of DNA length.
    An EcoR1 insert containing dsc for FCi R and isolated from the described lambda gtiO clone is ligated with the digested EcoR1 plasmid vector pCEM-4, designated LE392. This vector contains two promoters SP6 and T7 in the opposite orientation, the DND K for FccR can be immediately transcribed into mRNA. DNA is digested with WatH1 and the resulting DNA is used as a template for mRNA synthesis using SP6 RNA polymerase. The resulting RNA is injected into Xenopus oocytes. After two days of incubation, the oocytes are lysed and the presence of Fc R is detected by the method of Enim using two anti-FCgR antibodies 3-5 and 8-30 that recognize different epitopes on FcЈp. As proof that pFcЈ R-1 contains the entire FcЈR coding sequence,
    51015
    Glu Gly Glu Glu Glu Glu Glu Glu Glu Glu Glu Glu Glu Glu Glu Glu Glu Glu Glu Glu Glu Glu Glu Glu Glu Glu Glu Glu Glu Glu Glu Glu Glu Glu Glu Glu Glu Glu Glu Glu Glu Glu Glu Glu Glu Glu Glu Glu Glu Glu Glu
    20 2530
    Arg Cys Cys Arg Gly Thr Gin Lie Val Leu Gly Leu Val
    CGG TGT TGC AGG CGT GGG ACT CAG ATC GTG CTG CTG GGG CTGGTG
    GCC ACA ACG TCC GCA CCC TGA GTC TAG CAC GAG GAG CCC GAGCAC
    35 4045
    Thr Ala Ala Leu Trp Ala Gly Leu Leu Thr Leu Leu Leu Leu Trr
    ACC GCC GCT CTG TGG GCT GGG CTG CTG ACT CTG CTT CTC CTGTGG
    TGG CGG CGA GAG ACC CGA GAG GAG GAG GAG GAG GAG GAG GAGACC
    50 5560
    His Trp Asp Thr Thr Gin Ser Leu Lys Gin LeuGlu Glu Arg Ala
    CAC TGG GAC ACC ACA CAG AGT CTA AAA CAG CTGGAA GAG AGG GCT
    GTG ACC CTG TGG TGT GTC TCA GAT TTT GTC GACCTT CTC TCC CGA
    657075
    CGG AAC Arg TTG AAC AG ATPAGG AAC AG TGGG ASC AG ATS AG AG ATS AG ATS AG ATS ATS AG ATS AG ATS ATS AG ATS ATS A TG A A A A A A A A A A A A A A A A A A A A Of A Of A Of A Of A All Ago
    808590
    Gly Asp Gin GGT GAC CAG CGGGG CG CAG AAA TCC CAG CG TCC CG ACG CG TG GTA
    95100105
    Glu Glu Glu Glu Glu Glu Glu Arg Ala Glu Gin Gin Glu Arg Alu Glu Glu Gin Glu A GA GTA CG A GTA CG A G A G A T A A G A C A T A A G A G A G A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A Aa Aa Aa Aa A H CI
    110115120
    Asp Lea Glu Lea Ser Trp Asa Lea Glu Ain Le Gin Ala Asp Lea GAC GGGG CTG ASCGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGG
    pDE2 vector is used, which contains both SV40 early promoters in the opposite orientation of each other, which ensures the expression of dna, in both directions. The DNA segment between both S40 early promoters is removed by hydrolysis with the EcoR1 endonuclease and replaced with the pFcЈR-1 / pDE2-FcЈ R-1 ddna insert. Cos7 cells are transfected with FcОR pOE2-dDNK. The analysis shows that 30% of the transfected cells confirm that the ddn encodes the Fc R. molecule
    6. Determination of the nucleotide sequence of ddnc for FcR.
    The complete nucleotide sequence and the sequence of amino acid residues associated with it are presented below. In addition, the coding sequence has the following formula:
    125130135
    Ser Gin Glu Alu Glu Ala Glu Ala Glu Alag Serg Ace Gla Agn Glu Ala Glu Ala Glu Ala Glu Ala Glu Ala Ack Tug Ack AG AG
    140145 150
    Asp Leu Leu Glu ArgLeu Arg Glu Glu Thr Lys Leu Arg Met
    GAT TTG CTG GAA AGACTC CGG GAG GAG GTG ACA AAG CTA AQG ATG
    CTA AAC GAG CTT TCTGAG GCC CTC CTC CAC TGT TTC GAT TCC TAG
    155160165
    Glu Gag GGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGTG
    170175-180
    Lys Trp lie Asn Phe Gin Arg Lys Cys Tug Tug Phe Gly Lys Gly AAG TGG ATC AAT TTC CAA CGG AAG TGC TAG TAG TGC GAG AAG GGC TTC ACC TAG Ata GTG AAG TCC ACC ATG ATG AAG CCG TTC CCG
    185190195
    Ala Arg Tyr Ala Cys Asp Asp MetGlu AAC AAG CAG TGG GTC CAC GCC CGG TAT GCC TGT GAC GAG ATG GAG TGG TTC GTC ACC CAG GTG CGG GCC ATA CGG ACA CTG CTG TAG CTT
    200205210
    Gly Gin CG CG CG GTC AGC ATC CAC ASC GC GAG CAG CG AAG GAC
    215220225
    ACG AAG CAT GCC AGC CAC ACC GGC TGG ATG ATG GGC CTG CGG AAC ACG AAC TGG TGG CCG AGG ACC TAG ASCGGGGG TTG
    230235240
    Lea Lys Gly Glu Glu Phe Glu Gat Gat TTG ATC ATG TGG GTG GAT GGG AGC CAT GTG AAC CTG GAC TTC CCT CTC
    245250255
    Asp Tyr Ser As A Trg Ala Pro Gly Glu Pro Thr Ser Arg Ser A Gin GAC TAC AAC AAC TGG GCT A CCG A GG A G A G A G A G A C A S G A G A G A G A S C G S E A G C A G A G A G A G A G C A S G A G A G A G C A G Of A C Of Of Of A G Of Of His
    260 265270
    Gly Glu Asp Cys Val Met Met Arg Gly SerGly Arg Trp Asn Asp
    GGC GAG GAC TGC GTG ATG ATG CGG GGC TCCGGT CGC TGG AAC GAC
    CCG CTC CTG ACG CAC TAC TAC GCC CCG AGGCCA GCG ACC TTG CTG
    275280285
    Ala Phe Cys Asp, Arg Lys Leu Gly Ala Trp Val Cys Asp Arg Leu GCC TTC TGC GAG AAG CTG GGC GGC TGG GTG TGC GAC CGG CTG CGG AAG ACG CTG GCA TTC GAC CCG CGG ACC CAC ACG CTG GGC GAC GAC
    290295300
    Ala Thr Cys Thr Pro Pro Ala Ser Glu Gly Ser Ala Glu Ser Met GCC ACA TGC ACG CCG CCA GCC AGC GAG GGT TCC GCG GAG TCC ATG CGG TGT ACG TGC GGC GGT CGG TCG CTT CCA AGG CGC CTC TAC
    305310315
    Gly Pro Asp Serg Arg Pro Asp Pro Asp Gly Arg LeG Pro Pro Gr GGA CCG GTA GGC CGC GGC GGC GGC GGC GGC CG GG GG
    Ser Ala Pro Leu His Ser TCT GCC CCT CTC CAC TCT TGA AGA CGG GGA GAG GTG AGA ACT
    A large amount of soluble with a molecular weight of 25 cd is found in the culture supernatant of RPM1-8866 cells. The N-terminal amino acid residue (Met) of soluble Fc, R is at position 150. The previous residue, arginine, is a common target for trypsin-like proteases. The C-terminal region (150-321) contains two cysteine groups (160, 163, 174 and 191 and 259, 273, 282 and 288), which probably form disulfide bonds and are a structure resistant to the action of proteolytic enzymes. Therefore, the C-terminal region (150-321) corresponds to soluble Fcr R, which is the product of the proteolytic cleavage associated with the FcgR membrane.
    7. Expression of FccR mRNA.
    Poly / A / 1 -RNA is obtained from cells of various types and analyzed for the expression
    Met ATG TAG
    Glu Gag GGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGTG
    Lys Trp lie Asn Phe Gin Arg Lys Cys Tug Tug Phe Gly Lys Gly AAG TGG ATC AAT TTC CAA CGG AAG TGC TAG TAG TGC GAG AAG GGC TTC ACC TAG Ata GTG AAG TCC ACC ATG ATG AAG CCG TTC CCG
    Ala Arg Tyr Ala Cys Asp Asp Met Glu ACC AAG CAG TGG GTC CAC GCC CGG TAT GCC TGT GAC GAC ATG GAA TGG TTC GTC ACC CAG GTG CGG GCC ATA CGG ACA CTG CTG TAG CTT
    GGGG CAG CTG GTC AGC PBX CAC AGC CCG GAG GAG CAG CAG GTC GAG
    ACG AAG CAT GCC AGC CAC ACC GGC TGG ATG ATG GGC CTG CGG AAC ACG AAC TGG TGG CCG AGG ACC A TAGGG TTG
    Lea Lys Gly Glu Phe He Trp A Lys Gly Glu Phe A TGG GTG GAT GAT GGG AGC CAT A GTG AAC CTG GAC TTC CCT CTC
    mRNA for FcЈ R. A main band of 1,700 bases long is found in B lymphoblastoid cell lines (RPM1-8866 and RPM1-1788), regge In the FL # 8-2 cell line and in two Fcформ R + L transformants of the cell, but not in Fc-R cells, including two lymphoma lines, T cell lines (CEM), and in transformants cells that express another B cell antigen (CD20). In addition, mRN K dl is not detected in normal T cells, whereas normal B cells express the same amount of Pc-P mRNA as B lymphoblastoid lines.
    8. Obtaining a vector containing a DNA sequence encoding a water-soluble receptor fragment.
    The water soluble portion of the receptor contains at least the following sequence:
    Asp Serg Gag Gag Gag GG GG CCC GC GG GG CCC ACC AGC CGG AGC GG CG GGG
    Gly Glu Glu Asp Cys GAG GAG TGG GTG ATG GGG GGG TCC GGT CG TGG AAC GGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGG
    Ala Phe Cys Asp Arg Lys Leu Gly Ala Trp Val Cys Asp Arg Leu GCG TGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGG
    Ala Thr Cys Thr Pro Pro Ala Ser Glu Gly Ser Ala Glu Ser Met GCC ACA TGC ACG CCG CCA GCC AGC GAG GGT TCC GCG GAG TCC ATG CGG TGT ACG TGC GGC GGT CGG TCG CTT CCA CG AGG CTC CG AGG TAG
    Gly Pro Asp Serg Arg Pro Asp Pro Asp Gly Glag Arg Lea Pro Thr Pro Gpc Gt Gag Cg Gg Cg Cg Gtg Ggg Ggg
    Ser Ala Pro Le Ser Ser TC GTC CCT CTC CAC TCG TGA AGA CGG GGA GAS GTG AGA ACT
    The membrane enclosing region does not function as a signal sequence in conventional recombinant processes. Therefore, to isolate a water-soluble protein from a suitable host, it is necessary to use a eukaryan signal sequence. Such a signal sequence may additionally be used in the position before the DNA coding for the water soluble portion.
    A new recombinant water-soluble fragment is obtained, having at least one 0-glycosylation site, as well as new plasmids containing these DNA sequences, and psFc R-1.
    A DNA sequence is obtained in which no DNA is present for at least part of amino acids 1-148 franmete, so that there is only a sequence that encodes a part of the membrane protein, and a part that encodes a water-soluble fragment of the entire DNA receptor.
    A portion of the DNA sequence encoding amino acids 1-148 is replaced with a suitable DNA fragment encoding the eukaryotic signal sequence.
    EcoR1 BamH1 insert dDNK5R-2, length 1.2 kb. is obtained by digesting pBSF-2.38 with Hindlll and VatSh. The obtained fragment, containing all BSF-2 dna, is digested with Hinfl and the 3 ends are processed
    five
    0
    five
    0
    five
    0
    five
    fragment maple DNA polymerase. After digestion with Kpnl, the resulting 110 bp Kpnl-Hinf fragment containing the dominant BSF-2 sequence is cloned into pGEM4 after digestion with Kpnl and Smal. One of the selected clones is propagated and designated as pBSF2-L8. pBSF2-L8 is digested with the help of the WatH1 and the 3 ends are treated with the Klenow DNA polymerase fragment. Overcooked Hindlll and Pstl, FcЈR DNA is cloned into digested WatH1 and Pstl pBSF2-L8. One of the selected clones is propagated and labeled as.
    In order to compare the biological activity of proteins obtained by clones of pFcЈR-I, pZPc R-1, p ANFcЈ R-1 and pANFcrR-2, the plasmids are subjected to linearization by digestion using suitable enzymes, for example, and psFo. R-1 with BamH1, a p A NFc € R-1 and p A NFcg R-2 with EcoR1. The resulting fragments were used as a template for mRNA synthesis using SP6 RNA polymerase. The mRNA obtained is injected into the oocytes of xenopus leavis. After two days of incubation, the activity of FC, R in mutant supernatants and oocyte lysates was determined by Enim method using anti-FcjR antibodies 3-5 and 8-30, which recognize two different epitopes on FCgR. In PB lysates and culture supernatant
    io
    oocytes into which transcripts are injected. pANFcЈR-1 npANFcЈR-2, activity is not detected, whereas FcЈR activity is determined in supernatants and PBS lysates after injecting pSFcgR-1 fragments.
    The water-soluble FcЈR fragment from oocytes exhibits the ability to bind immunoglobulin E using a modified enim method, which consists in using anti-Fc R antibodies 3-5, immunoglobulin E and human AP-anti-immunoglobulin. At the same time, the supernatant of oocytes into which pSFc R-1 mRNA was injected was incubated on antibody-coated 3-5 plates, which were then successively incubated with human immunoglobulin E and AP-anti-immunoglobulin E. The Fc R released from the oocytes formed a complex with immunoglobulin E. As a control, binding to the untransformed supernatant from oocytes, buffer and supernatant from RPM1-8866 cells is carried out.
    Oocyte supernatant is subjected to further experience in determining the ability to inhibit the formation of rosettes between fixed ox RVS coated with human immunoglobulin E and SKW6-CL4 cells carrying FCg R on their surface.
    Express the Fc FR sequence under the control of the promoter of the bacteriophage Lambda / Pi /.







    EBI-496:
    5 GAACTGCAGGTGAGCTCTGGTTTCGTTTGCAACACTTGCCCGGAAAAATG
    EBI-497:
    3 CTTGACGTCCACTCGAGACCAAAGCAAACGTTGTGAACGGGCCTTTTTACCTAG 5
    Sau FOR
    in order to recover the corresponding formula codons
    GluLeuGlnValSerSerGlyPheVfelCysAsnThrCysProGluLygTrp 5 GAACTGCAGGTGAGCTCTGGTTTCGTTGGACACACTTGCCCGGAAAAATGE
    3 CTTGACGTCCACTCGAGACCAAAGCAAACGTTGTGAACGGGCCTTTTTACCTAG 5
    ZaIZA
    sixteen
    The temperature-sensitive allele of this repressor gene may include a vector that contains the complete PCR sequence. If the temperature is raised to 42 ° C, the repressor is inactivated and the promoter is expressed at its maximum level.
    The promoter-operator systems or their parts can be used, for example, arabinose operator, colicin-Ech operator, galactose operator, alkaline phosphatase operator, trp operator, kislose A-operator, tac-operator and others.
    Plasmid pRH 100 is digested with the power of Sstl. Then the 3-lugs are removed and the linearized plasmid is dephosphorylated. After extraction with phenol and chloroform, electrophoresis, electroelution and precipitation, the linearized vector is ligated with the insert obtained as follows.
    The plasmid pGEM4-FcЈR obtained by digesting the EcoRI insert of LE 392 plasmid with Hindlll and reintroducing it into
    5 pGEM4 long fragment EcoR1 and Hindlll, digested with EcoR1 and Hindlll. Then, the protruding 5-ends are blunted with a Klenow fragment of DNA polymerase 1, after which the 5-phosphate groups are removed. After
    Q purification of the resulting fragment is hydrolyzed with SauAI and a larger fragment is isolated. Since, as a result of this operation, not only the 5 region, but also the nucleotides for the first N terminal amino acids of the water soluble part are removed, two oligodeoxynucleotides of the formula
    17
    (without the ATG codon, as it is contained in the promoter-linker system of the plasmid pER 103).
    Inserts are ligated with a linearized DNA vector and transformed into E. coli HB101, ampicillin-resistant colonies are selected. Plasmids are examined by restriction analysis. Plasmid selected and denoted as RN 246.
    In addition to prokaryotes, yeast cultures can be used.
    The genes of the water soluble portion of the human Fc € R receptor with amino acids 150-321 can be expressed in yeast, if the ADH1 promoter is used together with the ADH1 terminator. Yeast expression vector can be obtained by obtaining:
    a) a plasmid containing a yeast ADH1 terminator;
    b) a plasmid containing a yeast ADHI promoter and a yeast AOSH terminator;
    c) a plasmid containing a yeast ADHI promoter, a gene encoding a yeast dominant MF a sequence, a multi-cloning site and a yeast ADH1 terminator;
    d) a plasmid containing the ADHI promoter, the sequence encoding the water-soluble part of the human FC ... R receptor of small affinity, and the AOH1 terminator;
    e) transformation of the obtained plasmid DNA into a yeast vector.
    You get a plasmid containing the expression cassette without the dominant MFa sequence, and a plasmid containing the expression cassette with the dominant MFa sequence.
    The operations are carried out as follows.
    a) an ADH1 terminator was isolated from plasmid plD14. The ADHI terminator is subcloned into the plasmid pUC18 at the Hindlll sites and
    Title
    MF I TCGAGCCTCATATCAATGAGATTCCCATCTATTTTCACTGCTGTTTTGTT (50-meR)
    KP 2 AGCAGCGAACAAAACAGCAGTGAAAATAGATGGGAATCTCATTGATA fGA GGC (53-MSR)
    MF 3 CGCTGCTTCCTCCGCTTTGGCTGCTCCA oTCAACACTACTACTGAAGACG AAACTGCTCAAATTCCAGCT (70-mer)
    72753318
    Sphl and get the plasmid pWS 214S4. After hydrolysis of pWS 214S4 with Sphl and Sail restrictases, the smaller fragment was isolated by agarose gel electrophoresis. 5The fragment containing ADHI terminator is ligated with vector DNA obtained by digesting Bluescribe M13 with Sail and Sphl restrictases, followed by purification by electrophoresis on 10 agarose gel.
    The ligated DNA is transformed into E. coli IM101 and ampicillin resistant colonies are selected. The plasmid of these colonies is designated pRH241. This plasmid contains 15 containing approximately 340 bp. fragment with AON1-terminator.
    b) AOH1 promoter isolates the plasmid pY-IDB-HulFN-omega-T, i.e. plasmids for obtaining human interferon 20 omega 1. The plasmid is digested with Sphl restriction enzyme, then the 3-spike is removed using DNA polymerase 1 in the presence of dGTP and re-hydrolyzed with Xho1 restriction. Agarose gel electrophoresis 25 isolates a fragment of 400 bp. and it is doped with an adapter pair of the formula
    EB 1-410: 5 AATTGGAAGGATC 3 EB 1-429: 3 CCTTCCTAG-p5
    At the sticky end are ligated with an adapter pair of the formula EB 1-418: 5 p-TCGAGCACGTGGTAC 3 EB 1-424: 3 CGTGCAC5
    The ligation reactions were carried out simultaneously. After purification by electrophoresis in agar-35 gels, DNAs are ligated with plasmid pRH241 DNA at EcoR1 and Krp1 sites and transformed into E. coli. Colonies are examined for the presence of a plasmid with the correct construction, which is designated as pRH242. before c) Chemical synthesis of the MFa peptide is carried out using pre-synthesized oligonucleotides:
    Sequence
    thirty
    172753320
    MF 4 CAGCTTCAGCTGGAATWGAGCAGTTTCGTCTTCAGTAGTAGTGTTGACT
    GGAGCAGCCAAAGCGGAGGA {7O-mvr)
    MF 5 GAAGCTGTCATCGGTTACTCTGACTTGGAAGGTGACTTCGACGTTGCT (48 -wet)
    MF 6 GCAAAACAGCAACGTCGA.GTCACCTTCCAAGTCAGAGTAACCGATGA (48 -met)
    MF 7 GTTTTCCCATTCTCCAACTCCACTAACAACGGTTTGTTGTTCATTAAC ACTACTATTGCATCGATTGCT (69th IT))
    MF 3 CCTTAGCAGCAATCGATGCAATAGTAGTGTTAATGAACAACAAACCGTTG TTAGTGGAGTTGGAGAATG {69-mer)
    MF 9 GCTAAGGAAGAAGGTGTTTCTTTGGACAAGAGGCCTCTGCAGGAATTCT (49-Uej)
    MF 10 CTAGAGAATTCCTGCAGAGGCCTCTTC-TCCAAAGAAACACCTTCTT
    (46-A / ep)
    Oligonucleotides MF2 - MF9 are phosphorylated. After stopping the reaction by heating, the following mixtures are obtained: MF1 and MF2; MF3 and MF4; MF5 and MF6; MF7 and MF8; MF9 and MF10. After heating and cooling, the five solutions are combined and ligated. To the resulting solution was added the linearized DNA of the plasmid pRH 242 obtained by digestion with Xho1 and XHa1 and subsequent purification by electrophoresis. The solution is ligated and transformed into E. coli IM101. Plasmids of the resulting colonies were examined by digestion with Xho1 and XHa1. Moreover, those plasmids that contain an insert of approximately 290 bp in length. cloned from M13mp8, followed by sequence analysis. Plasmid containing
    ATG GAA TTG CAA GTT TCC TCT ACT TGT CCA GAA AAG TG
    CTT AACGTT CAA AGG AGA CCA
    ASA GGTCTT TTC ACC TAG ISau3A
    The correct insertion is selected and designated pRH243.
    d) The sequence encoding the water-soluble portion of the human FcЈR receptor of small affinity is isolated from plasmid pGEM4 by digestion with Hindlll and EcoR1 and the sticky ends are supplemented using E.N. coli Klenow DNA polymerase 1 fragment. The large fragment is subjected to phosphosphorylation and purification, and then hydrolyzed with SauSA. Since this removes not only the 5 region, but also the nucleotides of the first 18 N-terminal amino acids of the water-soluble fragment, starting from amino acid 150, they synthesize two oligodeoxynucleotides
    21172753322
    in order to restore the complete gene using the yeast codon of the formula
    Met 5 TCGAGCTCATATACA PBX
    3 CGAGTATATGT TAG
    Xhol
    155160165
    Glu Leu Gin Val Ser Ser Gly Plu Val Cys As The Thr Cys Pro Glu GAA TTG CAA GTT TCC TCT. GGT TTC GTT TGT AAC ACT TGT CCA GAA CTT AAC GTT CAA AGG AGA CCA AAG CAA ACA TTG TGA ACA GGT CTT
    Trp TG ACC TAG Sau3A
    3 5
    The resulting oligonucleotides are renaturised and ligated with a SauSA and EcoRf fragment using T4 DNA ligase.
    The insert is ligated with DNA, plasmids pRH242 at sites XA1 and Xho1 and transformed into E. coli IM101. Plasmids of the obtained colonies are examined by restriction analysis. The plasmid containing the correct insert is selected and designated as pRH244.
    e) The sequence encoding the water soluble portion of the human low affinity Fc R receptor is isolated from
    EBI-430:
    5 ATGGAATTGCAAGTTTCCTCTGGTTTC ATG GAA TTG CAA GTT TCC TCT GGT TTC GTT TGT AAC ACT TGT CCA GAA AAG TG
    EBI-437:
    3 TACCTTAACGTTCAAAGGAGACCAAAG TAG CTT AAC GTT CAA AGG AGA CCA AAG CAA ACA TTG TGA ACA GGT CTT TTC ACC TAG 5
    Sau3A
    i.
    in order to restore the complete gene using the yeast codon of the formula
    I
    50met
    5 ATG
    3 TAG
    Glu Lea Gin Val Ser Ser Gly Plu Val Cys Cr GnGTA TGT AAC A TGT AAC A TGT AAC A TGT ASC A TGT A TGT A TGTA
    I
    plasmids pGEM4-FCgR by digestion with Hindlll EcoR1 and the ends are supplemented using the Klenow fragment of DNA polymerase 1 from E. coli. The smaller fragment is subjected to dephosphorylation and purification. Since Hindlll cuts DNA FcЈR approximately 50 p. O. after the first amino acid, the insert is hydrolyzed with SauSA. Since this not only removes the 5-region (towards the end), but also the nucleotides of the first 18 N-terminal amino acids of the water-soluble fragment, two oligonucleotides of the formula
    Trp TG LSS TAG Sau3A
    31 5
    The oligonucleotides are renatured, a fragment of SauSA to EcoR1 is added and ligated with DN K ligase T4. The resulting fragment is purified by electrophoresis, electroelution and precipitation by known methods.
    The insert is ligated with plasmid pRH243 DNA at EcoFM sites, and Stul and transformed into E. coli IM101. The resulting colines are examined by restriction analysis. The plasmid containing the correct insert is selected and designated as PRH245.
    e) The plasmids pRH244 and pRH245, consisting of promoter ADH1, MFa dominant gene (only in the case rRN245), water-soluble gene and terminator FcgR ADH1, gidrolizuyutrestriktazamiShpsLN and BamHI and ligated with a yeast plasmid rYuV207ili YEp13.
    Plasmid LE392 or pGEM4 (pFct R-1) was digested with Hindlll and EcoRL. DNA fragment starting at nucleotide 584, cloned into plasmid pGEM4, at Hindlll and EcoRL sites. One of the selected clones was propagated and designated as pANFc R-1.
    Plasmid LE392 or pGEM4 (pFcЈ R-1) is hydrolyzed with EcoR1 and a fragment containing approximately 1.7 kb in length of DNA (Fc-R) is obtained. The resulting fragment is partially digested with SauSA in order to remove the DNA sequence encoding the cytoplasmic domain, for example, for amino acids 1-23, and the DNA fragment, starting from nucleotide 254, is ligated with a 26-dimensional linker of the formula
    5 -GATCTGAGTCATGGTACCATGACTCA-3 in order to restore the PBX start codon at the b-end and the restriction site Kpp1. The ligated fragment is cloned into plasmid pGEM4 at the sites of Krp1 and EcoRL. By hybridization, clones are selected from which the region of the putative cytoplasm domain is absent. One clone is selected, propagated and designated as p A NFCgR-2.
    General materials and methods .:
    Monoclonal anti-Fc R antibodies 3–5 (yi) and 8–30 (/ g) were obtained by hybrid5
    dization of P3U1 myeloma with mouse spleen cells immunized with RPM1-8866 cells. Antibody 8-30 expands, recognizes the epitope adjacent to the Fc иммун receptor immunoglobulin binding site, and can block the binding of immunoglobulin to lymphoblastoid cells 8866. Antibody 3-5 recognizes another epitope and cannot effectively block the binding of immunoglobulin to its receptors . These antibodies precipitate, under reducing and non-reducing conditions, polypeptides with a molecular weight of 46 cd and 25 cd. Monoclonal antibodies are purified by precipitation with 50% saturated ammonium sulfate and subsequent gel filtration using separose 6B (in the case of immunoglobulin M)
    Q or ion exchange chromatography using Sephadex (in the case of immunoglobulin C1). Polyclonal mouse immunoglobulin G is isolated in the same manner.
    Pre-cleaned material
    5 FcЈR is fed to a column equilibrated with water containing 0.1% trifluoroacetic acid and eluted with a linear gradient of acetonitrile containing 0.1% trifluoroacetic acid. At the same time, aceto-nitrile is used in the amount of 0.5 ml / min with a linear gradient from 0 to 65% over 60 minutes. The eluted material is frozen and lyophilized before being tested for activity by the method of Enim.
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    The synthesized oligodeoxynucleotides are purified by polyacrylamide gel electrophoresis.
    PRI me R A. Obtaining plasmids pY-IDB-HulFN-OMeraL
    a) Obtaining the yeast ADH1 promoter.
    8 μg of the plasmid RÖZUZ is digested with BamHI and HHY1 in 500 μl of solution. A promoter length of about 1450 p. separated on a 1% agar gel and precipitated. The fragment is suspended in 40 μl of TE buffer (10 mM Tris, 1 mM ethylenediaminetetraacetic acid (hereinafter EDT), pH 8). RESOZ plasmid is obtained by introducing into PUC18 digested BamHI and Xho1 plasmid AH 11 about 1450 bp long.
    b) Preparation of the vector ryuV207.
    10 µg of pHUV207 are linearized with BamHL in order to prevent subsequent re-binding, the 5-terminal phosphate groups are removed by phosphatase treatment of the calf intestine. The linear form is separated in 1% agar,
    25
    gel from uncut plasmid. The resulting DNA vector is dissolved in 20 µl of TE buffer.
    c) Expression vector for interferon type omega 1.
    50 μl of the plasmid pRHW12 are linearized with Hindlll, treated with the Klenow fragment of DNA polymerase 1 and purified by agar gel electrophoresis. To obtain connecting ends with the promoter, a linker Xpo1 is attached to the ends of the linear pRHW12.
    By treating with 100 units of Hpo1, Xho1-specific adhesive 5-tabs are free. Supplied with Xhol-ends, linear pRHW12 is purified by agar gel electrophoresis, 5 µl of promoter is added (from step a), ligated and precipitated. The DNA is suspended and cut with the aid of WatH1. DNA is obtained by purification in agar gel.
    d) Ligation of fragments.
    The resulting expression vector is obtained by leading a fragment of a nanoparticle (promoter and interferon gene of the omega 1 type) with the vector ryu207.
    d) Transformation.
    E. coli HB101 cells were transformed by adding a solution of ligated DNA and cultured on LB agar plates containing 100 µg / ml ampicillin. Twelve of the clones obtained are selected and the plasmids contained therein are isolated. After analysis of the plasmids using a restriction analysis and electrophoresis on an agar gel, a plasmid having the correct design is selected. It is designated as pY-IDB-HulFN-omega 1.
    PRI me R B. Design of the expression plasmid rRNA.
    The plasmid pER103 DNA was digested with the Hindlll endonuclease, the b-terminal phosphate residues were removed with calf intestinal alkaline phosphatase (FTC). The DNA is precipitated by the addition of a 0.1 vol% ZM sodium acetate solution with a pH of 5.5 and ethanol. The DNA precipitate is centrifuged and washed with 70% ethanol solution.
    Synthetic oligonucleotides o (AGCTTAAGATGAGCT) and o (SATSTTTA) are phosphorylated by T4 polynucleotide kinase (PNA).
    The plasmid and phosphorylated oligonucleotide solution is mixed and heated to 70 ° C for 5 minutes, then cooled to 0 ° C and ligation buffer (500 mM Tris, pH 7.5), 100 mM magnesium chloride, 200 mM dithiothreitol (DTT) is added , 1 mM at ATP, 500 μg / ml of serum albumin
    72753326
    cattle (ACC), T4-DNA ligase. The reaction is carried out at 4 ° C for 40 hours. It is stopped by heating at 70 ° C for 10 minutes.
    The ligation product is digested with the SaC1 endonuclease, then ligated and transfected into E. coli HB101 cells. Bacteria are applied to LB agar plates containing 50 µl / ml ampicillin.
    12 of the bacterial colonies are randomly selected, plasmids are isolated and cut with the endonuclease Sad.
    The presence of a linear DNA molecule with a length of 4400 p. confirms that month 5 that recognition SaC1 has been inserted into the plasmid. One of the plasmids is randomly selected. The plasmid, designated as pRNAH, is digested with endonucleases EcoR1 and WatH1, divided into 1% agar
    20 gel and a smaller fragment are isolated from the gel. This EcoR1-BamH1 DNA fragment is approximately 460 bp long. subjected to analysis.
    ExampleB Construction of plasmid pANFc R-1.
    25 days K is digested with Hindlll in 50 μl and then with EcoR1. An EcoR1-Hindlll fragment is obtained containing a region of 1 kb in length. soluble dcn FcgR. Digested DNA is subjected to
    30 by electrophoresis in 1% agar gel, the Hindlll-EcoR1 fragments are isolated by electroelution followed by precipitation in 70% ethanol. PGEM4 is digested with Hindlll and EcoR1, extracted with phenol and precipitated with ethanol. Fragments
    35 are ligated and transfected into E.coli (MC1065). One clone is selected, propagated and designated as p ANFcf R-1.
    EXAMPLE G. Construction of plasmid pANFcR-2.
    40 pFcg R-1 is digested with the restriction enzyme EcoR1, subjected to agar gel electrophoresis (1%). EcoR1 fragment of approximately 1.7 T. p. is isolated by electroelution followed by precipitation with ethanol at 45–80 ° C for 1 hour. The fragment is digested with restriction enzyme Ac1 and partially with restriction enzyme SauSA, followed by extraction with phenol and precipitation with ethanol to obtain the Sau3A-EcoR1 fragment. DNA fragment
    50 is ligated with a synthetic linker (S -GATCTGAGTCATGGTACC- ATGACTCAGATCGTGCTG-3). DNA is extracted with phenol and precipitated with ethanol. The fragments are dissolved, digested with Krp1 strictase, and extracted with phenol, followed by precipitation with ethanol. The excess linker is removed by gel chromatography (Biogel A 50 m). pGEM 4 is digested
    27
    with restriction enzyme Krp1, then EcoRL Fragments associated with synthetic linkers are ligated with Krp1 and EcoR1 with the pGEM4 fragment and transfected in E. coli (MC1065). & iHPANFcf R-2, which lacks only the cytoplasmic domain, is isolated and propagated using the well-known colony hybridization method. PRI me R 1.
    a) Isolation of crude Fc R from a cultured short-sludge fluid.
    RPM1-8866 cells are cultured in RPM1-1640 medium containing another 10% fetal bovine serum, 100 units / ml penicillin and 100 μg / ml streptomycin, at a density of 1x10 ° cells / ml and cell viability of 95-99%. The cells are harvested, centrifuged at 5000 rpm for 15 minutes, washed three times with Hank's BBS solution and cultured in serum-free medium for 48 hours at the same density. The culture supernatant is collected and 1 mM phenylmethylsulfonyl fluoride (PMSF), 0.02% sodium azide and 200 units / ml aprotein are added. The culture fluids are stored at 4 ° C during concentration, which is carried out by filtration. Then the material, which is concentrated 200-300 times, is centrifuged in order to remove insoluble material. This gives crude FcЈ R.
    b) Isolation of FC R from cell lizatov.
    RPM1-8866 cells (2x109 cells) were washed four times with PBS buffer (containing 0.5% NP-40 surfactant) and lysed in 10 ml of lysis buffer and 1 mM PMSF for 45 minutes on ice with occasional stirring. An additional 10 ml of lysis buffer is added and the reaction is continued on ice for 30 minutes. Lysate centrifuged pr 37000 rpm for 45 min at 4 ° C. The lipid layer is carefully collected, the supernatant is collected and stored at -70 ° C.
    PRI mme R 2. Immunopurification.
    The culture supernatant concentrate (see Example 1a), equivalent to 5-10 liters of culture, is sequentially adsorbed onto 2 ml of a Sepharose gel bound to bovine serum albumin bound to human transferrin Sepharose gel and bound to a mouse immunoglobulin Sepharose gel each time for 1 h at 4 ° C. The effluent collected from the last gel is then fed to 2 ml of anti
    1727533
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    S
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    Fcf R (3-5) bound to sepharose. They are monoadsorbed for 4-16 hours at 4 ° C. The gel is fed to the column, the effluent is collected and the gel is successively washed with 150 ml of buffer A (10 mM Tris-HCl, pH 7.5, 0.5 M sodium chloride, 0.5% NP-40), 150 ml of buffer B (10 mM Tris-HCl, pH 7.5, 0.1% NP-40), 150 ml of buffer B (YuMM Tris-HCl, pH 7.5), eluted with 25 ml 2.5% by volume acetic acid and immediately neutralized in 2 M Tris-HCI with a pH of 8.0. The resulting material is stored at -80 ° C, if it is not directly purified by liquid chromatography. Then, the eluate is fractionated by reverse phase liquid chromatography on a C4 column using a linear gradient of 0-65% acetonitrile containing 0.1% trifluoroacetic acid.
    PRI me R 3. Analysis using an enzyme-linked immunosorbent (Enim method).
    FcgR activity is measured by determining its ability to bind to monoclonal antibodies 3-5 and 8-30. Microtiter plates are first coated with 100 µl of a sample of monoclonal antibodies 3-5 (in bicarbonate buffer) with 0.1 M sodium bicarbonate, pH 9.6, and then incubated overnight at 4 ° C, washed four times with phosphate buffer containing 0.05% Tween 20, followed by the addition of 100 µl of the test sample diluted with a buffer of pH 8.1 (0.05 M Tris-HCl, 1 mM magnesium chloride, 0.15 M sodium chloride, 0.05% by volume Tween 20.0.02% sodium azide and 1% ACC). Microtiter plates are incubated at room temperature for 2 hours and washed. 100 µl of pre-titrated and diluted goat-anate-mouse-immunoglobulin M and alkaline phosphatase conjugate are added. Plates are incubated at room temperature for 2 hours and washed, 100 µl of substrate buffer (0.05 M sodium bicarbonate, pH 9.8, 10 mM magnesium chloride hexahydrate) containing 1 mg / ml of disodium phosphate, and - Limetrate every 30 min for 2 h at 405 and 620 nm.
    PRI me R 4. Hydrolysis of the receptor using lysilendopeptidase and separation of peptides.
    Purified FcЈR is digested with Achromobacter lyticus lysyl endopeptidase in 50 mM Tris-HCl buffer with a pH of 9.5 and obtained by chromatography on oligo-dT-cellulose. Radio-labeled DNA is synthesized from poly / A / + - RNA using P-dCTT. Labeled DNA
    35 ° C for 6 h with a weight ratio of enzyme to substrate equal to 1: 500. Lyophilized peptide fragments, purified by liquid chromatography.
    Separation of peptides by reverse phase liquid chromatography.
    The separation of the peptides is carried out by liquid chromatography on a C4 column. The peptides were eluted under the following conditions: a linear gradient of a mixture of 2-propanol and acetonitrile in a volume ratio of 7: 3 in the range from 0 to 35% for 1 hour in 0.1% trifluoroacetic acid, consumption 1, 0
    Met-Glu-Leu-Gin-Val-Ser-Ser-Gly-Phe-Val-,
    Gly-Glu-Phe-Ile-Trp-Val-Asp-Gly-Ser-His-Val-Asp-Tyr-Ser-Asn-Trp-Ala-Pro-Gly-Glu-Pro-Thr-,
    Lys-His-Ala-Ser-Hi s-Thr-Gly-Ser-Trp-Ile-Gly-Leu-Arg-Asn-Leu-Asp-Leu-LysLys-Trp-Ile-Asn-Phe-Gln-.
    P p and measure 5. Obtaining the oligonucleotide formula
    Z-TT ACC TAG TTЈ AA GT -5 A
    The oligonucleotide is synthesized using synthesizer A 1, demethylated with a mixture of thiophenol, triethylamine and dioxane in a 1: 2: 2 ratio at room temperature for 90 minutes and washed with methanol (10x1 ml of methanol). After purification of the demethylated oligonucleotide on a porous glass and removal of the protective group with concentrated ammonia, the product is dried at room temperature for 1 hour and at 55 ° C for 16 hours.
    Further purification was carried out with a 20% acrylamide gel in the presence of 8 M urea, with elution with TBE buffer (10.9 g of tri (hydroxymethyl) aminomethane, 5.5 g of boric acid and 9.3 g of ethylenedinitrile tetraacetic acid disodium salt) in 1 l).
    Electrophoresis was carried out in a gel (40x25x0.01 cm) at 50 watts. The 17-mer band is cut, eluted with water and desalted on a Sephadex C 25 column with water. Fractions containing 17-mer are collected and
    0
    five
    ml / min Fractionated peptides are collected by determining the absorbance at 215 nm.
    Determination of amino acid residues and their sequence.
    The analyzes are carried out after the hydrolysis of the peptide fragments in 5.7 HCI at 110 ° C for 22-24 hours in evacuated closed tubes. The amino acids of phenylthiohydantoin (FTG) are determined by reverse phase liquid chromatography. The following amino acid sequences are determined:
    thirty
    dried Yield: 285 µg (optical density 260: 7.7).
    PRI me R 6. The establishment of L-cell
    transformants
    One million LtK cells are transfected with 150 ngvirus Herpes simplex from the tK gene and 20 µg of high molecular weight genomic DNA from RPM1-8866 cells. After
    storing in a HAT medium for 10 days takes cells. Cells I, which are resistant to HAT, are harvested, etched with biotinylated anti-Pc, .P (8-30) and avavidin bound to fluorescein isothiocyanate (ITF), and are classified for several cycles. transfection. In independent transfections, two transformant lines are obtained: L-V-80-30 and L-V1-8-30.
    Example. Cloning dCNA Fc, R
    Sample 1.
    RNA is isolated from the L-V-8-30 transformant by a known method using guanidine and cesium chloride. Poly / A / -RNA
    31
    subjected to renaturation of excess poly / A / -RNA of untransformed LtK cells and fed to the oxyapatite column. Single stranded DNA is collected and used as sample 1.
    Sample 2.
    The 17-mer oligonucleotides, complementary to the mRNA sequence that encodes the amino acid fragment Lys-Trp-lfe-Asn-Phe-GIn, and containing a mixture of 24 possible sequences, are radiolabeled x 32 P-ATP using T4 polynucleotide kinase. Double-stranded DNA is synthesized from poly / A / + - RNA obtained from RPM1-8866 cells. After treatment with methylase EcoR1 and DNA polymerase T4 double stranded dna more than 1000 p. clone into EcoR1 site lambda gt10 using EcoRI-linkers. Two sets of phage replica filters were prepared. One set of filters is hybridized with 17-mer P-labeled oligonucleotides (sample 2). Another set of filters was hybridized with 32P-labeled DNA, specific for FcfR positive L cells (sample 1), at 68 ° C overnight at 6xSSC (1xSSC: 0.15 M sodium chloride, 0.015 M trisodium citrate, pH 7.0 ), then washed with 0.1xSSC and 0.1% sodium dodecyl sulfate. The spots that hybridize with both samples are isolated.
    Example Expression of DNA FcfR.
    For the expression of DNA in pGEM4 using the SP6 promoter, mRNA is synthesized using SP6 RNA polymerase using digested WatH1 pPc R-1 as a template. 10 ng of mRNA is injected into one oocyte, and mouse BSF-1 mRNA is similarly obtained as a control experiment, which is injected into another oocyte. After incubation for 2 days in Bart's medium at 20 ° C, the oocytes are lysed in 50 µl of lysis buffer (10 mM Tris-HCfc pH 7.5.0.5 M sodium chloride, 0.5% NP-40). Oocyte lysates are tested for Fc R activity using the enim method, which uses two anti-PcЈ antibodies (3-5 and 8-30). As a control experiment, a concentrated supernatant obtained from RPM1-8866 cells was used.
    To express Fcg R DNA in Cos-7 cells, the pFc R-1 insert is cloned into the EcoR1 site of the pDE2 vector. 5x10 Cos-7 cells were plated on 60 mm tiles the day before transfection, which was performed using 2 µg of plasmid DNA in 1 ml of buffer consisting of 25 mM Tris-HCf (pH 7.5), 137 mM chloride sodium, 5 mM potassium chloride, 0.6 mM
    ten
    72753332
    sodium hydrogenphosphate, 0.5 mM magnesium chloride and 0.7 mM calcium chloride, and 500 µg of dextran supplied with diethylaminoethyl groups. After an hour of incubation at 37 ° C, the solution is replaced with DMEM containing 10% FCS and 150 mM chloroquine, incubated at 37 ° C for 3 hours and then replaced with fresh DMEM containing 10% FCS. After 48 hours, the cells are treated with phycocyanin-associated anti-Fc.R antibody (3-5) and biotinylated immunoglobulin E, they are developed with ITF-avidin and analyzed.
    PRI me R 9. The definition of the nucleotide f 5 sequence dDN K Fc, - R.
    The pPcЈR-1 insert is digested with the enzymes Hindlll and Pvull. DNA is subcloned into M13 and analyzed.
    20 I
    I'll try it on. Analysis of mRNA RSG R.
    Poly / A / + - RNA from different cells is separated on an agar gel (1%), fed to a nitrocellulose bundle, and hybridized with an insert of pFc-R-1 labeled in nick translation. For BSF-1 induction, 100 million B or T cells are cultured in the absence or in the presence of 10 µg / ml of immunoglobulin E and 50 µg / ml of recombinant human BSF-1 10 µg of total RNA
    30 extracted and analyzed.
    Example Expression of the water soluble portion of the human Fc receptor ,. small affinity in yeast.
    Step 1. Obtaining plasmids PjDB-244 and pjDB-245.
    a) Obtaining plasmid pRH 241 containing yeast ADH1 terminator.
    35
    Getting a vector.
    10 μg Bluescribe M13 + are double digested with 20 units of Sail and 20 units of Sphl. The reaction is stopped by adding 1/25 volume of 0.5 M
    ethylenedinitrilotetraacetic acid disodium salt and heating at 7 ° C for 10 minutes. The cut vector is purified by agar gel electrophoresis (1% agarose, 1xTVE buffer consisting of
    6.05 g / l of tris-oxymethylaminomethane, 3.1 g / l of boric acid, 0.37 g / l of EDTA and containing 0.5 μg / ml of ethidium bromide at 4 V / cm). After detection of the DNA band using ultraviolet light (254 nm), the DNA is isolated by electroelution
    using paper DE81 and cleaned by precipitation with ethanol. DNA was dissolved in 10 µl of TE buffer (10 mM Tris, pH 8.0, 1 mM EDTA).
    33
    Receiving insert.
    10 µg of pWS214 4 are digested with 20 units of Sphl and 20 units of Sail. A smaller fragment containing an ADH1-terminator is isolated by agar gel electrophoresis, electroelution and precipitation. The obtained DNA is dissolved in 10 µl of TE buffer. 1 μl of vector DNA and 1 μl of the DNA insert are ligated with UT-DNA ligase in 10 μl of solution. 3 µl of the ligase mixture is used to transform E. coli IM101 SupE, thi, del / lac-proAB /, F traD36, proAB, tectg, lacZ-del M15, lmbda-minus). After ampicillin selection, the plasmids are isolated and digested with Sphl and Sa (l. The fragments are separated on an agar gel. One of the plasmids is selected and is designated pRH 241.
    b) Obtaining plasmid pRH 242 containing the yeast ADH1 promoter and yeast ADHI terminator.
    Getting a vector.
    Plasmid pRH 241 is double digested with EcoR1 and Krp1. The smaller fragment is removed from the vector portion by agar gel electrophoresis, electroelution and precipitation. The resulting fragment was dissolved in TE buffer.
    Receiving insert.
    Plasmids pV-jDB-Hu-jFN-omega 1 (see example A) are cut with SphT restriction enzyme. S-terminus is removed by E.coli polymerase 1 in the presence of dGTP. The DNA is hydrolyzed with Xho1, the fragments are isolated by agar gel electrophoresis, electroelution and precipitation. The blunt end of the fragment length of 400 p. O.
    Title
    MF 1 TCGAGCCTCATATCAATGAGATTCCCATCTATTTTCACTGCTGTTTTGTT
    (50 mer)
    MF 2 AGCAGCGAACAAAACAGCAGTGAAAATAGATGGGAATCTCATTGATATGA GGC (53-mer)
    MF 3 CGCTGCTTCCTCCGCTTTGGCTGCTCCAGTCAACACTACTACTGAAGACG AAACTGCTCAAATTCCAGCT (70-mer)
    MF 4 CAGCTTCAGCTGGAATTTGAGCAGTTTCGTCTTCAGTAGTAGTGTTGACT GGAGCAGCCAAAGCGGAGGA (70-mer)
    MF 5 GAAGCTGTCATCGGTTACTCTGACTTGGAAGGTGACTTCGACGTTGCT (48 - "er).
    1727533
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    containing the ADH1 promoter, is ligated to the adapter pair EB1-410: 5 ATTGGAAGGATC 3 EB 1-429: 3 CCTTCCTAG-P5.
    The sticky end of the fragment is modified using an EB1-418 adapter pair: p-TCGAGCACGTGGTAC 3 EB1-424: CGTGCAC51
    After both adapters are simultaneously ligated, the fragment containing the ADH1 promoter is purified by agar gel electrophoresis.
    The vector and inserts are ligated, as a result of the ligation of the insert into the vector, the sites EcoR1 and Krp1 are destroyed. Ligase mixture transform E. coli IM101. Colonies are examined for the presence of a plasmid with the desired construct. One plasmid is selected and designated pRH, 242.
    c) Preparation of the pRH 243 plasmid containing yeast ADHI-npoMOTOp, the gene encoding the yeast factor MFa dominant peptide, the multi-cloning site and the yeast AOH1 terminator.
    The dominant peptide gene MF is chemically synthesized using a yeast codon.
    Getting a vector.
    Plasmid pRH 242 was digested with Xhol and XBa1. The fragment was purified by agar gel electrophoresis, electro-elution and precipitation.
    Receiving insert.
    The following oligodeoxynucleotides are obtained:
    I
    Sequence
    MF 6 GCAAAACAGCAACGTCGAAGTCACCTTCCAAGTCAGAGTAACCGATGA (48-mer)
    MF 7 GTTTTGCCATTCTCCAACTCCACTAACAACGGTTTGTTGTTCATTAAC ACTACTATTGCATCGATTGCT ()
    MF 8 CCTTAGCAGCAATCGATGCAATAGTAGTGTTAATGAACAACAAACCGTTG
    TTAGTGGAGTTGGAGAATG (69-uep)
    MF 9 GCTAAGGAAGAAGGTGTTTCTTTGGACAAGAGGCCTCTGCAGGAATTCT (49-iet))
    MF 10 CTAGAGAATTCCTGCAGAGGGCTCTTGTCCAAAGAAACACCTTCTT
    (46-iiep)
    60 pmol of each oligonucleotide, with the exception of MF 1 and MF 10, are phosphorylated using T4 polynucleotidinase (PNA). 60 pmol of MF 1 and MF 2 are combined. The solutions of MF 3 and MF 4, MF 5 and MF 6, MF 7 and MF 8, MF 9 and MF 10 are also combined. The combined solutions are heated at 100 ° C and slowly cooled to room temperature. Then all the solutions are combined, add 20 units. T4 ligases and the ligation reaction are carried out at 14 ° C for 16 hours.
    0.5 µl of vector DNA and 7 µl of the ligation mixture solution are combined and ligated with 5 units. T4 ligase. E. coli IM101 is transformed with a ligase mixture. Plasmids are isolated from some colonies and analyzed.
    EB-491: V TS0A0STSATATASMT60- ATC GAA TTS SMSTT TSS TOT
    COT TTC & TT TGT LAS ACT TG-T CCA GAAALS TGASH-95: 3 C AOTLTAT & TTLSS TAG G-TT AAS CTT CAAASS AHA SSA
    LAO SAL ASA TTG TG-A ASA & L1 CTT TTSASS TAGs in order to restore the complete gene using the yeast codon of the formula
    155160165
    Glu Leu Gin Val Ser Ser Gly Plu Val Cys Asylum Cr Gys Tt Gt Gtc Ttgt Atag AtagTa Atag Ata GTA Ata Ata GTG
    using Xho1 and XA1. Plasmids containing an insert of size 290, i.e., are further characterized by cloning the insert in MTZmrZ and subsequent analysis using the Sanger method. The plasmid containing the expected insert is designated pRH 243.
    d) Preparation of plasmid pRH 244 containing the ADHI promoter, a sequence encoding a water-soluble Fc R fragment and an AOSH-terminator.
    Receiving insert.
    The pGEM4 plasmid containing the larger Hindlll / EcoR1 DNA fragment was cut with EcoR1 and Hindlll restrictases and treated with the Klenow fragment, dephosphorylated, purified and separated on an agar gel. Two linker oligonucleotides of the formulas are synthesized.
    $ cm / ZZH-) Jtei5 TCGAGCTCATATACA PBX
    3 CGAGTATATGT TAS
    Xhoi
    Trp TG LSS TAG Sau3A
    3
    five
    25 pmol of each oligonucleotide is resuspended and added to about 3 µg of SauSA fragment (5-phosphate / EcoP1 / inserted, dephosphorylated) and ligated using T4-DNA ligase. The resulting Xho1- / EcoR1 / fragment is purified by agar gel electrophoresis.
    Getting a vector.
    The pRH 242 plasmid was cut with endonucleus XBa1, the ends were blunted by the Klenow fragment. The DNA was cut with Xho1 and the larger fragment was separated by agar gel electrophoresis.
    The vector and insert are ligated (ligation of the EcoRI-embedded space to the XBa1 embedded place leads to the restoration of both the EcoRI site and the XHa1 site). The ligase mixture is used to transform E.coll IM101. Plasmids were isolated from the colonies, which were examined for the presence of the water gene Glu Leu Gin Val Ser Ser A Gly Phe Val Cys Asn Thr Cys CGTA GTA TGTA GTA TGT AAC ACT TGT CCA ATA GTA AAG GTT CAA AGG AGA CCA AAG CAA ACA TTG TGA 4 JA GGT CTT
    Trp TG ACC TAG Sau3A
    3
    five
    synthesize two oligonucleotides of the formula
    EBI-430:

    5 ATGGAATTGCAAGTTTCCTCTGGTTTC ATG GAA TTG CAA GTT TCC TCT GGT TTC GTT TGT AAC ACT TGT CCA GAA AAG TG
    EBI-437:
    3 TACCTTAACGTTCAAAGGAGACCAAAG TAG CTT AAC GTT CAA AGG AGA CCA AAG CAA ACA TTG TGA ACA GGT CTT TTC. ACC TAG 5
    Sau3A
    soluble fragment using some restriction enzymes. One of the plasmids is selected and the Xho1-Xba1 fragment is subjected to sequence analysis in order to confirm the correct link between the ADHI promoter and the human gene. This plasmid is designated pRH244.
    d) Obtaining the plasmid pRH 245 containing the yeast AOH1 promoter, the dominant gene of the yeast factor MFa, the gene of the water-soluble Fc R fragment and the yeast AOH1 terminator.
    Getting a vector.
    Plasmid pRH 243 is digested with EcoR1 and Stu1. The larger fragment is purified by agar gel electrophoresis, electroelution and precipitation.
    Receiving insert.
    Plasmid pGEM4 / FcЈR / is double digested with EcoR1 and Hindlll, followed by dephosphorylation. The insert is digested with SauSA and the larger fragment is isolated. In order to restore the complete region encoding a soluble fragment of the formula
    Met
    5f ATG 3 TAS
    0
    five
    0
    five
    25 pmol of each oligodeoxynucleotide is renatured and added to 3 µg of Sau3A-EcoR1. The ligation reaction is carried out in a solution of a total volume of 20 µl using T4-DNA ligase. The resulting DNA is purified by agar gel electrophoresis, electrical elution and precipitation.
    1 μl of the vector fragment and 1 μl of the insertion fragment are ligated. The ligase mixture is used to transform E. coli IM101. Plasmids were isolated from some colonies, which were examined for the presence of the gene for a water-soluble FccR fragment using several restriction enzymes. One of the plasmids is selected and the Cta1-Xba1 fragment is subjected to sequence analysis in order to confirm the correct link between the MFa factor site and the gene for the water-soluble Fc.R fragment. This plasmid is designated pRH 245.
    e) Obtaining yeast vectors.
    The pRH 244 and pRH 245 plasmids were constructed using an E. coli vector (Btuescribe), which facilitates the analysis of the inserts. Both plasmids are cut with Hindlll and WatH1 and are ligated into the pjDB207 yeast vector containing the 2c replication start and the leu 2 selective marker.
    Getting a vector.
    Plasmid plDB207 was double digested with Hindlll and WatH1. The large fragment was isolated by agar gel electrophoresis, electrically eluted and precipitated.
    Receiving insert.
    Plasmid pRH 244 and pRH 245 are double digested with Hindlll and WatH1.
    The vector and inserts are ligated in solution and transform E. coli IM101. The DNA of the obtained colonies is examined for the correctness of the design. Two plasmids are selected, one of which is designated as ryUV-244 (containing expression
    EBi-4%:
    to
    & / iACTGCAG I & AGGTCTSaTrrTGGril1GCAACACTlieCGC & GA, uiAAT & 3:
    W-; 1-97:
    CITGAC & TGGACLUaAGAGGAAAGCAAAG & TTST & AACGGGGCTTTTTACGTAS which restore the reading skeleton, starting with the second codon (Gfu) of the FcgR soluble fragment, renaturate the cassette without the MF sequence, without making the MF, you will need to go to the object, you will need
    Both plasmids were isolated and used to transform a yeast strain WS21-3 (a, Ien2, his3, gp3, pp4).
    Step 2. Preparation of plasmids 289a1 and 289b3.
    Plasmid YEpG digested with restrictase Hindlll and BamHl. The linearized vector was isolated by agar gel electrophoresis. 1 μg of each plasmid pRH244 and pRH245 is digested with Hindlll
    and BamHl. From pPH244, a fragment with a length of 1,650 bp was isolated, and from pRH245, a fragment with a length of 1900 bp. 50 ng of linearized vector and 200 ng of fragments are ligated and used to transform E. coli
    HB101. Ampicillin resistant strains are selected.
    Plasmid colonies are examined for correct construction using restriction enzymes. Get two
    plasmids that designate 289a1 (from PRH244) and 289b3 (from pRH245).
    Example12. Expression of the soluble fragment in E. coli. Getting a vector.
    The pRH 100 plasmid (cm, Example B) was digested with Ssf1, the 3 ends were removed with DNA polymerase 1. The linearized plasmid was dephosphorylated. Receiving insert.
    20 μg of pGEM4-FcgR containing the larger Hindlll-EcoR1 DNA fragment for FccR is double digested with EcoR1 and Hindlll. The 5-ends are blunted with a Klenow DNA Polymerase 1 fragment. 5 phosphate groups are removed using calf intestinal phosphatase. After purification in an agar gel, the fragment containing the soluble fragment gene is hydrolyzed with restriction enzyme ZaIZA and
    the fragment is isolated.
    50 pmol of each oligonucleotide
    From 1
    S & c / jrf
    are boiled down and slowly cooled Oligonucleotides and DNA insertion are ligated using T4-DNA ligase.
    1 μl of the linearized vector DNA and 5 μl of the insert DNA are combined and ligated. Half of the material is used to transform E. coli HB101 (G, hsdS20 / rb-, mb /, rec A13, ara-14, proA2, -fecYI, gat K2, rpsL20 / 3m-stable /, xyt-5, mtf-1, SupE44, lmbda minus). Plasmids of ampicillin-resistant colonies are isolated and examined for correct construction using restriction enzymes. One plasmid was selected and analyzed for the connection between the Trp promoter and the gene for the soluble FcR fragment. After this final analysis, the plasmid is designated pRH 246.
    Example 13 Construction of the plasmid pSFcЈR-1.
    a) 350 µg of pbSF2-38, which contains DNA for BSF-2 in Sma1 together pGEM4, are digested with 700 units of EcoR1 and WatH 1 in 500 µl of buffer (100 mM sodium chloride, 50 mM Tris-HCt, pH 7.5, 10 mM magnesium chloride, 1 mM DTT) at 37 ° C for 2 hours. The overcooked DNA is purified by electrophoresis on a 1% agar gel. An EcoRI-BamHI fragment containing DNA for BSF-2 of 1.2 kb was isolated. 20 μg of this fragment is hydrolyzed by Hinfl units. Extracted with phenol and precipitated with ethanol. A fragment with a blunt end and a length of 127 p. extracted with phenol, digested with 40 units of Kpnl, incubated with 2.5 units. bacterial alkaline phosphatase, and then subjected to electrophoresis in agar gel (1%). The resulting 110 bp fragment containing the dominant BSF-2 sequence is electrically eluted and precipitated with ethanol. Fragment length 110 p. are ligated with digested Kpp1 and Sma1 plasmid pGEM4 and transfected into E. coli (MC1065). From the obtained colonies, a clone is selected that contains only one pre-evidence sequence, it is designated pBSF2-L8.
    b) Plasmids LE-392 or pGEM4 (pFc R-1) are digested with 150 units of H ind 111 and subjected to electrophoresis on an agar gel (1%). The Hindlll fragment containing the soluble FcЈR region is electroeluted from the gel, precipitated with ethanol, and dissolved. The Hindlll fragment is incubated at 20 ° C for 30 minutes in the presence of 8.2 units. Klenow fragment and 1 mM dNTP in medium of 10 µl 1 x nickel translation buffer in order to fill the 3 ends, extracted with phenol and precipitated with ethanol. Hindlll fragments filled with Z-ends hydrolyzed by restriction enzyme
    0
    five
    0
    five
    0
    five
    0
    five
    0
    five
    Psfl and phosphorus. pBsF2-L8 is hydrolyzed with the restriction enzyme WatH1. extracted with phenol and precipitated with ethanol. It is then dissolved, digested with Pstl nuclease, extracted with phenol and precipitated with ethanol. The digested Pstl fragment containing the FcЈ R-encoding soluble region and the digested Pstl fragment of the pBsF2-L8 fragment are ligated and used to transfect E. coli (MC1065). Clone is selected, designated psFc R-1. The multiplied plasmid contains seven bases from multiple cloning in pEM4 between the dominant BSF-2 sequence and the Fc R. sequence.
    Example 14: Expression of DNA for Fc, R.
    Plasmid psFc4R-1, containing modified DNA for Fc-R, is digested with restriction enzyme BtH1. mRNA is synthesized using the SH6 RNA polymerase using a linearized plasmid DNA as a template. Approximately 9 ng of mRNA is injected into Xenopus leavis oocytes, which are incubated at 20 ° C in modified Bart medium containing 100 μg / ml penicillin and 1 μg / m / streptomycin. After two days of incubation, the supernatant is collected and the oocytes are homogenized in PBS buffer medium containing 1 mM PMSF. PBS litases are centrifuged at 15,000 rpm for 10 minutes. The centrifugate is again extracted with NP-40 co-ligation of membrane-bound receptor (0.5% NP-40.0.1 M sodium chloride, 0.05 M Tris-HOf, pH 7.5).
    PRI me R 15. FcЈR assay in oocytes using sodium drdecyl sulfate and polyacrylamide gel.
    The 10 oocytes into which the mRNA was introduced are incubated at 20 ° C for 24 hours in 100 μl of modified Bart medium containing 150 μC of 353-methionine. The labeled oocytes are lysed in 1 ml of lysis buffer (0.5% NP-40, 0.1 M sodium chloride, 0.05 M Tris-HCf, pH 7.5) and centrifuged at 15,000 rpm for 10 minutes. The clarified lysates and supernatant are purified on Sepharose 4B beads with bound mouse immunoglobulin, followed by incubation in the presence of Sepharose 4B beads, which are bound to 3/5 antibody, which is associated with immunoglobulin G1. Incubation is carried out in ice with frequent shaking. The beads are washed, the products are eluted with sodium dodecyl sulfate-containing buffer by boiling for
    2 minutes. Samples were analyzed using sodium dodecyl sulfate and polyacrylamide gel (9%).
    Example 6. Definition.
    The activity of Fc R is determined by the method of enim, for which two different monoclonal anti-Fc R antibodies are used (3-5; with immunoglobulin 1; 8-30; with mouse immunoglobulin), which react with two different epitopes on and then add 100 µl of samples diluted with buffer (0.05 M Tris-HCf, pH 8.1, 1 mM magnesium chloride, 0.15 M sodium chloride, 0.05% tween, 20.1% cattle serum albumin and 0.02 % sodium azide). Plates are incubated at room temperature for 2 hours, washed four times with washing buffer, after which 100 ml of antibody 8-248 is added, to which alkaline phosphatase is bound (0.75 mg / ml). After 4 hours of incubation, the plates are washed 4 times. The enzymatic reaction is initiated by the addition of p-nitrophenyl phosphate in substrate buffer.
    Example17. Determination of FcЈR binding to immunoglobulin E.
    Plates equipped with a coating of antibodies 3-5 are incubated with the sample for 2 hours, washed, and human monoclonal immunoglobulins (PS myeloma) are added. After two hours of incubation at room temperature, the plates are washed and incubated with alkaline phosphatase associated with monoclonal anti-human immunoglobulin, developed by the addition of n-nitrophenyl phosphate in a static buffer.
    Example 18. The formation of the outlet of immunoglobulin E.
    Fc, R on lymphocytes is determined by analysis using fixed from RBCs provided with a human immunoglobulin coating. The number of rosettes of the forming cells is determined after subtracting the number of non-specific binding to cattle serum albumin coated fixed ox RBCs. In order to inhibit immunoglobulin E sockets, 25 µl of carrier cells (5 x U ° / ml) are mixed with a specific volume (for example, 100 µl) of the test sample or control medium and incubated at 4 ° C of 1 h. Calculate the number of sockets with OX RBC. In the first and third experiments, RPM1-8866 cells were used as carrier cells, and in the second experiment, SKW6-C4 cells. The supernatant was used as a control medium.
    control oocyte fluid, i.e. untransformed oocytes.
    An analysis using sodium dodecyl sulfate and polyacrylamide gel (a) 5 shows that the psFcЈR-1 product from oocytes, which is recognized by both antibodies 3-5 and 8-30 and has immunoglobulin binding activity, yielding broad protein bands and IQ product pANFc -R -1 from oocytes that does not have an N-terminal transmembrane region and can be recognized by antibody 3-5, but not by antibody 8-30 (b) and which does not show immunoglobulin E activity. These results suggest that the p D product, which does not have a signal sequence, is not properly processed and that the treatment is suitable, as in the psFcg clone R-1,
    2Q creates an epitope that is recognized by antibody 8-30 and has immunoglobulin binding activity.
    The expression of the water-soluble portion of the Fc-R receptor is carried out by cultivating 25 the corresponding strain of E. coli, yeast, or cells of another organism under conventional conditions, followed by concentration and purification of the resulting water-soluble fragment.
    The yeast strain WS21-1 transformed with the plasmid 289b3 (WS21-1 / 289e) was cultured for 40 h in the medium of KS NN to an optical density (546 nm) of about 45 (weight of dry cells 13 g / l).
    5 After cell separation by centrifugation, the yield of FcЈ R is 2.5 units / ml FcЈ R (1 unit / ml Fcf R corresponds to the activity of the supernatant equal to 1x105 RPMI cells / ml).
    0
    FORUMAWLAH AND ISLANDS
    The method of obtaining the water soluble part of the human Fc small affinity receptor, which consists in isolating the water soluble portion of the binding factor E-factor FctR immunoglobulin from RPM1 8866 lymphoblastoid cells, purifying it by chromatography, hydrolyzing, then isolating the fragments are in them
    Q amino acid sequence, on the basis of which oligonucle T A is synthesized
    leotide 3 -TTTTASS TA G TT G AA GT-5,
    A g &
    in parallel, from the RPM1-8866 cells, 5 poly (A) + - PH K is isolated, ddn more than 1000 lp in length is synthesized, which is cloned into the EcoR1 site-lambda gt10, then hybridization of the dc Fc, rR + L with the synthesized oligonucleotide is performed, selected the insert
    45
    The 1600kb ddn, which is cloned into the EcoR1 site LE392 or pGEM4 plasmid, is hydrolyzed by the Pstlll endonuclease obtained by DN, the resulting fragment is 1.0 kb long and filled with the Klenow DNA polymerase fragment, then the DNA is cloned into the pBSF2 vector -L8, treated with the endonuclease VatH1 and Psf1, receive the expression plasmid psFc R-1 for FcЈ R, simultaneously the Hindlll-EcoR1 insert fragment from the plasmid LE392 is cloned into the vector pGEM4, get pCEM4-Pc P, which is treated with EcoRI and Hindlll endo - nucleases, 5-overhangs are blunted with fragments Klenow DNA polymerase 1 and four deoxynucleotide triphosphates, 5-phosphate groups are removed, the resulting fragment after purification is treated with ZaIZA, the larger fragment is ligated with oligodeoxynucleotide
    72753346
    EB1496 and EBT497 using T4-DNA ligase, then DNA is ligated with linearized vector DNA, which is obtained by treating plasmid pRH 100 with Sstl endonuclease, removing 3-protruding ends and dephosphorylation, and get the plasmid pRH246 or simultaneously with the yeast plasmid YE. Hindlll and BamHI restrictase and linearized
    10 vector is ligated with the Hlndlll-BamHI fragment with a length of 1900 LP of plasmid pRH245 using T4 DNA ligase; an expression plasmid 289B3, previously obtained by plasmids pRH246 and psFc R-1, is obtained
    15, E.cofi HB101 is transformed, and the 289b3 expression plasmid is transformed into a yeast strain WS21-1, and the transformed strains are cultivated, followed by isolation and purification of the target product with
    20 using chromatographic methods.
    权利要求:
    Claims (1)
    [1]
    Claim
    A method of obtaining a water-soluble part of the human Fc 6 low affinity receptor, which consists in isolating the water-soluble part of the immunoglobulin binding E-factor Fc t R from lymphoblastoid cells RPM1 8866, purifying it by chromatography, hydrolyzing it, then extracting fragments, determining the amino acid sequence in them, on the basis of which oligonukTa A leotide 3'-TTc T ACC TA G TT G AA GT-5 'is synthesized,
    AGG in parallel from the RPM1-8866 cells isolate poly (A) + -PH K, synthesize dDNA longer than 1000 bp, which is cloned into the EcoR1 site-lambda gt10, then hybridize the Fc, -R + L dDNA with the synthesized oligonucleotide, select the dDNA insert 1600 kb long, which is cloned into LE392 or pGEM4 plasmid site EcoR1, the DND obtained with Pstlll is hydrolyzed, the obtained 1.0 kb fragment is filled with a Maple DNA polymerase fragment, DNA is digested with Pstl, the resulting fragment is cloned into pBSF2-L8 vector, treated with endonucleases and Psf 1 get express ionic plasmid psFc ^ R-1 Fc £ R, 111 simultaneously H ind-EcoR 1 fragment of the insert of the plasmid LE392 was cloned in the vector pGEM4, prepared pGEMA4-Fc.R, which was treated with EcoRI and Hindlll endonucleases 5 -vystupayuschie ends blunted using the Klenov fragment of DNA polymerase 1 and four deoxynucleoside triphosphates, 5-phosphate groups are removed, the obtained fragment is treated with Sau3A after purification, the larger fragment is ligated with oligodeoxynucleotides
    EB 1496 and EB 1497 using T4 DNA ligase, then the DNA is ligated with a linearized vector DNA, which is obtained by processing the plasmid pRH 100 with Sstl endonuclease, removing the 31 protruding ends and dephosphorylation, and plasmid pRH246 is obtained or the yeast plasmid YEp13 is hydrolyzed Hindlll and BamHI restriction enzymes and the linearized 10 vector are ligated with the HlndllbBamHI fragment of length 1900 bp of plasmid pRH245 using T4 DNA ligase, expression plasmid 289b3, previously obtained with plasmids pRH246 and psFc ^ R-1, transform H bacteria E. plasmid 289Z - yeast strain WS21-1, transformed strains are cultivated, followed by isolation and purification of the target product 20 by chromatographic methods.
    Editor M. Tsitkina Compiled by T. ZaboykinaTehred M. Morgenthal Corrector M. Demchik
    Order 1284 Circulation Subscription
    VNIIIPI of the State Committee for Inventions and Discoveries under the State Committee for Science and Technology of the USSR 113035 * Moscow, Zh-35, Raushskaya nab ,, 4/5
    Production and Publishing Plant Patent, Uzhgorod. Gagarina St., 101
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    引用文献:
    公开号 | 申请日 | 公开日 | 申请人 | 专利标题
    EP0248211B1|1986-04-30|1992-03-11|Junji Yodoi|Novel protein and a method for production thereof|
    ES2033747T3|1986-07-22|1993-04-01|Ciba-Geigy Ag|PREPARATION OF POLYPEPTIDES RELATED TO LINKAGE FACTORS.|
    GB8727045D0|1987-11-19|1987-12-23|Research Corp Ltd|Immunoglobulin e competitor|
    US5693758A|1987-11-19|1997-12-02|501 Research Corporation Limited|Immunoglobulin E competitor|
    WO2011068853A2|2009-12-01|2011-06-09|Boston Medical Center Corporation|TREATMENT OF IgE-MEDIATED DISEASE|
    法律状态:
    优先权:
    申请号 | 申请日 | 专利标题
    EP86111581A|EP0257114A1|1986-08-21|1986-08-21|Human low affinity Fc epsilon-receptor, the isolation and purification thereof|
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