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    • 专利摘要:
    Mature human leukocyte interferon species and their preparation by recombinant DNA technology as well as the means used in this process, i.e. recombinant DNA molecules coding for the amino acid sequences of said interferons, vectors capable of expressing said interferons in microbial host organisms and host organisrris transformed with these vectors.
    公开号:SU1414319A3
    申请号:SU813302642
    申请日:1981-06-30
    公开日:1988-07-30
    发明作者:Фан Нормен Геддель Девид；Пестка Сидней
    申请人:Ф.Хорфманн-Ля Рош Унд Ко,Аг (Фирма)；Генентех,Инк. (Фирма)；
    IPC主号:
    专利说明:

    4ib SLE
    WITH

    CM
    The invention relates to the field of recombinant DNA technology, t, e "to the methods used in recombinant DNA technology, and to products obtained by these methods.
    Example: Two microorganisms are used; Eocoli x1776 and E.coli K-12 pcs mm (end of A.thi, hsr, bsn-L),
    Human leukocyte interferon (LelF) mRNA obtained from human leukopa 5 and taken from patients with chronic myelogenous leukemia Tako are cell lines, called KG-tj, derived from patients with acute myelogenous leukemia „
    In KG-1 cells, leukocyte interferon mRNA is induced by Sendai or Newcastle viruses, cells are harvested 5 hours later after induction, and RNA is prepared using the guanidine thiocyanate-guanidine hydrochloride method. To obtain 12 S fractions of poly (A) mRNA, oligodeoxythymidine d T-cellulose chromatography and self-sustained gradient ultracentrifugal groaning are used.
    5 µg of mRNA is used to obtain double-stranded cDNA by the chain method. The indicated cDNA fractions are sized by electrophoresis on 6% polyacrylamide gel and 230 ng of material with a Mii size in the range from 500 to 1500 V, P. vyde.g. are electroelution. 100 and g of this kDN K are attached to deoxy cytidine residues (dc) and redenatured with 470 ng of plasmids pH R3225 which were linked to the deoxyguanosine (dG) residue; ш by (Pst I) site and used to transform E.col x177bo. Tetracycline resistant, ampicillin sensitive transformants are obtained. Four sets of deoxy oligonucleotide probes are prepared for each sequence, containing three (T-IA, B, C, D) or one (T-13A, Bj C D) KaxgiaK oligonucleotide.
    mRNA is obtained from 12 S RNA ,, KG-l induced by Sendai virus, or entirely poly (L) mRNA from non-H-g,
    leukocytes, P-labeled cDNAs are prepared in a known manner, He. the labeled product is isolated with gelfiltration at a column filled with 10 ml of Sephadex C-30, treated
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    No about J 3N NaOn For 30 minutes at 70 ° C, for destruction of the RNA, neutralize with HC1 and carry out hybridization.
    To identify pL1-pL30 clones, a fast plasmid isolation procedure according to Birnboim is used.
    You get 1 μg of plasmid. DNA from every 500 individual Secoli K-12 transformants of strain 294, Each DNA sample is denatured and applied to nitrocellulose filters in triplicate following the procedure of Kafatos et al. (See above)
    Three groups of nitrocellulose filters containing 500 plasmid samples 5 are hybridized with the stimulated cDNA prepared with the T-1 group of information carriers (primers), T-T3, laid with the stimulated cDNA, non-stimulated cDNA, prepared using both groups of information carriers
    Clones are considered positive if they hybridize stronger than one or both of the probes stimulated with cDNA than the fully unstimulated Probe Bto selected 30 positive clones () out of 500 for further analysis.
    Transformants of E.coli x1776 are screened by the method of colony hybridization using as a probe.
    7
     P-labeled stimulated mRNA. Unlabeled mRIC from unstimulated cells is mixed with a probe at a ratio of 200: 1 to compete with unstimulated mRNA present in
    32.
    P-labeled drug. Hybridization of labeled mRNA will preferably occur in columns of xJ containing stimulated sequences,
    Three classes of transformants are obtained;
    yi 2-3% of hybridized colonies -
    mRNA is very strong; 10% hybridized-nmx; significantly less than the 1st Kless; the rest is not giving a definable signal hybridnaacin
    Investigate positive colonies (classes 1 and 2) for the presence of interferon-specific sequences using an analysis that depends on the hybridization of the interferon mRNA specifically into the DNA plasmid. Forward, 60 strongly positive colonies (class 1) per 100 - (l of medium ME supplemented with tetracycline (200 µg / ml),
    diaminopimelic acid (100 μg / ml), thymidine (20 μg / ml) j, and d-biotin (1 μg / ml).
    Medium M9 contains 6 g / l, 3 g / l KNgP04, 0.5 g of NaCl, and 1 After autoclaving, add 1 ml of sterile 1M MgSO4 and 10 ml of sterile 0.01 M CaCl. Collect 10 cultures and isolate plasma DNA from test pools as described by Clevell Mot. Biochemistry 9, 4D28-440 (1970). 10 µg of each plasmid DNA pool was cleaved with Hind III, denatured and covalently bound to a DVM (diazo benzyloxymethyl) paper. On each filter, 1 µg of purified mRNA from stimulated cells is hybridized; Non-hybridized mRNA is removed by washing. Specifically, the hybridized mRNA is eluted and translated into the societies of Xenopus larvae. In this trial, all 6 pools were negative. 5 pools of 10 colonies each and 1 pool of 9 colonies are made from 59 weakly positive colonies (class 2). Plasmids are prepared from pools and examined as described above. The medium 6 of the tested pools was tested alone (K10), hybridized to interferon mRNA. subject to significantly higher initial levels of each time. In order to determine the specific interferon cDNA clone, plasmids of DNA are prepared from 9 colonies of the K10 pool and examined individually. Two of the nine plasmids (No. 101 and No. 104) bind in; Terferon mRNA is significantly better than at the indicated initial levels. One Bgl II restriction fragment was isolated from plasmid No. 104, Co4O.
    holding 260 V. p. labeled P using the procedure described by Taylor et al., and using as a probe for independent screening 400 E. coli 294 transformants using the in situ colony screening procedure.
    9 colonies (pb 31-pL 39) are identified that hybridize to varying degrees with this probe.
    In addition, a labeled 260 B, p, fragment for independent screening of 4,000 E. coli 294 transformants was used in the same way. Fifty colonies are identified that hybridize to varying degrees with this probe. One contains the LeIF G fragment, one contains the LelF H fragment, and one contains fragment 19CH.
    ment, designation LeIF HI, very similar to LeIF N. The resulting hybrid plasmids denote pLEIF H, etc.
    A plasmid of DNA was prepared from all 39 potential LeIF cDNA clones and re-screened with the same 260 Pile DNA probe using the Kathathos-et al. Hybridization procedure. (see above). Three plasmids (pL 4, pL31 pL 34) give very strong hybridization features (signals), four (pL 13, pL 30, pL 32, pL 36) are modestly hybridized and three (pL 6, pL 8 L 14) are weakly hybridized the probe.
    Also, 39 potential LeIF cDNA of recombinant plasmids are screened using P-labeled synthetic undecamers (individual T-1 primary information primer pools or individual T-13 information primers) directly as hybridizing probes. Hybridization conditions are chosen such that exactly paired bases must be required for detectable hybridization signals. Consequently, a plasmid of DNA from 3 clones was prepared according to the standard procedure for the purification of lysate (Clevell et al., See above) and purified by column chromatography on Biorad Agarose A-50.
    Samples of 3 µg of each preparation are made linear with the Eco RI treatment, denatured in alkali and applied to 2 separate nitrocel polosny filters of 1.5 µg per spot (Kapatos with g / cm above), individual synthetic deoxyol7 phosphorus of the primer information and primer information pools using () ATP as follows: 50 pCmol of oligonucleotide and 100 pcMol (Cfr) ATP, (2500 Cyu / mmol) are combined in 30 µl of 50 mM Tris-HCl, 10 mM MgCl, 15 1 Hb- mercaptoethanol. Add 2 units. T4 polynucleotide kinase and after 30 min at 37 ° C, P-labeled primers were purified by chromatography in columns with 10 ml Sephadex ® G-50, Hybridization was carried out using 10 CPM pool of T-13C primers or 3 10 CPM pool of Primer T- 1C at 15 ° C for 14 hours in 6x SSC (IxSSC 0.15 M NaCl, 0.015 M sodium citrate, pH 7.2, 10x Denhardt solution (0.2% bovine serum albumin, 0.2% polypiM
    nilpyrolidone, 0.2% ficoll). Filters are washed with 5 JIHH (3 times) at О С 6x SSC, dried and irradiated on x-ray film,
    At the same time, the DNA plasmid from clone 104 gives significant hybridization with the T-1C information primer pool and T-ISC information primers, but does not provide definable hybridization with other underammers. Several of the 39 potential LeIF plasmids (pL 2,4,13j 17,20,30,31,34) also hybridize with both of these probes. Pst 1-digestion pL 31 shows the size of the cDNA insert, which should be about 1000 ip.
    It was found that the first / ATC-translational initial codon consists of 60 nucleotides from the 5th end of the sequence and then 188 codons. After the TGA terminal triplet, there are 342 non-translated nucleotides at the H end, followed by a poly (A) -sequence, The putative signal peptide (apparently, included in the secretion of the finished LeIF. From leukocytes has a length of 23 amino acids, 165 amino acids constituting the ready LeIF, have a calculated molecular weight of 19390, LeIF5 encoded pL 31 denote LeIF A, SAU tricot endonuclease area of races olozhen between codons 1 and 3u) FA. Two synthetic deoxy oligonucleotides include an ATC-translational initial codon, reconstruct the amino acid 1 codon (cysteine) and create an Eco RI sticky end. These oligomers were bound in 34 v, p „SAU Za-A all fragment pL 31. Obtained as a result of 45 V. p. the product is ligated into two additional DNA fragments to construct 86.5 bp. synthetic (natural hybrid gene that encodes LIFAu, which binds to Eco RI and Pst I. (restriction M11 sites). Such a gene is included in the pR R322 melody Eco RI and Pst I regions to produce the PLIF AI plasmid.
    The construction of a tryptophan control element containing, E. coli trp promoter, operator and trp leader of the ribosome-binding region, but not containing ATC sequences for initiating translation,
    The pGMI plasmid carries tryptophan E. coli opera, containing a deletion of & L E1413, and expresses the protein.
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    comprising the first 6 amino acids of the trp leader and approximately the last third trp E polypeptide (later referred to in combination as LE), as well as the trp D polypeptide in its entirety j, all under the control of the trp system promoter operator, Plasmid (20 μg) is treated with restriction enzyme PVU II which cleaves the plasmid into five plots. The gene fragments are then combined with Eco RI linkers consisting of a self-complementary oligonucleotide with the sequence pCATGAATTCATG, providing the Eco R 1 cleavage site for subsequent cloning into a plasmid contain eso. R 1-site, Processed with 20 μg of DNA fragments 5 obtained from pGMI, 10 units of Td DNA lgase in the presence of 200 pM 5 -phosphorylated synthetic oligonucleotide pGATGAATTCATG and in 20 μl T DNA-ligase buffer (20 kM Tris,
    pH 7.6, 0.5 mM ATP, 10 1G MgCL ,, 5 mM dithiotreitol) 4 C for HO4Hi Then the solution is heated for 10 min at 70 ° C until the ligation is stopped, the Gsc is cleaved off by digestion with Eco R I and
    the fragments, now with the ends of Eco RI, are separated using electrophoresis on a 5% polyacrylate sdn gel (PAGE), and with the widest fragments isolated from the gel with the first stain
    ethidium bromine, localization of the fragments with ultraviolet light, and cut out the regions of interest from the gel. Each gel fragment is placed with 300 μl of 0.1 X TBE in the dialyzer
    tank and subjected to electrophoresis at 100 V for one hour in 0.1 TBE-buffer (TBE-buffer contains: 10.8 g Tris-base and 5.5 g of boric acid 0.09 g Na - EDATUK 1 liter of water). The aqueous solution is collected from a dialyzer tank, extracted with phenol, extracted with chloroform. A 0.2 M solution of sodium chloride is made and the DNA is extracted in water after ethanol precipitation contains the trp gene.
    promoter operator with sticky ends. The Eco RI is identified by the procedure below, which causes the fragments to be inserted into the plasmid sensitive to tetracycline, which, upon insertion of the promoter operator, becomes resistant to tetracycline,
    Stasmid rVRL expresses pitsklinova resistance and content7 1
    The tetracycline resistance gene is living. Therefore, plasma is sensitive to tetracycline. The plasmid is made tetracycline resistant by introducing a promoter-operator system into the EcoRI region,
    The pBRHI is cleaved with EcoRI and the enzyme is removed by extraction with phenol, followed by extraction with chloroform and collected in water after precipitation with ethanol. The resulting DNA molecule in the individual reaction mixtures is combined with each of the three fragment ™ DNA tones obtained above, and ligated to the T DNA ligase, as described above. Use the DNA} contained in the reaction mixture. To transform E. coli K-12 strain 294 according to a standard procedure and the bacteria are placed on LB (Luria-Bertani) plates containing 20 µg / l ampicillin and 5 µg / l tetracycline. Several tetracycline-resistant colonies are selected, the DNA plasmid is isolated and the presence of the desired fragment is proven by restriction enzyme analysis. The resulting plasmid is designated pB RH trp.
    The cleavage product with Eco RI and You HI of the viral genome of hepatitis B is prepared in the traditional way and cloned into Eco RI and Wat H I plasmid p G H 6 plasmids to form the pH S 32 plasmid. Then the X plasmid is digested, extracted with phenol, chloroform, precipitated with ethanol and treated with 1 μl of E. coli DNA polymerase I Klenow fragment (Boehringer-Man - nheim) in 30 μl of polymerase buffer (50 mM potassium phosphate pH 7.4 mM MgClg 1 mM | | -mercaptoethanol) S containing 0.1 mM d TTP and 0.1 mM, d MFR, for 30 min at 0 ° C, then 2 h at 37 C. Taka treatment It is designed to fill 2 of 4 nucleotides complementary to the forward protruding 5 end of X la I cleavage region,
    5 CTAGA .5. CTAGA
    3 T 3 TST Two nucleotides, dC and dT, were included and gave an end with two 5 nucleotides protruding. Such a linear residue of the plasmid pH S 32 (after extraction with phenol and chloroform and collected in water after precipitation with ethanol) is cleaved with Eco R Ij to separate the wide plasmid fragment from the smaller Eco R I-XbaI fragment with 19
    using PAGE and isolated after electroelution of X. This DNA fragment from pH S 32 (0.2 μT;) is bound, under conditions similar to those indicated, to Eco R 1 -Tar, 1-fraction of tryptophan operen (OjOl μg ), originating from pB RH trp, in the method of doping a fragment of an enzyme nz rp s 32 to a fragment
    Echo In I - Tag 1, as described above, for stepping forward Tag I end is connected with Fast Forward for late X Xa Ij although it is not perfectly paired by Watson-Crick
    - E CTAGA - - CTTAGA - AGC TST - - - AGCTCT - A part of this ligation reaction mixture, transformer1 | Toth, into E. coli 294 cells, is heat treated and placed on LB plates containing ampicillin. 24 colonies are selected, grown in 3 ml of LB (Luria-Bertani) medium and the plasmid is isolated. It was found that 6 of them have an Xbal-region rendered by E. coli, catalyzed by DNA by repeated pairing and replication.
    - TCTAGA - - TCTAGA - - AGCTCT - AGATCT Found that these plasmids are cleaved with both Eco R I and Hpa I and give the expected restriction fragments, One plasmid, designated pTgr 14, is used to express heterologous polypeptides,
    The pHGH 107 plasmid contains the gene for human growth hormone (HGH), composed of 23 amino acid codons derived from synthetic DNA fragmentsJ and 163 amino acid codons 5 obtained from complementary DNA obtained by reverse transcription of informational HGH RNA, This gene5 although no codons rge sequence hGH 5 contains ATG-broadcasting initial codon. This gene is isolated from 10 μg of pHGH 10 after treatment with Eco R I, followed by treatment of E. coli with the DNA polymerase clinoza fragment and d TTP and d ATP. as stated above. After extraction with phenol and chloroform and precipitation with ethanol, the plasmid is processed into Bai H1o
    The fragment containing the HGH gene is isolated using PAGE followed by electroelution. The resulting DNA fragment also contains
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    the first 350 nucleotides of the tetracycline resistant structural gene but lack the tetracycline promoter-operator system, so that with sequential cloning into the expression plasmid, plasmids containing the insert can be detected by the restoration of tetracycline resistance. Since the Eco R I end of the fragment was filled in with Klenov's polymerase I, the fragment has one blunt and one sticky end, which provides a clear orientation upon subsequent incorporation into the expression plasmid.
    . An expressive plasmid of pTgr 1Dl is then prepared to prepare a fragment containing the hGH gene prepared earlier, pTgp 14 is cleaved with XbAI, and the obtained sticky ends are filled using the complete procedure of I Klenow, using d ATP, d TIP, dG IP and d PAGE. After extraction with phenol and chloroform and precipitation with ethanol, the resulting DNA is treated with Wat HI and the resulting plasmid fragment is isolated using PAGE and electroscopy. The fragment originating from pTgr 14 has one blunt and one sticky end, which allows recombination with a clear orientation with a fragment containing the hGH gene described previously
    The fragment of the hGH gene and the fragment of pTgr 14 u, Xba-Bam HI are combined and ligated under conditions similar to those described above. The filled Xbal and Eco R I ends linked together by a blunt end to recreate the Xbal and Eco R I sites: a filled Xbal filled with Eco R I initiation of the HHR-TCTAG AATTCTATG- -TGTAGAATTCTATG- + gene
    -AGATC TTAAGATAG -AGATGTTAAGATACXba I Eco R I
    This construct also recreates the tetracycline resistance gene, Since the pHGU 107 plasmid expresses tetracycline resistance from the promoter located above the HGH gene (lac promoter), this design is designated pHGH 207, it allows the expression of the tetracycline resistance gene under control tryptophol promoter operator; The ligation mixture is transformed into E. coli 294 and colonies are selected on LB plates containing 5 μg / l of tetracycline.
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    Plasma1 rNOI 207 is cleaved with Eco RI using PAGE and electroelution, and a fragment containing the trp promoterJoperator and trp is the leading ribosomal binding site, but lacking the ATC sequence to initiate translation. This DNA fragment is cloned and the Eco R 1 pLelF A region, the expression plasmids containing the above-modified trp regulon (E. coli trp operatic, from which the depleted sequence was removed for controlled expression levels) are grown to a predetermined level in the nutrient medium 3 containing an additive of tryptophan in an amount sufficient to suppress (repress) the promo promoter-operator system, then tryptophan is removed to de-repress the system and induce the expression of the target product. For this, 250 µg of plasmid pL 31 are cleaved with pst I and isolated 1000 V, p, insert by electrophoresis on a 6% polyacrylamide gel.
    Approximately 40 µg of the insert was eluted from the gel and divided into three aliquots to further break up the sample. 16 µg of this fragment was partially cleaved with 40 units, BPH II- within 45 min at 37 G and the reaction mixture was purified on a 6% polyacrylamide gel . Collect approximately 2 μg of the target 670 in. P. fragment.
    Another sample (8 μg) of 1000 v.r. pst I inserts are restricted to A v all and Bgl II, 1 μg of the indicated 150 Vp is collected. fragment after gel electrophoresis.
    16 g microcopy 1000 v., P, part D) Au and Z and A V all. After electrophoresis on a 10% 7% polyacrylamide gel, approximately 0.25 "x (10 pmol) of the 34 V, p fragment is collected.
    Two of the indicated deoxy oligonucleotides 5 -d AATTCATGTGT (fragment 1) and 5 - d-GATCAGACATG (fragment 2) are synthesized according to the phosphorotriether procedure.
    Fragment 2 is phosphorylated as follows.
    Dry with 200 μl (pcmol) (p) - ATP (Qraersham ZOOOQ / mmol) and resuspend in 30 μl of 60 LTfl Tris-HG l (pH 8) j 10 mM Mr.S, 15 kM W-mercaptosistenol) containing- { its 100 pcol LIC fragment and 2 units
    . 14
    4 polynucleotide kinases. After 15 minutes at 37 ° C, 1 µl of 10 mM ATP is added and the reaction continues.
    15 minutes. The mixture is then heated at 70 ° C for 15 minutes, combined with 100 pMolar 5 -OH fragment 1 and
    10 pmol 34 in, r. SAU Za-A v all ragmenta. Binding is carried out for 5 hours at 4 ° C in 50 µl of 20 mM Tris-HC (pH 7.5), 10 mM MgCl4. 10 ml of dithiothreitol, 0.5 Mli ATP and 10 units. T4 DNA ligase. The mixture is subjected to electrophoresis on a 6% polyacrylamide gel and electro-eluted is collected with 45 rp. product. 30 ng (1 pmol) 45 Bp are combined. product with 0.5 µg (5 pcmol) 150 in.R. A-V aII-VRd of the 2nd fragment and 1 μg (2 pmol) 670 v, p. Bgl Il-Pst of the 1st fragment. Ligation is carried out at 20 ° C for
    16h using 20 units of T4 DNA ligase. The ligase is inactivated by heating at 65 ° C for 10 minutes. The mixture is then digested with Eco R I and Pst I to remove polymers from the gene. The mixture is purified
    at 6% PAGE. About 20 ng, (0.04 pmol) of 865 vp are isolated. product. Half of this product (10 ng) is ligated into p3R 322 (0.3 µg) between the Eco R I and Pst I sites. The transformation of E. coli 294 gave 70 tetracycline resistant ampicillin-sensitive transformants. The DNA plasmid, isolated from 18 of these transformants, cleaved with Eco RI and Pst I, 16 out of 18 plasmids have an Eco RAO I-Pst 1 fragment of 865 bp. in length. 1 µg of one of them is cleaved, pLelF, A 1, Eco RI, and ligated into a 300 bp, EcoRI fragment (0.1 µg), containing the E „c611 trp promoter and the leading ribosomal binding site, prepared as described above . Transformants containing the trp promoter are identified using a P-trp probe in conjunction with the process; (Grunshchsteyna-Hogness colony screening, Asymmetrically located Xbal site in the trp fragment allows you to identify recombinants in which the trp-npoMOTop is oriented in the direction of LeIF A -gene.
    The definition of activity LeIF A.
    Extracts are prepared for IF assay as follows.
    1 ml of the culture is grown in its bouillon containing 5 mg / ml of tetracycline to an A550 value of about 1.0. Then it is diluted with 25 ml of medium M9;
    5 μg / ml tetracycline. 10 ml samples are collected by spectrifugation when ASSQ reaches a value of 1.0 and granulated cells are suspended in 1 ml of a 15% sucrose solution, 50 mM Tris-HC (pH 8.0), 50 mM EDTA. 1 mg of lysozyme is added and after 5 minutes of incubation at 0 ° C, the cells are destroyed by exposure to ultrasound. Centrifuged samples for 10 minutes (15,000 rpm) and interferon activity in the upper layer are determined by comparison with the standards of LeIF using
    cytopathic effect (CPE) inhibition. To determine the number of TF molecules per cell, LeIF uses a specific activity of about 4-10 U / mg clone pLelF A trp 25, in which
    The trp promoter is inserted in the desired orientation, yielding high levels of activity (up to 2, u / l). The trp 25 strain 294 / pLeIF A obtained by E. coli K-12 behaves like
    authentic human LeIF, it is resistant to treatment at pH 2 and is neutralized by rabbit anti-human leukocyte antibodies. Such an interferon has an apparent molecular weight of approximately 20,000.
    Isolation of additional leukocyte interferons with DNA, DNA from LeIF with a DNA-containing plasmid is cut with pst 1, isolated
    by electrophoresis, and is labeled with the P isotope. The resulting radiolabeled DNA is used as a probe for screening additional E.coli 294 transformants obtained by a method identical to that described in Part C using the in situ colony screening method proposed by Grundsey and Hogness ( see above). Colonies that hybridized in various quantities with a probe, plasmid DNA from such colonies and ten hybridized colonies referred to above, were synthesized with Pst I and characterized in three different ways. First, the samples of the restriction endonuclease degradation enzyme with the Bgl II, PVU II and Eco R 1 enzymes. Such analysis allows to classify at least eight different types (LeIF A, LeIF B, LeIF C, LeIF D, LeIF E , LeIF F, LeIF G, LeIF H), which corresponds approximately to the position of various restriction sections relative to
    13U
    currently known pre-sequence and coding sequence.
    Secondly, some of the DNAs are tested for hybridization selection in order to establish the ability to selectively remove LeIF and CIC from poly-Aj containing KG-1 cellular RNA. According to this analysis, LeIF A, B, C and F give positive results. Third, the latter fragments are inserted into the expression plasmid E, coli294 (transformed with the plasmid and expressed fragments. The products of this expression are pre-interferons, give positive results in the CPE assay for the activity of interferon, although they have marginal activity in the case of LeIF F-fragment.
    In the sequence of an isolated fragment containing the mature LeIF B gene, the first fourteen nucleotides of types A and B were identical. Accordingly, a fragment from pLelF A 25 carrying the trp promoter operator, the ribosome attachment site and the beginning of the LeIF A (B) gene is in the gota section and is linked to the remaining part of the B sequence in the expression plasmid,
    In order to obtain approximately 950 bp, SAU. For the Pst fragment, several stages are necessary due to the presence of one or more alternating SAUs.
    1. The following fragments are identified; ) 0110 v.r. from SAU Over to Fco R I, c) 7i 132 c, p. from Eco R I to Xa,
    c) 700 century p. from Cha to Pst.
    2. Fragments (1a and 1c) are ligated and cut with XBa and Bgl II
     in order to prevent self-polymerization through the SAU Za and XBa terminal terminals (the corresponding SAU site is located inside the Bgl II site, the Bgl II is cut in order to leave the SAU Z - sticky end). 242 ip are available. fragment.
    3. The product from steps (2) and (1c) is ligated and cut with Pst I and Bgl 11 in order to prevent self-polymerization. Approximately 950 vol. fragment from SAU to Pst. This fragment contains part of the LeIF B gene, which is not characteristic of LeIF A.
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    4. Approximately 300 in „p. a fragment from Hind III to SAU Gamma containing the trp promoter operator, the riboso attachment site, the ATC initial signal and the cysteine codOn LeIFA are derived from pLelF A 25,
    5. Approximately 3600 century p. Pst I - Hind III fragment is isolated from
    Q pB R 322 “This fragment contains replicon and coded tetracycline, but is not resistant to ampicillin.
    6. Fragments, obtained at the stage of the 5 x 3 j4 j5 ligirtuto and the resulting plasmid is transformed into E. coli K-12 strain 294.
    Transformants are miniscreened and plasmid samples are digested
    0 with Eco R I, the products of this reaction are three fragments with the following characteristics: Eco R I - Eco R I trp promoter fragment, internal Eco R I - Eco R I - Eco R
    5 fragment of pL4 and protein translational initial signal - Eco R I fragment of pL4.
    According to CPE analysis, bacterial extracts from clones obtained from
    In this way, usually have about 10-10 units. interferon activity per liter with Ajgg 1. One of these clones obtained by this method is
    3g E.coli 294 / pLeIF B trp 7 „
    Direct expression of additional mature leukocyte interferons (LeIF C, D, F, H, I, and L). Additional gene fragments
    0 full lengths that contain other LeIF types can be converted to a sequence and placed into expression vectors for expression, as is the case for LeIF A.
    45
    A short length of synthetic DNA that terminates at the 3'-end of the coding
    the helix, with the translational initial ATC signal, is then ligated, for example, by ligating the blunt end to the resulting cross-linked gene for mature interferons, the gene is inserted into the expression plasmid and regulated by the promoter and its associated ribof-lbl attachment site.
    According to a method similar to that described above, the gene fragments encoding LeIF C and LeIF D, respectively 51
    accordingly, configured for direct bacterial expression, the expression strategy for such additional leukocyte interferons in each case involves the use of approximately 300 bp. the fragment (Hind III-SAU Za) containing the trp promoter operator, the ribosome attachment site, the ATC initial signal and the cysteine codon LeIFA from pLelF A 25. To it are added gene fragments from additional interferon genes encoding their corresponding amino acid sequences higher than the original cysteine inherent in all the sequences of mV Each plasmid obtained as a result is used to transform E. coli K-12 strain 294.
    From pLelF C, select the following fragments: a) 35 century p. SAU 3A-SAU 96, c) 900 century p. SAU 96 - Pst 1, c) isolate approximately 300 bp. the fragment (Hind 111 - SAU Za) from pLelF A 25, similarly to that described in part 4 d), approximately 3600 in. of the fragment is separated according to part 5
    Build.
    1. Ligate (a) and (c). The cleavage is carried out with Bgl II, Hind III and approximately 335 bp. product.
    2 Conducted triple ligation (1) + (b) (d) and transformation of E. coli using the resulting plasmid.
    Corresponding Clown | J thus obtained is E. coli K-12 strain 294 / pLeIF C trp 35
    LeIF D.
    From pLelF D there are: a) 35 v, p, SAU CONA - A V all, c) 150 v, p. A v al Bgl II, c) approximately 700 v, p, Bgl II - Pst I
    From pLelF A 25, the following are included: d) 300 Bep. Hind III - SAU Pro.
    From pB, R 322 was isolated; e) approximately 3600 century d. Hind III - Pst lo
    Build. ,,
    1. Ligirzpot (a) + (b), cut with Bgl II and clean 185 century p. product (1).
    2. Ligate (1) (d), cut with Hind HI, Bgl II and purify approximately 500 bp. product (2)
    2. Ligate (2) + (c) + (e) and transform with E. coli using the resulting plasmid.
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    five
    The corresponding KjiOHj obtained in this way5 is E. coli K-12 strain 29 4 / pLeir D trp 1 1. LeIF F containing the fragment can be crosslinked for direct rapid expression through reassembly5 simplified by complete homology of a н nnocolon7 1-13 LeTF B and LeIF F trp promoter-containing fragment (a) with appropriately con fi gured ends obtained from HGH 207 as described above, via Pst I and Xa X-cleavage followed by release of approximately 1050 in the „p. fragment. The second fragment (b) is obtained as a larger fragment obtained from Pst I and Bgl II by splitting the plasmid RNA Y 10,
    Fragment (a) contains approximately half of the gene encoding ampicillin resistance, fragment (b) - the gene residue and the complete gene encoding tetracycline resistance. Fragments (a) and (b) are combined through T4 ligase and the product is treated with XBa T and Bg II to suppress dimerization to form fragment (c) containing the trp promoter operator and genes for tetracycline and ampicillin e
    Fragment (d) containing approximately 580 V. p., Obtained by splitting pLelF F under the influence of And Ya TI and Bgl II. It contains amino acid codons 14-166 LeIF F.
    Fragment (e) (49 c. P) is obtained by cleaving pLelF B under the influence of XBa I and A V allo Fragment (e) encodes amino acids 1-13 LeIF Fv
    Fragments (c), (d) and (e) are triple ligated in the presence of LT4 ligase. The cohesive ends of the Relevant fragments are that the composite plasmid is in a state of proper circulation and, at the same time, the tetrasin resistance gene is under the control of the trp the promoter operator together with the mature LeIF F gene, so that bacteria transformed with the desired plasmid are selected from plates containing tetracycline. The clone thus obtained is E. coli K-12 strain 294 / pLeIF F trp 1.
    The complete LeIF H gene can be configured to express as a mature leukocyte interferon according to the following.
    171D
    1. Plasmid pLelF H is hydrolyzed in the presence of Pae II and RS al with the release of 816 V, p. a fragment extending from the signal peptide of amino acid 10 to 3 non-coding region.
    2. A fragment is denatured and subjected to synthesis with a Kpenova DNA polymerase 1 fragment using synthetic deoxyribo-oligoiucleotide primer 5 -ATGTGTAATCTGTCT
    3. The resulting product is cleaved with SAU Zero and 452 ip are recovered. a fragment representing amino acids 1-150.
    4. As a result of LeIFH cleavage with the help of SAU Za and Pst.1 and isolation of the resulting 500 bp. fragment receive the gene encoding amino acids from 150 to the end of the coding sequence.
    5. The fragments allocated in steps (3) and (4) are ligated to form a fragment.
    1166
    met cys asp stop PBX TGT .. + i. GAT TGA - ... - Pst I
    SAU For encoding 166 amino acids LeIF-H,
    6.pLelF A trp 25 is cleaved with Xba I, blunt ends are ligated with DNA polymerase 1, and the reaction product is cleaved with Pst I. The resulting large fragment can be extracted and ligated
    with the product from step (5) with the formation of expression gshazmida capable of expressing mature LeIF H after transformation of E. coli K-12 strain 294 or another bacterial host.
    The L-phase Charon 4A recombinant library of the human genome is screened for leukocyte interferon genes. The radioactive LeIF probe obtained from the cDNA of the LeIF A clone is used to screen 500,000 colonies. Six LeIF genomic clones are obtained as a result of this screening. As a result of subsequent screening and purification of the colonies, one of these clones, H LeIF 2, is selected for further analysis.
    Using this method, other probes can be used in order to successfully isolate additional LeIF clones from the human genome. They're in
    . NE 18
    Turn5 can be used to produce additional leukocyte interferon proteins of the invention.
     1. 2000 century p. The Eco R I fragment of the clone / XH LeIF 2 is cloned into pBR325. At the Eco R I site, the resulting plasmid is LeIF I
    Q is cleaved with Eco R I and 2000 bp is recovered. fragment. Deoxyoligonucleotide d AATTCTGCAG (the Eco RI-Pst I converter) is ligated to 2000 century p. Eco R I fragment and obtained
    The 5 product thus cleaved with Pst I to form a 2000 fragment containing the Pst 1-terminus. This product was digested with SAU 96 and 1100 bp. the fragment is isolated.
    0 2. Plasmid. PLelF C trp 35 split pot with Pst I and Xb al. Enlarges a large fragment
    3. A small XB al - Pst I fragment from pLelF C trp 35 is cleaved with
    5 with the help of XbA and SAU 96. There are 40 cp. Hb al - SAU 96 fragments,
    4, Fragments isolated in steps (1), (2) and (3), are ligated to form an expression plasmid
    0 pLelF I trp 1. LeIF J.
    ,one. The plasmid pLelF J contains 3.6 (: Hind III fragment of human genomic DNA, which includes the LeIF J gene sequence, and is allocated 700 cp. Dde I - RSa I fragment. 2. The plasmid pLelF B trp 7 is cleaved with Hind III and Dde G and isolate 350.c, p. Hind III-Dde I fragment,
    Q 3 ", The RB plasmid R 322 was digested with Pst Ij, the ends were blunt by incubation in the presence of DNA polymerase I (maple fragment), then digested with Hi, III and large (i 3600 B, p) was isolated fragment,
    4. The fragments isolated in steps (1) (2) and (3) are ligated to form an expression plasmid.
    p pLelF J trp I,
    Interferon Purification, 1. Frozen cell granules containing expressed leukocyte interferon are manually crushed with the use of appropriate equipment to reduce the particle size. Partially thawed cells are suspended in 4 vol. Buffer Aj containing 0.1 M Tris (pH 755-8.0) 3
    35
    45
    55
    nineteen
    10% (w / v) sucrose 0.2 M MaCl, 5 mM EDTL, 0.1 mM P11SF and 10-100 -sH Mp, C1. This suspension is kept at a temperature of approximately A. The C, the resulting suspension is passed through a homogenizerJ working under pressure of 6000 lb / in - j, after which it is passed through the device again but at a pressure of 1000 lb / in. The effluent from the homogenizer from two passes is cooled in an ice bath,
    2, Polyethyleneimine is slowly added to a homogeneous medium to a concentration of about 0.35% and the resulting system is allowed to stand for 30 minutes. The solids are removed by centrifugation or filtration. The temperature at this stage is controlled or carried out rather quickly, with the result that the upper layer (filtrate) is maintained at a temperature of less than 10 ° C. The upper layer (filtrate) is concentrated by ultrafiltration to approximately 1/10 of the initial volume. Small particles or nebula
    in the retained phase can be removed on the appropriate filter, for example on a microporous membrane.
    3, The clarified solution is loaded directly onto a column with a monoclonal antibody at a feed rate of 5–8 cm / h (for example, 25–40 ml / h to a column with a diameter of 2.6 cm). After loading, the column is washed with approximately 10 vol. 25 mM Tris-HC1 pH 755-8.55 including NaCl (0.5 M) and a surfactant such as Triton-X-100 (0.2%) or its equivalent. After washing, the column is rinsed with approximately 10 v of solution5 containing 0515 M NaCl
    and a surfactant such as Triton X-100 (0.1%) or its equivalent. The column is eluted with a 0.2 M solution of acetic acid containing a surfactant such as Triton-X-100 (0.1%) or its equivalent Fraction j giving a protein peak from a column with a monoclonal antibody (according to UV spectroscopic or another ana1920
    Lizu) 5 obedii. the pH of the system is adjusted to about 4.5 with a 0.1 N solution of NaOH or 1 ,, O M tris bases,
    4 o A joint and interferon peak is loaded onto a cation exchanger, such as Eat, an Qi 52 cellulose or its equivalent5 which equilibrates with an approach U (m); m with buffer, as well as ammonium acetate, pH 4.5 (50 mM), after loading the column washed with a balancing buffer until the Y-spectrum gives an even straight
    in the analysis of eluent, as a result of Th. A small amount of effluent is eluted from the column. Then, the column is eluted with 25 mM ammonium acetate and 0s12 M sodium chloride or a combination thereof, which optimizes the regeneration of interferon and results in the formation of a lyophilized filter cake.
    权利要求:
    Claims (1)
    [1]
    Invention Formula
    The method of obtaining human leukocyte interferon with a partial sequence of C s-Ala-trp-Glu-Val-Val-Arg-Ala-Glu-I le-Met-Arg-v-Ser-j involving the transformation of EtColi strain 294 ATCC 31446 plasma , dami selected from the group pLelF A 25, pLelF B trp 7,
    pLelF C trp 35, pLelF D trp 11,
    pLelF F trp I, pLelF I trp 1, pLelF J trp I, cultivation of the transformers obtained, followed by extraction and purification of the resulting polypeptides.
    Priority signs: 01 07о80 with a partial sequence of Cys-Ala-trp-Glu-Val-Val-Arg-Ala-Glu-Ile-Met-Arg-Serr, pLelF A 25, pLelFB trp 7, Escherichia coli ATCC 31446 ;
    08 „09о80, with pLelF С trp 35 pLelF D trp 11, pLelF F trp I. 10,11o80 with pLelF I trp 1, pLelF J trp 1,
    21 "04; 81 in the extraction and purification of the obtained polypeptides.
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    法律状态:
    优先权:
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    US16498680A| true| 1980-07-01|1980-07-01|
    US18490980A| true| 1980-09-08|1980-09-08|
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