• 专利附录
  • 专利说明
  • 权利要求
  • 类似技术
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  • 优先权
    • 专利摘要:
    A method for the recombinant production of zymogen forms of human protein C is described. These zymogen forms differ from native zymogen protein C in their increased sensitivity to activation by thrombin and thrombin/thrombomodulin. DNA compounds, vectors, and transformants useful in the method are also disclosed.
    公开号:SU1739854A3
    申请号:SU884613143
    申请日:1988-12-22
    公开日:1992-06-07
    发明作者:Ульрик Бэнг Нильс；Джозеф Эрлих Хармут；Вилльям Гриннелл Брайан；Бетти Ян Сау-Чи
    申请人:Эли Лилли Энд Компани (Фирма)；
    IPC主号:
    专利说明:

    The invention relates to biotechnology and can be used to produce zymogenic forms of human protein C.
    Protein C - vitamin K - dependent plasma protein plays an important role in the regulation of blood coagulation. Protein C concentration is low in thrombotic conditions, such as disseminated intravascular thrombosis, and in diseases that lead to thrombosis, such as large
    10 20
    5 -ATS TGG CAG STS ASA AGC STS STO H2N-MET TRP GLN LEU THR SER LEU LEU
    five
    trauma, major surgery and cancer.
    To facilitate understanding of the invention and activating protein C, the coding sequence and the corresponding amino acid sequence of human protein C are presented below. This amino acid sequence and its corresponding parts also characterize the native human protein C for the purposes of the proposed method.
    3040
    CTG TTC GTG GCC ACC TGG GGA ATT LEU PHE VAL ALA THR TRP GLY ILE 1015
    V |
    CA O 09 SJ
    N

    CO
    50 60 70 8090
    TCC GGC ACA CCA GCT CCT CTT GAC TCA GTG TTCTCC AGC AGC GAG CGT
    SER GLY THR PRO ALA PRO SER SER VAL PHESER SER SER GLU ARG
    20 25 30
    -1739854
    100110 120 130 140
    GCC CAC CAG GTGCTG CGGATS CGC AAA CGT GCC AAC TCC TTC CTG GAG
    ALA HIS GLN VALLEU ARGILE ARG LYS ARC ALA ASN SER PHE LEU GLU 35 40 45
    150 - 160 170 180190
    GAG CTC CGT AGC AGC AGC CTG GAG CGG GAG TGC ATA GAG GAG ATC TGT GLU LEU ARG CGG
    200 210220 230 240
    GAC TTC GAG GAGGCC AAG GAA ATT TTC CAA AAT GTG GAT GAC ACA CTG
    ASP PHU GLU GLA LYS GLU ILE PHN GLN ASN VAL ASP ASP THR LEU 65707580
    250 260 270 280
    GAC TAG TGG TCC AAG CACGTC GAC GGT GAC CAG TGC TTG GTC TTG CCC
    ALA PHE TRP SER LYS HYSVAL ASP GLY ASP GLN CYS LE VAL LEU PRO
    8590 95
    29Q 300 310 320 330 TTG GAG CAC CCG TGC.GCC AGC CTG TGC TGC GGG CAC GGC ACG TGC ATC LEU GLU HIS CYS GLY HY GL THR CYS ILE 100105110
    340 350 360 370 380
    GAC GGC PBX GGC AGC TTC AGC TGC GAC TGC CGC AGCGGCTGG GAG GGC
    ASP GLY ILL GLY SER PHY SER CYS ASP IG ARG SERGLYTRP GLU GLY
    115120125
     390400410420 430
    CGC TTC TGC CAG CGCGAGGTG AGCTTC CTC AAT TGC TCG CTG GAC AAC
    ARG PHE CYS GLN ARGGLUVAL SERPHE LEU ASN CYS SER LEU ASP ASN
    130135140
     440 450 460 470 480
    GGC GGC GGC TGC ACG CAT TAC TGCST GAG GAG GTG GGC TGG CGG CGC TGT
    GLY GLY CYS THR HYC TYR CYSLEU GLU GLU GLY COLY TRP ARG ARG CYS 145-101515160
    490 500 510 520
    GAC GAC CG CG CAG CG CAG CG SER CG CG CG CG TGT GGG GAC CG CG CAG CG CAG CG CAG CG CAG CG CAG
    165 170175
    530. 540 550 560 570
    CCC GCA GTG AAG TTCCCT TGT GGG AGG CCC TGG AAG CGG ATG GAG AAG
    PRO ALA VAL LYS PHEPRO CYS GLY ARG PRO TRP LYS ARG MET GLU LYS 180185190
    580590 600 610 620
    AAG CGC ACTCAC CTG AAA CGA GAC ACA GAA. GAC CAA- GAAGAC CAA GTA
    LYS ARG SERHIS LEU LYS ARG ASP THR GLU ASP GLN GLUASP GLN VAL
    195200 205
    630640 650 660670
    CGG CGG CGG ATC CGG CGG AAG ATG CGG CGG ASC CGG CGG ASC ASC A TG A A S A A A A A T A S A A C A A C A A C A A C A A C A A C A A C A A C A A C A A C A A C A C A C C A C C A C C A C C E C C E C C E C S C A C А S C A C S C S C A C S C A S C A S C E C A S S C A S C A S C E C A S S C A S C E C A S S S S C A S C A S C A S S S S S S S S C A S S S S S S S S C S C S S S S S S S S S S S S S S S S S S S S S S S S S S S3 Of D3 C 6
    51739854
    680 690 700 710 720 TGG, CAG, GTG, GTC, CTG, CTG, GAC, TCA, AAG, AAG, AAG, CTG, GCC, TGC, GGG, GCA, GG
    730 740 750760
    GTG CTC PBX CAC CCC TCC TGG GTG CTG ACA GCG GCC CAC TGC ATG ATC GAT VAL LEU
    245250255
    770 780 790 800 810
    GAG TCC AAG AAG CTC CTT GTC AGG CTTGGA GAG TAT GAC CTG CGG CGC
    GLU SER LYS LYS LEU LEU VAL ARG LEUGLY GLU TYR ASP LEU ARC, 260,265,270
    820830 840 850 860
    TGG GAG AAG TGGGAG CTG GACCTG GACATS AAG GAG GTC TTC GTC CAC
    TRP GLU LYS TRPGLU LEU ASPLEU ASPILE LYS GLU VAL PHE VAL HIS
    275280285
    870 880 890 900 910 AAC AAC AAC AAC AAC AAC AAC AAC AAC AAC AAC AAC ASC AAC AAC AAC ASC AAC AAC ASC AAC ASC AAC AAC
    920 930 940 950 960 CTG GCC CAG ASC GCC CTC ASC CTC TCG CAG ASCA ATA GTG CCC ATC TGC CTC LE CU LE 3 305310315320
    970 980 990 1000
    CCG GAC AGC GGC CTTGCA GAC CGC GAGCTC, AATCAG GCC GGC CAG GAG
    PRO ASP SER GLY LEUALA GLU ARG GLULEU ASNGLN ALA GLY GLN CLU
    325330335
    1010 10201030 1040 1050
    ACC CTC GTG ACG GGC TCGGGC TAC CACAGC AGC CGA GAG AAG GAG GCC
    THR LEU VAL THR GLY TRPGLY TYR HISSER SER ARG CLU LYS GLU ALA.
    340345 350
    1060 1070 1080 1090 POOP AAG AGA AAC CGC ACC TTC GTC CTC AAC TTC PBX AAG ATT CCC GTG
    1110 1120 1130 1140 1150 CCG CAC AAT GAG TAC AGC GAC GTC ATC AGC AAC ATG GTG CTA
    116011701180 1190 1200
    ATG CTG TGT GCG GGCATC CTC GGGGAC CGG CAG GAT CCC TGC GAC GGC
    MET LEU CYS ALA GLYILE LEU GLYASP ARG GLN ASP ALA CYS GLU GLY
    385 390395 400
    121012201230 1240
    GAC AGT GGG GGG CCCATG GTC GCCTCC TTC CAC GGC ACC TGC CG CTC ASP SER GLY GLY
    405410. 415
    1250 1260 1270 1280 1290 GTG GGG CGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGYGYGYGY
    1300 1310 1320 1330 1340 GGC GTT TAC ACC AAA GTC AGC CGC TAC CAC CAC TGG ATC CAT GGG CACGY GLY VAL TYR
    1350 1360 1370 1380 PBX AGA GAG AAG GAG ASC ASC ASC ASC A TGG GCA CCT TAG-3
    The DNA sequence is derived from c-DNA clones created from human hepatic m-RNA encoding human protein C Two of these c-DNA clones are used to construct a DNA molecule that includes both the coding sequence of protein C and parts of DNA encoding untranslated mRNA at the ends and 3-coding region. This DNA molecule is introduced into the Pscl site of the plasmid pBR322 to construct the plasmid pC77. The pHC7 plasmid includes the coding sequence described above, as well as the following additional sequences:
     TGG AGG GGG GGG GGG GGG GGG GGG CTA TCA TGG CGG CAG GGC GAA CTT GCA CTA TST CCA CGA CCC GCC CST ASA GGT GCC ACT GCC TCC AGA-31
    he-pro is a sequence of amino acid residues 1-42, encoding a signal peptide and a propeptide of human C protein, important for the directed secretion and fr-carboxylation of protein C:
    LC - Amino acid residue sequence 43-197 of protein C constitutes the light chain (LC) of both the double-stranded zymogen (formed from single-stranded zymogen by cleavage of the KR-dipeptide) and the activated forms of protein C}
    KR - sequence of amino acid residues 198 - 199 human protein C; it is believed that these residues are split off possibly in two stages, including first cleavage (on residues 197–198 or 199–200), and then the action of carboxyl peptidase or amino-i peptidase to form a double-stranded protein C;
    AP is a sequence of amino acid residues 200 - 211 of protein C, including an activation peptide cleaved from zymogenic
    and
    5-CGA CCC TGC CTG-CAG GGC TGG GCT TTG GCA TGG CAA TGG CAA TGG ATG GGA CAT TAA AGG GAC ATG TAA CAA GCA CAC CCC CCC CCC CCC CCC CCC CCC CCC GCA GCA G-3
    r r
    respectively, at the ends of the 5- and 5-strands of the coding sequence for the released human protein C Due to the complementary nature of the base pairs of DNA, the sequence of one strand of the double-stranded DNA molecule is residual to determine the sequence of the second strand. Plasmid pNC7 was isolated from E. coli K12 RR 7 / pNC7 deposited as NRRL B-15926,
    Protein C can be represented schematically as follows:
    five
    0
    five
    forms C to form activated protein C; The ANS — amino acid residue sequence 212–461 of protein C, once post-translationally modified, constitutes the activated heavy chain (ANS) of the active protein C; NS - the desired chain of the double-stranded form of zymogen C, once post-translationally modified, consists of amino acid residues 0 200 - 461, AR and ANS „
    Zymogen C is a precursor of the sarin proteae, synthesized in the liver and present in the blood. . For full biological activity, protein C requires post-translational modifications, for which vitamin K is necessary. Double-stranded, linked by a disulfide bridge, zymogen C is formed from single-chain zymogen by proteolysis. This limited proteolysis is considered to include cleavage and deletion of amino acid residues 198 and 199. Activation of double-stranded 5 zymogen includes proteolytic cleavage of the ARG-LEU peptide bond (residues 211 and 212). This last cleavage releases decaliptide (residues 200–211), an activating peptide constituting the amino terminus of the larger (heavy) chain of the two-chain zymogen molecule. Protein C contains about 23% carbohydrates. Protein C also contains a large amount of uncommon amino acids, including J-carboxyl-glutamic acid and -oxy-aspartic acid, J1-arq sig glutamic acid (ja) is formed by jf-glutamyl carboxylation of glutamic acid residues using microsomal carboxylase.
    Amino acid residues in the described proteins and peptides have the abbreviations given in table.
    T a b l and c a
    200 201 202 203 204 205 296 207 208 209 210 211 ASP-THR-GLU-ASP-GEN-GLU-ASP-GLN-VAL-ASP-PRO-ARG,
    to
    one
    ten
    15
    20
    73985410
    Several methods have been described for obtaining the native zymogen of human protein C. The zymogen form of human protein C, the resulting technology of recombinant DNA, is identical to the zymogenic forms of human protein C, naturally present in human blood, and is activated in the body only naturally, including the thrombin-thrombomodular complex - The present invention proposes zymogenic forms of human protein C, which can be activated in vivo by thrombin alone at a clinically significant rate. In addition, these zymogenic forms are more easily subjected to thrombin / brombomodulin activation than the native zymogen of human protein C
    The invention also provides recombinant DNAs that encode a pre-prepeptide containing a signal peptide to direct secretion and
    25 propeptide from Y-carboxylated protein o
    Recombinant DNA. The invention also includes a sequence encoding a light chain of human protein C located in the translational reading structure immediately after the sequence coding for the pre-propeptide. The light chain of human protein C contains the amino acid sequences of 43-197 protein C, and the amino terminal parts of vitamin K-dependent plasma proteins, such as the amino terminal portion of the light chain of protein C, are responsible for the calcium binding activity of these proteins.
    Recombinant DNAs of the present invention also include a sequence encoding a LGS-ARG dipeptide (KR) located in
    45 of the translational reading structure immediately after the sequence encoding the light chain. The LYS-ARG dipeptide is located on the carboxy-terminal portion of the light chain,
    50 The zymogenic forms of this invention differ from the native zymogenic forms of human protein C, In native human protein C. There is the following approval peep 55:
    thirty
    35
    40
    where the numbers correspond to the position leading to the cleavage of amino acid amino acid residues in protein C. In residues 1 -42) to form the invention, the replacement of the motif form is described. Replacing the last ASP residue at position 209 with one residue at position 210 in the isolated human protein C with valine,
    of the following residues: PHE, GLY, TYR or TRP, which leads to the corresponding zymogenic form, which is more susceptible to cleavage by one thrombin, except for increased sensitivity to cleavage by the thrombin-thrombomodulin complex
    Other amino acid substitutions, along with substitutions at position 209, may
    except for one of the four substitutions described above at position 209, it leads to a new zymogen. Replacing the IQ residue of aspartic acid at position 214 of protein C, together with one of the substitutions described above at position 209 and with or without the replacement described for position 210, with a residue, also increases the sensitivity of the as-5 zimogen to the action of thrombin
    Thus, the preferred new zymogenic forms of human protein C are formed as a result of sec-acid residues in the isolated 20 Reactions and the treatment of molecules with the human protein G, however, the released human protein C must first be secreted (which is when HFcN-MET TRP GLN LEU THR SER LEU LEU LEU PHE VAL ALA THR TRP GLY ILE
    . SER GLY THR PRO ALA PRO SER SER VAL PHE SER SER SER GLU ARC
    ALA HIS GLN VAL LEU ARC ILE ARC LYS ARG ALA ASN SER PHE LEU GLU
    GLU, LEU ARG HY SER SER LEU GLU ARG HY CYS
    . ASP PHE GLU GLA ALA LYS GLU ILE PHE GLN ASN VAL ASP ASP THR LEU
    ALA PHE TRP SER LYS HIS VAL ASP GLY ASP GLN CYS LE VAL LEU PRO
    LEU GLU HIS PRO CYS
    GLY GLY ILLA GLY ILLA SERGY CYS GLY IGNY GLG
    GLY GLY GLS CLEAN THROW HYDG COL LE GLU GLY HYLA TRY ARG ARG ARG CGYA SERGUES GLY GLA CRO GLU HOLY LYSHY LYS ARG ASP HOLY LYS CENHY GLA ASPA GLNASAUA GL GLU GLA GLU GLU GLU GLU GLU GLU GLU GLU GLU GLU GL
    except for one of the four substitutions described above at position 209, it leads to a new zymogen. Replacing the aspartic acid residue at position 214 of protein C, along with one of the substitutions described above at position 209 and with or at the replacement described for position 210, with a new zimogen,
    Thus, the preferred new zymogenic forms of human protein C are formed as a result of the human C-protein secretion C with the following amino acid sequence:
    one
    13 ASN TYR SER.
    ALA GLN PRO ASP SER GLY LEU VAL THR ARC ASN ARC HIS ASA GLA LE CYS
    .LYS SER ALA THR LEU ALA GLY TRP THR PHY CYS SER GLY ILE PRO MET SER TRP LYS VAL GLU ALA
    plasmids pL APC
    The amino acid sequences encoded at positions 209, 210, and 214 in the preferred coding sequences are shown in Table 2
    Table 2
    39854 THR ASP
    SER, GLN, GLU, ARC, GLU, GLY, ARC, GL, LYS
    14 ASN ASP ILE ALA LEU LEU HIS
    ALA GLY GLU GLA SER.SER ARC GLU GLU GLA SER CYS GLY LEU LE HIS
    Plasmid pLPC-167G is an illustrative expression vector in which the codon of aspartic acid at position 209 of protein C is replaced with a glypine codon. The construction of plasmid pLPC-167G is described in Example 3. It is significant that the design involves site-specific mutagenesis of the sequence encoding protein C. Part of the sequence encoding protein C is inserted into the M13 tr18 phage and altered by site-specific mutagenesis. The mutagenized coding sequence is then cloned into a eukaryotic cloning vector to obtain plasmid pLPC-167G, identical to the plasmid
    15
    mid pLAPC, except that, there is an insert sequence, | coding activation peptide, c. wherein the codon for glycine is replaced by the codon for aspartic acid at position 209.
    In the case of compounds according to the inventive method, where the aspartic acid residue at position 214 is replaced by an asparagine residue, protein C derived from activation of the zymogenic form, is also a compound of this invention
    The DNAs of this invention can also be synthesized chemically or by a combination of restriction fragments, or a combination of methods known in the art
    Illustrative vectors of this invention, plasmids pLPC-167G and pLPC-1-67G contain a BK amplifier that stimulates transcription by adenoviral main late promoter. A large number of eukaryotic promoters, amplifiers and expression vectors are known in the art and can be used in the proposed method. The eukaryotic expression vector can also function without amplifying. an item. The essence of the present invention is not related to a specific enhancer or promoter used to stimulate the expression of the zymogen of protein C, but rather is determined by a new coding sequence and the corresponding proteins derived from such a sequence.
    However, the choice of vector elements, such as promoters, enhancers, and selective markers, can have a strong effect on the maximum protein concentrations produced by the host cell.
    The choice of host cells depends on the specific expression vector used to express DNA compounds coding for proteins C. Because protein C and protein C derivatives according to the proposed method undergo significant post-translational modification, some host cells are more preferable for use. with vectors according to this invention. It is known that
    15
    20
    25
    Jf-carboxylated proteins, such as I as human C protein. One such adenovirus transforming human embryonic kidney cell line is the 293 cell line available in ATCC by ATCC number CRL 1573.
    However, the advantages of obtaining JQ Y-carboxylated protein, such as the human zymogen protein C in the cell line transformed by adnovirus, are not limited to human kidney embryonic cells transformed with adeno virus. In fact, as a rule, adenovirus-transformed cells are excellent hosts. for producing Jf-carboxylated human protein C. One of the particularly preferred cell lines of this type is the AV 12-664 (hereinafter referred to as AV 12) cell line, available at the ATCC under accession number ATCC CRL 9595 Cellular whether audio received AV 12. 12 human adenovirus injection into the neck of the Syrian hamster and isolating the resulting tumor cells
    Expression of the coding sequences of human protein C contained in the vectors of this invention occurs in those host cells in which the promoter is associated with structural gene functions.
    2-type pSV vectors include segments of the SV 40 genome containing a specific eukaryotic promoter unit (ep), a sequence of insertion 40 (IVS) and polyadenomal (pA)
    site. In the absence of SV4 T-antigen, plasmid 2-type pSV vectors transform mammalian host cells and other eukaryotic cells by integrating host cells into the chromosomal DNA of host cells.
    The degeneracy of the genetic code allows the nucleotides to be replaced in the regions encoding the polypeptide, as well as in the translational stop signal without changing the sequence encoding the polypeptide. Such sequences can be derived from a known amino acid or DNA sequence.
    thirty
    35
    45
    50
    adenovirus-transformed human 55
    C and can be constructed according to conventional synthetic or site specific mutagenic techniques.
    Belonging embryonic renal cells are preferred for use in recombinant production.
    , |

    15
    20
    25
    73985416
    Jf-carboxylated proteins, such as I as human C protein. One such transformation by adenovirus, the human embryonic kidney cell line is the 293 cell line available in ATCC under the ATCC number CRL 1573.
    However, the advantages in obtaining JQ-carboxylated protein, such as human zymogen C protein in adenovirus-transformed cell lines, are not limited to human, renal embryonic cells transformed with adenovirus. In fact, as a rule, cells transformed with adenovirus are excellent hosts for obtaining Jf-carboxylated C human protein. One particularly preferred cell line of this type is AV 12-664 (hereinafter referred to as AV 12) cell line, foot at the ATCC under accession number ATCC CRL 9595 cell line was obtained AV 12. 12 human adenovirus injection into the neck of the Syrian hamster and isolating the resulting tumor cells
    Expression of the coding sequences of human protein C contained in the vectors of this invention occurs in those host cells in which the promoter is associated with structural gene functions.
    2-type pSV vectors include segments of the SV 40 genome containing a specific eukaryotic promoter unit (ep), an insertion sequence (IVS), and a polyadenomal (pA)
    site. In the absence of SV40 T-antigen, plasmid 2-type pSV vectors transform mammalian host cells and other eukaryotic cells by integrating host cells into the chromosomal DNA of host cells.
    The degeneracy of the genetic code allows the nucleotides to be replaced in the regions encoding the polypeptide, as well as in the translational stop signal without changing the sequence encoding the polypeptide. Such sequences can be derived from a known amino acid or DNA sequence.
    thirty
    35
    45
    50
    human dietary protein
    C and can be constructed by conventional synthetic or site-specific mutagenic techniques.
    17
    The main disadvantage of activated protein C, like any activated serine proteases, is its short half-life (T1 / 2) compared to the zymogen precursor. The reason for the shorter biological half-life of activated serine proteases, including activated protein C, is complex processes involving cellular and humoral mechanisms. Activated serine proteases also form complexes with serine protease inhibitors normally present in plasma. Activated protein C (APC) is complexed with the APC inhibitor, as well as with alpha-2-macroglobulin,. Inactive zymogens, including protein C zymogens of the present invention, do not react with serine protease inhibitors.
    The advantage of the proposed C zymogens is that they are better activated by thrombin than the native protein zymogen, because for the activation of zymogens in the presence of Ca there is no absolute requirement for the complexation of thrombin with thrombomodulino, which means that these zymogens C can be activated in places intravascular thrombin formation, in any zones of development of intravascular thrombus. Thus, these recombinant protein C zymogens can be used as preventive drugs and will only be activated at the sites where blood clots form. Since these thrombin-sensitive zymogens can be administered in a zymogenic form, they will not form complexes with protein C inhibitors and will have a biological half-life equal to that of the native protein C zymogen.
    Recombinant zymogen protein s. The present invention is suitable for the prevention and treatment of a wide range of diseases, including intra-vascular coagulation, including deep vein thrombosis, pulmonary emboli, peripheral arterial thrombosis, emboliemosis in the heart and peripheral arteries, acute myocardial infarction, thrombotic strokes and disseminated intravascular thrombosis.

    and 73985418
    Compared with activated protein C, the doses of zymogens of protein C according to this invention due to their increased 5 (personalized T1 / 2 can be significantly reduced in clinical use. With a homozygous deficiency of protein C, the dose of zymogen of protein C according to this invention is within 10 5 - 100 mg per treatment, and for heterozygous deficiency of protein C - in the range of about 2.5 - 50 mg per treatment
    A good therapeutic indication for activated protein C is the prevention of deep vein thrombosis and pulmonary embolism.
    The zymogens of protein C according to the invention can be used in the treatment of embolism resulting from blood clots in peripheral arteries, mainly in the carotid arteries.
    The zymogens of this invention 25 are also suitable for the treatment of acute myocardial infarction. In the acute phase of myocardial infarction, these zymogens can be administered together with a tissue plasminogen activator.
    thirty
    35
    40
    45
    50
    55
    one
    Example 1 ,, Plasma plasmid pLAPC.
    A. Isolation of the DNA fragment encoding the protein peptide C.
    Plasmid PHC7 contains the complete coding sequence of protein C, 1 l of L-broth (10 g peptone, 10 g NaCl, 5 g yeast extract) containing 15 μg / ml tetracycline, inoculated with Escherichia coli K12 RR1 / pHC7, incubated with shaking at 37 ° C until an optical density (O. U.) is reached at 590 nm of approximately 1 and 150 mg of chloramphenicol is added. Incubation is continued for 16 hours; the addition of chloramphenicol inhibits protein synthesis and further cell division, but allows plasmids to still replicate.
    The culture is centrifuged, the supernatant is discarded, the cell mass is washed with 40 ml of TES buffer (10 mM Tris-HCl, pH 7.5; 10 mM NaCl and 1 mM EDTA) and then precipitated again by centrifugation, the supernatant is discarded, the cell mass is frozen in a bath dry ice is ethanol and thawed. The crushed cell mass is resuspended in 10 ml of solution.
    25% sucrose 50 mM EDTA, add 1 ml of 5 mg / mg lysozyme solution; 3 ml of 0.25 M EDTA, pH 3.0 and 100 µl of 10 mg / ml of RNA-ase A and incubation in ice for 15 minutes with 3 ml of lysing solution (3 ml of 10% Triton-X 100; 75 ml of 0.25 M EDTA, pH 8.0; 15 ml of 1 M Tris-HCl and 7 ml of water) are added to lysozyme-treated cells, mixed and incubated on ice for 15 minutes. "Cells are frozen in a dry ice bath - ethanol , thawed, centrifuged in a gradient of cesium chloride. Plasmid band observed in UV light was isolated. Plasmid DNA was extracted.
    1 mg of plasmid pHC7 DNA is suspended in 1 ml of TE buffer (10 mM Tris-HClt pH 7.6 and 0.1 mMEDTA) and stored at –20 ° C.
    7 μg of the plasmid pHC7 DNA is hydrolyzed with the Sst 1 enzyme, and then with Sal 1 Sst I - Sal I reticectase, the fragment is extracted first with phenol, then with chloroform, collected by precipitation with ethanol, centrifuged and suspended in 15 μl TE / 10-buffer (10 mM Tris base, pH 7.6 and 0.1 mM EDTA).
    The mixture is subjected to electrophoresis on O, 6% low temperature gelation agarose gel for 2–3 hours at approximately 130 V and 65 mA in Tris-acetate buffer. The gel is stained in a solution of ethidium bromide and a DNA band of approximately 0.7 To.O. Extract from the gel as a small segment. The segment is melted by incubating at 72 ° C. A volume of about 400 µl is obtained with approximately 0.5 µg of the Ssc I-Sal I restriction fragment of the plasmid pC7 with a length of about 0.7 tPO. Additional DNA purification is performed by passing the DNA solution through a NaCS-pre rac column (BRL) in accordance with the instructions. The purified fragment is resuspended in 15 μl of deionized water.
    B. Construction of recombinant phage and removal of DNA encoding the activation peptide by site-specific mutagenesis
    About 1 µg of replicative form (RF) of phage M13 m P18 DNA is treated with Sst I and Sal 1 restrictases. Two fragments obtained as a result of cleavage are separated in a 0.6% gel from a low-temperature agorori gel.
    five
    Formation, a larger fragment is cut out of the gel and purified.
    O, 1 μg Sst I - Sal I fragment
    The plasmid pHC7-length of about 0.7 mp “o0 is ligated with 5 μl Sst I - Sal I - treated with M13 m p18 RZ DNA in 2 μl of UH ligase buffer, (0.5 M Tris-HCl, pH 7.8 ; 60.8 mM rfgCl; 0.3 M dithioate
    Reitol / DTT /), 2 μl of 1 mg / ml BSA,
    1 μl 25 mM ATP, 1 μl (about 400 units) of T4-DNA ligaey (NEB) and 2 μl of water.
    About 300 µl of E. coli K12 Ml01 culture grown overnight was used to inoculate 30 ml of 2X-TU-broth (TY-broth is 10 g / l tryptone, 10 g / l NaCl and 5 g / l yeast extract) and the resulting the culture is incubated at 37 ° C and aerated until the OOD reaches about 0.5, the culture is cooled in an ice bath for 10 minutes, collected by centrifugation and again suspended in 15 ml of cold 10 mM NaCl. The cells are collected again by centrifugation and suspended in 15 ml of cold 30 mM CaCl-j 200 µl of cells are separated, added to 9 µl of the above-obtained ligated DNA and incubated on ice 0 for approximately 30 minutes. Then the DNA-cell mixture is incubated at 42 ° C
    2 min. And added to 3 ml of top agar (TY-broth with 0.5% agar maintained as a melt at 45 ° C), also containing 50 μl of 2% X-Gal (X-Gal is 5-bromo-5 -chloro-3-indolyl- / 3-D-galactopyranoside), 50 µl of 100 mM, IPTG (IPTG is isopropyl-g-B-thiogalactopyranoside) and 100 µl of E. coli C12 B101 in the logarithmic growth phase. Then a mixture of top agar / cells is placed on TY agar plates and the plates are incubated overnight at 37 C.
    The next morning, four clear 5 colonies are separately used for seeding 2 ml of 2X TY-broth and the resulting cultures are incubated at 37 ° C and aerated for 6 hours. Then the cultures are centrifuged and 500 μl of the resulting supernatant (cell mass is used to obtain phage DNA for restriction enzyme assays are added to 500 µl of the culture (OD55Q-0.5) E. coli K12 C1101 and. 50 ml of 2X TY-broth. These cultures were incubated overnight at 37 ° C, the phage KF DNA was isolated from the cell mass using the simplified procedure of Example 1A without antibiotic in the culture medium and
    five
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    21
    ultracentrifugation steps are replaced by extraction with phenol and chloroform. Transformants containing DNA phage M13 m p18-HEI, identified by restriction enzyme analysis of their phage DNA.
    After overnight, the cultures were centrifuged and about 1 ml of a solution containing 20% polyethylene glycol (PEG) 6000 and 2.5 mM NaCl was added to 5 ml of the supernatant and incubated for 10 minutes at room temperature. The mixture is centrifuged, the resulting precipitate containing the single-stranded DNA of phage M13 m p18-PE1 is suspended again in 500 μl of TES buffer (20 mM Tris-HCl, pH 7.5; 0.1 MEDT; 10 mM Nad) DNA solution
    57-GGGCAGTCACCTGAAACCACTGATTGATGGGAACATGA-3V
    30 pmol of this fragment are individually treated with 5 units of T4-polynucleotide kinase in 10 μl of IX-kinase buffer (100 mM Tris-HCl, pH 8.3, 10 mM DDT; 100 mM MgClg.) Containing 1 μl 1 mM ATP, 30 min at 37 ° C and then incubated for 10 min at 65 ° C and frozen. Kinase-treated DNAs are used for the mutagenesis described below.
    In the first stage of mutagenesis, the mutagenic oligonucleotide and the universal primer M13 are renatured into single stranded phage DNA. Renaturation is carried out by adding 300 ng (0.5 μl) of single stranded phage M13mp18-HEI to 1 pmol (1.2 μl) of the universal primer, 1 pmm (0.3 μl) of mutagenic oligonucleotide, 2 μl of 1OX-renaturation buffer (100 mM Tris-HCl, pH 7.5; 1 mM EDTA and 500 mM NaCl) and 16 μl of water, incubate the mixture at 80 ° С 2 min and then 5 min at 50 ° Сf then allow the mixture to cool to room temperature.
    After renaturation of the oligonucleotides, phage DNA is made double-stranded, the primers are expanded with a DNA polymerase. This reaction is carried out by adding 3 μl of 1OX extension buffer (500 mM Tris-HCl, pH 8; 1 mM EDTA and 120 mM MgCl) 3 μl 10 X - ligase buffer; 1.5 μl 0.2 mM DTT; 3 μl of dNTP carry (0.5 mM each dNTP); 1.2 μl 25 mM ATP; 0.5 µl Klenow enzyme (5 units // µl, WWII); 1 μl of T4 DNA ligase (400 udo, NEB) and 19.8 μl of water to synergated DNA estimates, Expansion

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    extracted first with chloroform, zgN with that twice with TE-saturated phenol and then again with chloroform, Single-stranded DNA is precipitated with NaOAc and ethanol, centrifuged, washed with 70% ethanol.
    The precipitate is dried and dissolved in 80 µl water. This phage sample is used in the next stage of site-specific mutagenesis to remove the DNA encoding the activation peptide.
    The single-stranded DNA fragment used for mutagenesis when DNA encoding the activation peptide is removed is obtained on an automatic DNA synthesizer in the following form:
    carried out by incubation at room temperature for 30 minutes, then 4 hours at 37 ° C and overnight at 4 ° C.
    The reaction is stopped by extraction with phenol-chloroform and the DNA is precipitated with ethanol with sodium acetate (NaOAc). DNA is collected by centrifugation, suspended in 40 μl of Si buffer (0.3 M NaCl :; 0.03 M NaOAc, pH 4.5 and , 3 mM ZnCl2) and added to the DNA solution. It has been established that Si-processing does not have significant advantages, and in the design methods described in the following examples, Si-processing is completely excluded.
    The DNA solution is divided into two tubes into two equal parts, and 100 units are added to one of the tubes. (WWII) SI nucleases, Si-reaction is carried out by incubating for 5 minutes at room temperature and stopped by extracting the reaction mixture with a TE-saturated mixture of phenol-chloroform (50:50).
    The precipitated DNA is suspended in 60 μl of water and transformed into E. coli K12 IM101 o. Mutants are selected using radioactively labeled mutagenic oligonucleotide, AAACGACTCA-TTGA-3 and point hybridizations,. Several positively hybridized spots are selected and seeded in 2 ml of culture E, coli K12 IM101, which is in the logarithmic growth stage. The cultures are incubated at 37 ° C and aerated for about 6 hours and then used to produce single stranded DNA.
    Single stranded DNA is sequenced. Several phages are identified by a chain mutation. The phage in which the sequence encoding the activation peptide is deleted is the target phage M13 mpia-HE2. Mutation in the phage M13 of Gar18-HE2 causes a 36 bp reduction in size compared to the natural coding sequence, and this difference can be used to identify DNA containing the mutated region. The RF form of phage M13 tr18-HE2 is obtained by subsequent construction.
    C. Construction of shtazmida pLAPC from phage M13 tr18-HE2 and plasmid pLPC
    Sst I - Sal I fragment (about 0.7 t „n„ o,) KF forms of phage M13 tr18-NOT are cut off from phage and isolated by the method of Example 1A „
    Three DNA fragments are linked together to produce the plasmid pLAPC: Sst I - the Sal I fragment of the phage M13 mp18-HE2 (about 0.7 kb) and 2 DNA fragments of the plasmid pLPCo Since restriction enzyme sites are in the plasmid pLPC Sal Sst I and Eco RI, then the target Eco RI - Sal I and Eco RI - Sst I restriction fragments need to be obtained by two separate cleavages.
    Sst I - Eco RI cleaved DNA is suspended in water and placed in a 0.6% gel from a low gel temperature agarose to separate the DNA fragments.
    To obtain the EcoR I - Sal I fragment, about 15 µg of the plasmid pLPC is first treated with the restriction enzyme Ara to remove contaminants of similar size, restriction fragments. Then Sal I restriction enzymes, EcoR I, are added to the Apal-digested pLPC plasmid DNA solution and the resulting mixture is incubated. I-EcoR I, the digested pLPC plasmid DNA is first extracted with phenol, then chloroform,
    -
    collected by precipitation with ethanol and centrifuging. Suspended in water (placed on a 0.6% agarose gel and separated by electrophoresis.
    The restriction fragments EcoR I - Sal I (about 3.76 kb) and EcoR I - Sst 1 "(about 2.0 kb) are cut out from the gel and isolated.
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    . Jq jg
    20 2 25,
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    Each of the two purified restriction fragments was added to the Sst I-Sal 1-restriction fragment (about 0.7 kb) of M13 ph18-HE2 phage and ligated, resulting in the target plasmid pLAPC. Ppasmid pLAPC differs from plasmid pLPC in the absence of DNA encoding the activation peptide.
    Plasmid pLAPC is transformed into E. coli K12 RV308 and grown.
     Transformants Her coli K12 RV308 / / pLAPC confirmed by restriction enzyme analysis. Plasmid DNA is obtained from the E. coli K12 RV308 / PLAPC transformants in almost the same way as in Example 1A, except that 50 μg / ml of ampicillin and not tetracycline is used as the selective agent.
    PRI mme R 2. Construction of plasmids pLPC.
    The plasmid pLPC is used as an intermediate vector in the construction of the plasmid pLAPCo. The plasmid pLPC contains a DNA segment encoding a BK viral enhancer and the late promoter of adenovirus 2 leading to the expression of human protein C. Constructing plasmid pLAPC mainly leads to the replacement of the sequence encoding human protein C in plasmid pLPC, to another sequence coding for protein C, on which the DNA encoding the activation peptide has been removed.
    Example 2B describes the construction of the pVK peo1 plasmid obtained by inserting a VK-amplifier into the plasmid pdBPV-MMT neo. Example 2C describes the construction of the plasmid pLPcat obtained by inserting the late promoter of adenovirus 2 into the plasmid pSV2cat. In Example 2F, the construction of the plasmid pBLcat containing a VK-amplifier for enhancing the activity of a late adenoviral promoter is described. Example 2E describes the construction of plasmid rYZZ protein C expression vector, beginning with starting plasmid pHC7 through the construction of intermediate plasmid pSV2-HPC8 and the resulting construction of the intermediate plasmid pLPC, .soderzhaschey expression control sequence of the BK enhancer (adenovirus late promoter of plasmid pBLcat inserted into plasmid RYZZ to control the expression of human protein C)
    A. Preparation of VK virus DNA. VK virus is obtained from the Collection
    American Type Cultures, ATCC number VR-837. The host organism for the production of BK-viral DNA is a human embryonic kidney (PHEC) cell culture.
    The virus is isolated from the cells by three freeze-thaw cycles and the cell debris is removed by centrifugation. The virus is precipitated, harvested by adding PEG-6000, incubated for 24 hours at 4 ° C and centrifuged. The precipitate is dissolved in 0.1 X SSC buffer (1X SSC 0.15 M NaCl and 0.015 M sodium citrate, pH 7) at 1/100 of the initial volume. The virus suspension is centrifuged, and two bands are observed. The lower band containing the complete virion is collected and desalted on a Sephadex C-50 column.
    Sodium dodecyl sulfate is added to the solution of purified virions obtained after the column to a final concentration of 1%; Protease at a concentration of 100 µg / ml is added and the solution is incubated for 2 hours at 37 C. Then cesium chloride is added to the solution to a density of 1.56 g / ml and ethidium bromide to a concentration of 100 µg / ml. The solution is centrifuged. After centrifugation, the viral DNA band was isolated and extracted 5 times with isoamyl alcohol saturated with 100 mM Tris-HCl, pH 7.8 °. Then, the solution of BK-viral DN was dialyzed against TE buffer until the DNA absorbance ratio at 260 nm / 280 nm was equal to 1 , 75 - 1.90 DNA precipitated, bringing the concentration of NaCl to 0.15 M, 2 volumes of ethanol are added, the solution is incubated for at least 2 hours at -70 ° C and centrifuged for 10 mi at 12000 g. The resulting precipitate of BK-viral DNA is suspended in TE buffer at a concentration of 1 mg / ml.
    B. Construction of the plasmid rVK neol.
    E. coli K12 HB101 / pdBPV-11MT peo cells are obtained in lyophilized form from the American Type Culture Collection under the number ATCC 37224 “Lyophusized cells are placed on L-agar plates containing 100 μg / ml ampicillin, and incubated at 37 ° C to obtain isolates Comrade single colony.
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    1 l of L-broth containing 50 µg / / ml of ampicillin is inoculated with E. coli K12 HBlOI / pdBPV-NMT neo colony and incubated at 37 ° C until OoD.5g0 reaches approximately 1 g. 150 mg of chloramphenicolum is added to the culture Incubation continues for approximately 16 hours; the addition of chloramphenicol inhibits protein synthesis and further cell division, but allows plasmid replication to continue. PdBPV-MMT neo plasmid DNA is obtained from the culture according to the procedure described in Example 1A.
    The plasmid pdBPV-MMT neo DNA was digested with the restriction enzyme VatH1 "VatH1 - VatH1; the DNA fragment was precipitated, collected by centrifugation and suspended
    The VatS – VatNCH plasmid fragment of the pdBPV – MMT peo (1 μl) and the VagN1 – VatH1 fragment of the BK-viral DNA are ligated with T4-DNA ligase. The ligated DNA is transformed into E. coli K12HB101 cells.
    Transformed cells are placed on L-agar plates containing 100 μg / ml ampicillin. Transformants Eocoli K12 HB101 / rVKpeo1 and E.coli K12 / rVK peo2 are identified by restriction enzyme analysis.
    C Construction of plasmid pLPcat, an intermediate plasmid for constructing plasmid pBLcat.
    The virion DNA of adenovirus 2 (Ad2) is a double-stranded linear molecule about 35.94 tons in length, "p0o. The late promoter Ad2 can be distinguished in the AccI-Pwu II, restriction fragment (about 0.32 Ton.oJ of the Ad2 genome; this restriction the fragment corresponds to the sequence between nucleotides b755 and 6071 of the Ad2 genome; to isolate it, the Ad2 DNA is first digested with the restriction enzyme Bal Iri and Bal I, a restriction fragment of about 2.4. The OO containing the entire AccI – Pvu II sequence restriction fragment with a length of about 0.32 t.po. then Bal I restriction fragment for another. About 2.4 ToP.O., cleave AccI Pvu II to obtain the target fragment.
    Ad2 DNA (obtained from BRL) is treated with restriction enzyme Bal I, subjected to electrophoresis before separation, restriction fragments,
    Bal I - restriction fragment Ad2, about 2.4 kb in length, cleaved
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    first with the restriction enzyme Accl, then with the restriction enzyme Pvu II, applied onto a 6% polyacrylamide gel and subjected to electrophoresis before separation of Accl - Pvu II - a restriction fragment of about 0.32 tonnes containing the Ad2 promoter.
    In order to transform the Accl - Pvu II restriction fragment into Accl - Bel I - the restriction fragment Bel I - linkers are bound to the restriction fragment Accl - Pvu II with a length of about 0.32 TcPo. Because Bel I linkers have blunt ends, they only attach to the Pvu II end of restriction fragments. Bel I linkers have the following sequence:
    5 -CTGATCAG-3
    3 -GACTAGTC-5 about
    0.25 µg of Bel I-linker-treated kinase is added to the Accl-Bvu II restriction fragment solution of a length of about 0.32 tons of pnc, then 1000 units of T4 DNA ligase and 1 µl of 10X ligase buffer are added and the resulting mixture is incubated overnight at 16 ° C. Bel I linkers can only bind to the Pvu II end of the AccI-Pwu II restriction fragment. Subsequent DNA sequencing shows that 4 Bel I-linkers are attached to the Pvu II end of the AccI-Pvu restriction fragment But These additional Bel I linkers can be removed by Bel I cleavage and rebinding; however, additional Bel I linkers are not removed because they do not interfere with the proper functioning of the vectors containing these linkers.
    Eocoli R12 HB101 / pSV2cat cells were obtained in lyophilized form from ATCC as ATCC number 37155, and the plasmid DNA pSV2cat was isolated. The plasmid pSV2cat DNA is ligated first with the restriction enzyme Ace I, and then with the restriction enzyme Stu I.
    The Accl-Stu I plasmid pSVcat DNA fragment is mixed with the Accl-Pv “II Ad2 fragment having Bel I-linkers attached, ligated and transformed into Eocoli K12 HB101. Bound DNA is the target plasmid of pLPcat containing the Ad2 late promoter, located so as to control transcription and expression of the chloramphenicol acetyltransferase gene.
    D. Construction of plasmid pBLcat.
    The pVK puo plasmid DNA was digested with the Accl enzyme and an agarose gel was used to isolate a fragment of about 1.4 ToPoO. Containing a VK-amplifier 5 of a fragment of the fragment was digested with Pvu II restriction. The Pvu II fragment of DNA Q was isolated, purified and prepared for zyvanie
    The plasmid pLPcat DNA is first hydrolyzed with the restriction enzyme Accl and then with the Stuff restriction enzyme, and the plasmid pLPcat DNA is precipitated several times with ethanol to purify the Accl - Stu I-restriction fragment of about 4.81 TcPoO. 0 Tor Ad2.
    The Accl - Stu I fragment of the plasmid pLPcat is ligated with the Accl t Pvu II fragment (1.28 kb) of the pBK neol plasmid. Bound DNA is 5 target plasmid pBLcat,
    Bound DNA is used to transform E „CoC K12 HB101 cells. Transformants E. coli K12 HB101 // pBLcat are identified by restriction analysis.
    E. Construction of the plasmid RYZ3. The plasmid RYZ3 is the expression vector of human protein C.
    5.0 μg of DNA of the plasmid pHH / mixed with 50 units of Ban I restriction enzyme, 10 μl of 10X Ban I - reaction buffer (1.5 M NaCl; 60 mM Tris-HC1, pH 7.9; 60 mM McC12 and 1 mg / ml BSA), 35 μl of water and incubated, Split DNA plasmid rHC7 is subjected to
    electrophoresis in 3.5% full-acrylamide gel.
    A region containing Ban I is cut out of the gel - a restriction fragment of about 1.25 mp in length, 0 °, area
    squash into test tube and grind. 1 ml of extraction ferrum (500 mM ShNOAc, 10 mM MgOAc,
    1 mM EDTA, 1% SDS and 10 mg / ml t-RNA) are added to the particles and the tube is kept overnight at 37 ° C. The residues are removed by centrifugation and the supernatant is transferred to a new tube. The supernatant is passed through glass wool, 2 volumes of ethanol are added and mixed. The resulting solution is placed for 10 minutes in dry ice - eta29
    sex and DNA precipitated by centrifugation.
    The purified fragment is suspended in 10 µl of TE buffer and stored at -20 ° C. Ban 1 restriction fragment is modified by adding a linker to construct the pSV2-HPC8 plamid
    The required linker has the following structure:
    5 -AGCTTTGATCAG- З1
    3 -AACTAGTCCACG-5
    8 μg of Ban I-fragment length of about 1.25 T. p. add and mix with 500 pmol linker and ligate
    The DNA is isolated and dissolved in the Ara of the reaction buffer and hydrolyzed with Apal restriction enzyme. DNA precipitated The precipitated DNA is dissolved and hydrolyzed with restriction enzyme Hind III. Hind III - - Apal - restriction fragment was isolated by gel electrophoresis, 5 µg of the target fragment was suspended in 10 µl of TE buffer and stored at -20 ° C.
    The plasmid pHC7 DNA is hydrolyzed by restriction enzyme Psc I buffer (1.0 M NaCl; 100 m M Tris-HCl, pH 7.5; 1.00 mM MgCl2 and 1 mg / ml BSA) and 35 μl of water and incubated for 2 h at 37 ° C, subjected to electrophoresis in a 3.5% polyacrylamide gel, the target fragment of about 0.88 tyPoO, is cleaned, suspended in 10 µl of TE buffer and stored at -20 ° C
    5 μg of Pst I fragment approximately 0.88 in length. OO is mixed with 50 μl of the following linker, which is constructed on an automated DNA synthesizer:
    5 -GTGATCAA-3
    S -ACGTCACTAGTTCTAG-S,
    The mixture is ligated using T4 DNA ligase.
    The DNA is precipitated, dissolved and hydrolyzed first with Apal restriction enzyme, then with Bgl II restriction enzyme, applied onto a 3.5% polyacrylamide gel and the targeted Apal Bgl® restriction fragment with a length of about 0.19 ToPoO is isolated.
    The plasmid pSV2gpt DNA (ATCC 37145) was first hydrolyzed with the Hind III restriction enzyme and then with the Bgl 11 restriction enzyme. After Bgl II was digested, the reaction mixture was applied to a 1% agarose gel and the fragments were separated by electrophoresis. The gel is stained with ethidium bromide, a band corresponding to the target

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      The Hind III-Bgl II fragment is about 5.1 in length. OO, is cut out from the gel and placed in a dialysis tube and electrophoresis is continued until the DNA is released from agarose. The buffer containing DNA from the dialysis tube is extracted with phenol and chloroform and DNA is precipitated, the precipitate is suspended in 10 μl of TE buffer and approximately 5 μg of the target restriction fragment Hind III - Bgl II of plasmid pSV2gpt is obtained with a length of about 5.1
    T.P.Oo
    Hind III - Ara I fragment 1.23 tp long, O., Ara I - Bgl II fragment 0.19 To.r. and the Hind III - Bgl II fragment is mixed and ligated. The resulting DNA is the target plasmid PSV2-HPC8.
    E. coli K12 RRI cells (NRKL B-15210) are transformed with a ligase mixture and placed on L-agar plates containing 100 μg / ml ampicillin. Then the plates are incubated at 37 ° C. The presence of E0coli K12 RRI / pSV 2-HPC8 transformants is confirmed by a restriction enzyme assay,
    50 µg of the pSV 2-HPC8 plasmid is first hydrolyzed with Hind III and then with Sal I0. The mixture is applied to a 3.5% polyacrylamide gel and subjected to electrophoresis prior to separation of the target Hind III-Sal I restriction of about 0.29 ToPoo.
    The plasmid pSV 2-HPC8 DNA was first hydrolyzed with the restriction enzyme Bgl II and then with the restriction enzyme Sal 1. The mixture was applied to a 3.5% polyacrylamide gel and subjected to electrophoresis to separate the desired restriction fragment Sal I-Bgl II with a length of about 1.15 ToP.o,
    The p5Ґ2-Ј-globin plasmid DNA (NRRL B15928) is hydrolyzed with Hind III and Bgl 11 restrictases. After cleavage, the reaction mixture is applied to a 1% agarose gel and the fragments are separated by 50 electrophoresis. Cells were isolated from the gel.
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    left restriction fragment Hind III - Bgl II.
    Hind III - Sal I fragment of plasmid pSV 2-HPC8, about 0.29 tp0o length, Sal I - Bgl II fragment of plasmid pSV 2-HPC8, about 1.15 tbp length and 2 μl of Hind III - Bgl II of the pSV 2-th-globin plamid fragment are ligated with a T4-ligase. Saint dan
    DNA is the target plasmid RYZ3 Bound DNA is used to transform the E.sub.11 R12 RRI and the target E. coli K12 RRI transformants / / p3R3 are identified by their ampicillin-resistant phenotype and by restriction analysis.
    F. Construction of plasmid pLPC from plasmid RYZZ and pBLcat.
    The pBLcat plasmid DNA is hydrolyzed with the HindIII restriction enzyme, a fragment of about 0.87 ToP.o in length, which contains a BK-ushshtel and a late promoter Ad2, are separated in an agarose gel.
    The RYZ3 plasmid DNA is hydrolyzed with the Hind III restriction enzyme, the mixture is incubated for 2 hours at 37 ° C. The DNA is then diluted to 100 µl, treated with 0.06 units of calf intestinal alkaline phosphatase, extracted twice with phenol and once with chloroform, precipitated with ethanol and again suspended in 10 μl of TE buffer o
    The Hind III fragment (about 0.87. O. Long) of the PBLcat plasmid is added to the. Hind III-treated plasmid RYZ3 and ligated. Bound DNA constitutes the target plasmid
    5 -GACCAAGAAGACCAAGTAGGCCCGCGGCTCATTGATG-3
    The mutavised phage obtained by site-specific mutagenesis is named M13 tr18-HE4 „
    The construction of the plasmid pLPC-167 G is carried out similarly to the construction of the plasmid pLAPC described in Example 1 Co. However, the plasmid pLAPC is constructed using two restriction fragments from the plasmid pLPC. When constructing the plasmid pLPC-167G, these same fragments are obtained from the plasmid pLAPC. The reason for using the plasmid pLAPC as the source of the fragments instead of the plasmid pLPC is to facilitate restriction analysis in identifying transformant plasmids pLPC-167G. Since the plasmids pLPC and pLPC - 167 G are very similar in size, it is difficult to distinguish the plasmid pLPC from the plasmid pLPC - 167С „
    However, since the plasmid pLAPC is smaller than the plasmid pLPC-167G, then by obtaining two fragments from the plasmid pLAPC, it is easy to distinguish the parent (plasmid pLAPC) from the target plasmid pLPC 167G. In this way,
    Bound DNA is used to transform EoCdli K12 HB101, Transformed cells are placed on L-agar plates containing ampicillin, and ampicillin-resistant transformants DNA are checked by restriction analysis. Hind III restriction fragment of about 0.87 t "CoO" encoding BK Amplifier and Ad2 Late Promoter, can be inserted into Hind III - the cleaved plasmid pL133 in one of two orientations, only one of which gives the plasmid pLPCo
    Example of pp “Construction of plasmid pLPC-167 Go
    Plasmid pLPC-167 G is designed in accordance with site-specific mutagenesis and other techniques used in constructing plasmid pLAPC, as described in Example 1.
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    When constructing the plasmid pLPC - 167G phage M13 mp18-IIE1 (see example 1B), the site-specific mutagenesis is subjected to the following mutavizing oligonucleotide:
    When constructing the plasmid pLPC - 167G, the Sst I Sal I restriction fragment of about 0.7 t, n, o „phage M13 tr18-HE4 is bound to the EcoR I - Sal I restriction fragment of about 3.76 t. plasmid pLAPC and restriction fragment EcoR I Sst I length of about 2.0 plasmids pLAPC. Bound DNA is the target plasmid pLPC - 167G9 transforming Eocoli K12 RV308 Resultant E. coli K12 transformants
    RV308 / pLPC - 167G is used to produce large quantities. PLPC plasmid DNA - 167G "
    PRI me R 4. Construction of plasmid pLPC - 167F
    The pLPC-167F plasmid is constructed in much the same way as described in Example 1 for constructing the plasmid pLAPC using site-specific mutagenesis and other steps,
    The construction of the plasmid pLPC-167F phage M13 tr18-HE1 is subjected to site-specific mutagenesis using the following mutagenizing oligonucleotide:
    33 173985434
    5 -GACCAAGAACAACCAAGTATTCCGCGGCCTCATTGATC-3 J
    The mutagenized phage resulting from site-specific mutagenesis is termed M13 tr18-HE5.
    The final construction of the plasmid pLPC-167F is carried out analogously to the construction of the plasmid pLAPC described in Example 1C. However, the pLAPC plasmid is constructed using two restriction fragments from the plasmid pLPC0 In contrast, when constructing the plasmid pLAPC - 167F, the same two fragments are obtained from the plasmid pLAPC. The reason for using the plasmid pLAPC as a source of fragments instead of the plasmid
    pLPC is a facilitation of restriction analysis in identifying transformants of the plasmid pLPC - 167F. Since the pLAPC plasmid is smaller than the pLPC-167F plasmid, by obtaining two fragments from the pLAPC plasmid, the parent plasmid (pLAPC) can be easily distinguished from the target plasmid pLPC-167F
    Thus, when constructing pLPC - 167F, the Sst I - Sal I restriction fragment is about 0.7 kb in length. phage M13 tr18-HE5 is bound to an EcoR I restriction fragment - Sal I of the plasmid pLAPC of about 3.76 length. and with the restriction fragment EcoR I - Sst I length of about 3.76, etc., of O., and with restriction fragment EcoR I - Sst I length of about 2.0 top.o. plasmid pLAPC, Bound DNA is the target plasmid pLPC - 167F, which is transformed into Eocoli K12 RV308
    EXAMPLE 5 Construction of adenovirus-transformed human embryonic kidney cells of line 293 and adenovirus-transformed transformants of the Syrian hamster AV12 line with is. using plasmids pLPC - 167G and pLPC - 167F.
    Human embryonic kidney cells of line 293 were obtained from the American Type Culture Collection under the number of ATCC CRL 1573 ,, Adenovirus-transformed cells of the Syrian hamster AV12 line can also be obtained from the American Type Culture Collection where they are stored under the ATCC No. CRL 9595.
    Cells 293 are grown in minimal medium. Needle with 10% thermo-activated degraded serum at 37 ° C (medium is changed twice a week). The medium consists of D MEM with 10% of bodies of serum, 50 µg / ml of entamycin 0 and 10 µg / ml of Agua MERNUTSCH of Vitamin K Fitanedione. The cells are subcultured by removal of the medium, washed with Hank's solution, 0.25% trypsin (containing 0.2 g / l EDTA) is added 5 to 1-2 min, washed with fresh medium, sucked off and distributed into new vessels with a subculturing ratio of 1: 5 or 1:10.
    1 day before transformation, 0 cells are seeded in an amount of 0.7x10 cells per 100 mm Petri dish. Sterile plasmid precipitated with ethanol is dissolved in TE-buffer and used to produce 2X DNA-CaC1a, 5 containing 25 µg / ml transforming plasmid DNA (plasmids transformed with PLPC - 167F or pLPC - 167G usually use two plasmids, plasmid pLPC - 167F or 0 pLPC - 167G and a plasmid containing a selective marker) and 250 mM CaC12 ° 2XHBSS is obtained, containing 280 mM NaCl, 50 mM HepeS and 1.5 mM sodium phosphate with a pH adjusted to 7.05 - 5 7.15. A solution of 2X DNA-CaClg is added dropwise to an equal volume of sterile 2X HBSS and rinsed with bubbles during the addition of DNA. DNA-calcium-phosphate-0 precipitate is allowed to form without mixing for 30 to 45 minutes at room temperature.
    Then the sediment is gently mixed with a plastic pipette and 1 ml (per plate) of the precipitate is added directly to 10 ml of medium, growth covering the recipient cells. After 4 hours of incubation at 37 ° C, the medium is replaced with fresh one and the cells are incubated for another 72 hours. before selection. For 0 plasmids that do not contain a selective marker that functions in eukaryotic cells, such as plasmid pLPC-167F or pLPC-167G, the following plasmid mixture is used in the transformation technique: the expression vector of this invention not containing a selective marker; expression vector containing a selective marker that works in eukaryotic cells. Plasmids pSV2 - dhfr (ATCC 37146), pSV 2 - neo (ATCC 37149), PSV2 - Bpt (ATCC 37145) and pSV2 - hyg (NRRL B18039) were used for transformation. to hydromycin B. Thus, the method of co-transformation allows selection of cells containing plasmids with a selective marker.
    Plasmid pSV2 - neo gives -, resistance to neomycin (G418) o
    The transformation of cell lines 293 and AV 12 with a mixture of the plasmid pLPC - 167F or pLPC - 167G and the vector, which gives resistance to hydromycin, and the subsequent selection of hydromycin - resistant cells, a large number of transformants are obtained 0
    EXAMPLE 6 Selection of Transforman ™ (comrade with high secretion of
    The hydromica-resistant transformants obtained in Example 5 are grown in 100 mm tissue culture discs or densities of several cot cell clones per disc. The medium is decanted and the cells are washed twice.
    The nitrocellulose filters are placed in an agar layer after the removal of air bubbles, the plates are incubated for 1-3 hours at 37 ° C, then placed in PBS (50 mM Tris-HCl, PH 7.2 and 150 mM NaCl).
    In order to maintain cell viability, cells are coated on top with a mixture of 2 ml of 1.8% agar (47 ° C), 2 ml of DME salts (37 ° C) and 4 cl of DME salts with 20% bovine serum ( 37 ° C) and placed in an incubator at 37 C,
    All washes and reactions are carried out with the filter placed on the oscillating platform. First, the filters are blocked by incubation at room temperature5 - GCG CAG TCA CCTG AAACG ACTCATTGATGGGAAGATGA-3
    The method of constructing a plasmid DN plasmid zymogen-C from a human being, which is carried out. ment of 0.7 kV plasmidizing it with fragmentation of the phage MB mp18 with the image of mp18HE1; carrying out c mutagenesis from oligonucleotide
    with the formation of phage M13 tr8NE2 or
    5 -GACCAAGAAGACCAAGTAGGCCCGCGGCTGATTGATG-3 with the formation of phage tr18N4 or
    51 GACCAAGAAGACCAAGTATTCCCGCGGCTGATTGATC-3T
    EcoR I - Sst I fragment of the plasmid pLAPC and EcoR I - Sal I fragment
    with the formation of phage M13 mp18-HE5, ligation of the fragment SstI - Sal I
    0.7 kb of phage M13 mp18HE2 with
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    4 ° C
    in 5% milk in PBS. The filters are then washed 4 times in PBS (5 minutes per wash). 10 µg / ml of biotinylated polyclonal sheep antibodies against human protein C in 2.5% bovine serum albumin are added to the filter and incubated for 1 hour at 37 ° C.
    The filters are washed 4 times with PBS at
    Then avidin D and biotinylated peroxidase from a seaside spoon are prepared and added as described in the supplier's instructions. Filters are incubated with HRP-conjugated avidin D for 1 hour at 4 ° C (if a small amount of protein is secreted, then longer incubation times can be used ie night) o
    For the appearance of the color of the indicator, the incubator is added at room temperature until the color appears. The colonies secreting the greatest amount of human zymogen C are noted on the filters not only by the earlier appearance of color, but also by darker spots on the filters.
    After the filters are developed, they are again placed on the original plates to match the colonies with spots on the filter. The colonies secreting the greatest amount of human zymogen C are selected and used to obtain a zymogen.
    权利要求:
    Claims (1)
    [1]
    Invention Formula
    A method for constructing a recombinant plasmid DNA encoding the zymogen-C. From a human, consisting in isolating a fragment of 0.7 kV of the plasmid rHC7, ligating it with the Sst I fragment - Sal1 I of the mp mp18 phage to form M13 mp18HE1 - carrying out site-specific mutagenesis using oligonucleotide
    2.0 kV of the same plasmid or
    37 173985438
    the same fragments of phage M13 trHE4 and -carboxylated secreted
    pLAPC plasmids, or M13 trneZ and protein, human C light chain,
    Plasmids PLAPC or pLPC - 167F COOT-dipeptide lysine-arginine, arginine, respectively, followed by selection, - zine or arginine-arginine with the following
    plasmids encoding the polypeptide, including the amino acid and nucleotide signal peptide and propeptide sequence:
    i / vii A:; I s: and Ш1; I.N CYS HIS
    PRO ALAVAL LYS PIIE PRO CYS CLY ARG PROTRPLYS ARG MET CLU LYS
    LYS ARGSER HIS LEU LYS ARC ASP THR CI.UASPGLN GLU ASP GLN VAL
    Ri R2ARG LEU ILC R3 GLY LYS MET THRARCARG GLY ASP SER PRO
    TRP GLNVAL VAL LEU LEU SER SERYS LYSLYSLEU ALA CYS GLY ALA
    VAL LEUILE HIS PRO SER TRP VAL LEU THRALAALA HIS CYS MET ASP
    GLU SERLYS LYS LEU VAL ARG LEU GLYGLUTYR ASP LEU ARG ARG
    TRP GLULYS TRP GLU LEU ASP LEU ASP ILELYSGLU VAL PHE VAL HIS
    PRO ASNTYR SER LYS SER THR THR ASP ASNASPILE ALA LEU LEU HIS
    LEU ALAGLN PRO ALA THR LEU SER GLN THRILEVAL PRO ILE CYS LEU
    PRO ASPSER GLY LEU ALA GLU. ARC GLU LEUASNGLN ALA GLY. GLN GLU
    THR LEUVAL THR GLY TRP GLY TYR HIS SERSERARG GLU LYS GLU ALA
    LYS ARGASN ARG THR PHE VAL LEU ASN PHEILELYS ILE PRO VAL VAL
    PRO HISASN GLU CYS SER GLU VAL MET SERASNMET VAL SER GLU ASN
    PET LEUCYS ALA GLY ILE LEU GLY ASP ARGGLNASP ALA CYS GLU GLY
    ASP SERGLY GLY PRO MET VAL ALA SER PHEHISGLY THR TRP PHE LEU
    VAL GLYLEU VAL SER TRP GLY GLU GLY, CYSGLYLEU LEU HIS ASN TYR
    GLY VAL TYR THR LYS VAL SER ARG TYR LEU ASP ILL HIS GLY HIS
      .
    ILE ARC ASP
    .
    5 -GAC ACA GAA GAC CAA GAA GAC SLA STA
    R4 R5 CGG CGG ATC ACC AGG CGG CGG GTC AGC CGG CGG CGG CGG CGGGGGG ASC AAG AAG AO CTO GCC TGCGGG CCA, GTG CTC ATS CGG GTCG CG TGG GTG CGG ACG GCCG CG CG CTC ATS CGG GTCG CGG CGG TAG AAG AAG CTC TGG GAG AAG TAGG GAG TAGG AAG TAGG AAG TAGG AAG TAGG GAG A GAG A GAG A TAC AAG A GAG GTC TTC GTC CLC A CAC AAC A TAC AGC AAG AGC ACC ACC GAC LAC CAC ATS CCC GCC ACC CTC TCG CAG ACC ATA GTG CCC CCC ATC TGC CTC CCG GAC AGC GGC CTT GCA GAG CGC GAG CTC LCT CAG GCC GCC CAG GAC
    39 173985440
    ACC STS GTG ACG GGC TGG GGC SLS AGC AGC CGA GAG AAG GAG ACC AAG ACA AAC CGC ACC TTC STS CTC ALS TTC ATC AAG ATT CCC GTG GTC
    R
    CCG SLS LAT GAG TGC AGC GAG GTC ATG AGC LLS ATG GTG TCT GAG LLS ATG CTO TOT CCG GGC LTC CTC GGG GAG COG CAG GAT GCC TGC GAG GGC GAC ACT GGG GGG CCC ATG GTC GCC TCC TTC SLG GGC ACC TCG HUNDRED GTG AGC TGG GOT GAG GGC GGC GTC GTC CG CAC AAC TAG GTC GTT-TAG ACC AAA GTC ASC CGC TAC CAC GAC TGG ATC CAT GGG CAC ATS AAC GAC
    类似技术:
    公开号 | 公开日 | 专利标题
    US5516650A|1996-05-14|Production of activated protein C
    US5225537A|1993-07-06|Methods for producing hybrid phospholipid-binding proteins
    EP0319312B1|1993-11-03|Vectors and compounds for direct expression of activated human protein C
    JP2851423B2|1999-01-27|Activable fibrinolytic and antithrombotic proteins
    CA1340263C|1998-12-15|Expression of protein c analogues
    CA2096604C|2003-12-16|Protein c derivatives
    EP0191606B1|1993-01-27|Vectors and methods for expression of human protein c activity
    JP2614848B2|1997-05-28|Gene encoding human-protein C
    EP0234051B1|1991-12-11|Tissue-type plasminogen activator mutants; recombinant genetic information coding therefor and process for preparing said mutants; their use and pharmaceutical compositions
    WO1988009811A1|1988-12-15|Proteins and derivatives thereof
    US5358932A|1994-10-25|Hybrid protein C
    SU1739854A3|1992-06-07|Method for construction of recombinant plasmid dna, encoding human zymogen c
    HUT61592A|1993-01-28|Process for producing deoxyribonucleic acid molecules and vectors for expressing zymogen forms of human c protein
    US5302529A|1994-04-12|Plasmid coding for human protein C
    US5196322A|1993-03-23|Vectors and compounds for expression of zymogen forms of human protein C
    CA2071630C|2000-02-22|Hybrid protein c
    JP3004375B2|2000-01-31|Vectors and compounds for expression of glycosylation mutants of human protein C
    IE873299L|1988-06-05|Hybrid proteins
    JP3045307B2|2000-05-29|Cell culture method for producing activated protein C
    KR930003913B1|1993-05-15|Method for preparation of polypeptide having thrombin inhibiting activity
    USRE38981E1|2006-02-14|DNA sequence coding for protein C
    JP2518832B2|1996-07-31|Deformed tissue plasminogen activator
    WO1991009951A2|1991-07-11|Recombinant protein c with truncated light chain
    WO1992013080A1|1992-08-06|Microsomal endopeptidase
    同族专利:
    公开号 | 公开日
    EP0323149A3|1990-06-13|
    IL88758D0|1989-07-31|
    AU2732988A|1989-06-29|
    PT89285B|1993-08-31|
    HUT50499A|1990-02-28|
    JP2851287B2|1999-01-27|
    RU2018535C1|1994-08-30|
    DK723488A|1989-06-29|
    ES2065341T3|1995-02-16|
    DE3852625D1|1995-02-09|
    CN1035526A|1989-09-13|
    AU609919B2|1991-05-09|
    MX14312A|1994-01-31|
    EP0323149A2|1989-07-05|
    AR244804A1|1993-11-30|
    ZA889497B|1990-08-29|
    JPH022376A|1990-01-08|
    CA1340111C|1998-11-03|
    NZ227434A|1991-03-26|
    KR890010202A|1989-08-07|
    HU210863B|1995-08-28|
    DE3852625T2|1995-05-24|
    PT89285A|1989-12-29|
    GR3015609T3|1995-06-30|
    IE883849L|1989-06-28|
    EP0323149B1|1994-12-28|
    IE66337B1|1995-12-27|
    DK723488D0|1988-12-27|
    AT116368T|1995-01-15|
    IL88758A|1994-02-27|
    引用文献:
    公开号 | 申请日 | 公开日 | 申请人 | 专利标题
    US4775624A|1985-02-08|1988-10-04|Eli Lilly And Company|Vectors and compounds for expression of human protein C|JPH03501921A|1987-05-18|1991-05-09|
    US4992373A|1987-12-04|1991-02-12|Eli Lilly And Company|Vectors and compounds for direct expression of activated human protein C|
    GB8927722D0|1989-12-07|1990-02-07|British Bio Technology|Proteins and nucleic acids|
    US5358932A|1989-12-29|1994-10-25|Zymogenetics, Inc.|Hybrid protein C|
    JP3270462B2|1989-12-29|2002-04-02|ザイモジェネティクス，インコーポレイティド|Hybrid protein C|
    IL97311D0|1990-02-23|1992-05-25|Lilly Co Eli|Vectors and compounds for expression of glycosylation mutants of human protein c|
    US5270178A|1990-02-23|1993-12-14|Eli Lilly And Company|Vectors and compounds for expression of zymogen forms of human protein C|
    MY110664A|1992-05-21|1999-01-30|Lilly Co Eli|Protein c derivatives|
    US5618714A|1993-12-15|1997-04-08|Eli Lilly And Company|Methods for producing protein C|
    WO2001036462A2|1999-11-19|2001-05-25|Eli Lilly And Company|Protein c derivatives|
    法律状态:
    优先权:
    申请号 | 申请日 | 专利标题
    US13800987A| true| 1987-12-28|1987-12-28|
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