Classifications

• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
  • C12N9/14—Hydrolases (3)
  • C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
  • C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
  • C12N9/64—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue
  • C12N9/6421—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue from mammals
  • C12N9/6424—Serine endopeptidases (3.4.21)
  • C12N9/6456—Plasminogen activators
  • C12N9/6459—Plasminogen activators t-plasminogen activator (3.4.21.68), i.e. tPA
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P7/00—Drugs for disorders of the blood or the extracellular fluid
  • A61P7/02—Antithrombotic agents; Anticoagulants; Platelet aggregation inhibitors
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Y—ENZYMES
  • C12Y304/00—Hydrolases acting on peptide bonds, i.e. peptidases (3.4)
  • C12Y304/21—Serine endopeptidases (3.4.21)
  • C12Y304/21069—Protein C activated (3.4.21.69)
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N2291/00—Indexing codes associated with group G01N29/00
  • G01N2291/02—Indexing codes associated with the analysed material
  • G01N2291/025—Change of phase or condition
  • G01N2291/0256—Adsorption, desorption, surface mass change, e.g. on biosensors

Definitions

• the present invention relates to a human cell line, and specifically to a human cell line capable of expressing mutant human tissue-type plasminogen activin (TNK-TPA).
• the present invention also relates to a method for constructing the cell line, and its application in a method for producing a human gene tissue-type plasminogen activin. Background technique
• Human tissue-type plasminogen activin can locally dissolve thrombus in blood vessels by activating plasminogen, and reopen the blocked blood vessels.
• Genetech is the only company in the United States that can use genetic engineering technology to carry out large-scale recombination and development of human t-PA genes and successfully develop rt-PA drugs.
• the company's first-generation product, rt-PA has become the drug of choice for treating thrombotic diseases in European and American countries.
• rt-PA has become the drug of choice for treating thrombotic diseases in European and American countries.
• TNK-TPA is one of the most studied mutants, such as Keyt BA, Paoni NF, Refino CJ, etc., published in the journal of the American Academy of Sciences (April 1994) "a fast-acting and more effective human tissue type fiber "Lysogenogen activin", which discloses the DNA sequence of TNK-TPA (A Faster-acting And More Potent Form of Tissue Plasminogen Activator.
• T103N adds a glycosylation site, increases the specificity of TNK-tPA binding to fibrin and reduces its clearance rate in the body;
• Q117N destroys the binding of the K1 region to mannan oligosaccharides, and also destroys liver KUPFFER cells
• KHRR296-299AAAA disrupts the site of action of the serine protease inhibitor family (PAI-1) and prolongs the half-life.
• the present inventors found two families with normal phenotypes carrying additional double satellite chromosomes (BM).
• the small chromosomes were stable between the second and third generations of the two families, respectively. No harm to human body. Seventeen such pedigrees have been reported all over the world, but no one has proposed the idea of using this small chromosome to assemble human gene vectors.
• the applicant proposes a method for isolating the original small chromosome and assembling a human-derived gene vector.
• FISH technology to find out that the small chromosome originated from the short arms of human chromosomes 0 and G, namely the short arms of chromosomes 13, 14, 15, 21, and 22, because the short arms of group D and G are the nucleus.
• the inventors first constructed a small chromosome-specific PUC19 library using microdissection and screened it to obtain a single copy fragment.
• the fragment was confirmed by fluorescence in situ hybridization (FISH) to originate from the small chromosome and group 0 and G chromosomes.
• Arm further screened the human PAC genomic DNA library with this single copy fragment as a probe, and obtained a small chromosome-specific DNA fragment (BMSF) of about 120 kb, which was confirmed by FISH from the small chromosome and the short arms of group D and G chromosomes ( figure 1 ).
• BMSF small chromosome-specific DNA fragment
• Sequencing analysis of the sequence characteristics of BMSF found no genes related to important physiological functions, so it proved to be safe to use this as a target site.
• the applicant further used the above-obtained about 120 Kb or a smaller specific fragment as a guide sequence of a target gene to construct a gene vector.
• the gene guide sequence is selected from a DNA sequence having no important physiological function-related genes on the short arms of human D and G chromosomes or a DNA having homology with the DNA sequence.
• the sequence is used as the guide sequence of the shellfish gene, and the gene vector constructed therefrom has specificity, and can introduce the target gene into the short arms of group D and G chromosomes in a human cell to obtain a cell line that meets the purpose of the present invention.
• various forms of vectors can be constructed according to the prior art, and the target gene TNK-TPA is loaded into the constructed vector to obtain a vector-TN-TPA recombinant.
• the method of constructing a vector and loading a gene of interest into a vector is a conventional method.
• the target gene TNK-TPA can be introduced into a host cell by a conventional method.
• a specific approximately 120 kb DNA sequence cloned from the short arms of human D and G chromosomes is specifically given, and a smaller specific 3.8 kb DNA fragment is selected as the target gene guide sequence.
• the target gene was further incorporated into this vector to obtain a vector-TNK-TPA recombinant, in which the insertion site of the target gene TNK-TPA was 5910.
• a new cell line is obtained by introducing the target gene of TNK-TPA into the chromosomes of groups D and G in HT1080 cells according to the method described above.
• the cell line was deposited with the China Type Culture Collection Center (in Wuhan University, Wuhan, China, postal code: 430072) on 18. August 2000 (18.08.00), and its deposit number is: CCTCC C200006.
• the name of the deposit is: human fibrosarcoma cell line, which is a human transgenic cell.
• the preserved culture is a pure culture, and the culture conditions are: DMEM (high sugar) containing 10% calf serum, PH: 7.0-7.4, 37 degrees Celsius.
• the obtained cell line can directly replace the CHO transformant in the prior art and is used for preparing T K-TPA. After purifying the expression product obtained by expressing the cell line provided by the present invention, it was confirmed to be the target protein by Western blot and amino acid sequencing.
• FIG. 1 is a FISH map of 120kb PAC clone DNA
• FIG. 2 is a structural diagram of a gene vector provided by the present invention (the full length of the gene vector sequence: 11162 bp); wherein pGEM -7 (8267-11162): a vector replication element and a prokaryotic screening system; TK (112-2840): true Negative selection genes for nuclear cells, this gene uses the TK promoter and TK polyA signal; Neo (4342-5910): eukaryotic cells are positive selection genes, which uses the sv40 promoter and sv40 polyA signal; GLS (2841-4341, 5911- 8267): the target gene guide sequence; Cloning site (5910): the target gene insertion site;
• Figure 3 is the FISH mapping of the exogenous TNK-TPA target gene in positive cell clones, demonstrating that the TNK-TPA has been directed to the AD, G short arms of the chromosome group by using this vector;
• FIG. 4 is the Western result of the purified product of TNK-TPA, 1 to 4 are the purified products of TNK-TPA, and “1” indicates a negative control;
• Example 1
• Hybridization Soak the colony lattice membrane in 2 X SSC for 10 minutes, and gently wipe the bacteria on the membrane surface. Debris, 65 ° C, 5ml hybridization pre-hybridization, more than 30 minutes. According to the liquid scintillation value, take 1.2 X 10 6 cpm / ml of the hybridization solution and take the probe solution at 100 ° C for boiling and denaturation for 10 minutes, add 5 ml of fresh hybridization solution, and hybridize with the colony lattice film at 65 ° C for more than 12 hours.
• D- 32 P-dATP random primer method was used to label 260bp single-copy probe P8-7— G-50 column (medium particle size) for purification-4 ° C for use-7 PAC membranes immersed in 2XSSC for 10 minutes ⁇ 55 ° C Pre-hybridize for 3 hours, degenerate the probe at 100 ° C for 10 minutes, add 4.6X 10 5 cpm / ml to 50ml of purchased hybridization solution, and hybridize with PAC membrane at 65 ° C for 1 hour.
• Pulse electrophoresis conditions electrode buffer: 0.5 X TBE, High stregth Analytical Grade Agarose: (Bio-Rad, Low Melting point Agarose LMP) 1%, Switch Time: 2 seconds ⁇ 15 seconds electrophoresis time: 18 hours, voltage: 6V / cm, angle: 120 ° temperature: 14 ° C
• TPCF and TPCR to amplify the TN-TPA gene and expression elements (CMV promoter and BGHpoly A signal), and put Avrll restriction sites on both ends; the primer sequence is:
• TpcF ATgCAT ClAggggAggTCgCTgAgTAgTg
• TpcR TgCATg £ C14ggTACCCCCTAgAgCCCAg
• TNK-TPA / FIX gene and expression elements CMV promoter and BGH poly A signal
• Avrll Digestion of TNK-TPA / FIX gene and expression elements (CMV promoter and BGH poly A signal) with Avrll and ligation with pNS2 vector digested by hel.
• the vector-TNK-TPA recombinant was obtained in Example 2, and the target gene was introduced into the host cell HT1080. After screening, the desired cell line was obtained and expressed in vitro, confirming its site-directed introduction and efficient expression.
• the electric shock conditions are set to a voltage of 260V and a capacitance of 550 ⁇ P.
• the electric shock time is generally 11 to 13 milliseconds.
• the electric shock cells were transferred into a 75 cm 2 culture flask, and 14 ml of the medium containing the double antibody was added, and the cells were cultured at 37 ° C and 5% C02 for 24 to 48 hours.
• G418 was added to the medium for screening at a final concentration of 300 ⁇ 1, and the solution was changed once every 2 to 3 days. G418 was added again during the change, and normal cells were used as a control.
• TKK-TPA gene was electroporated into HT1080 cells, and positive and negative screens were used to obtain positive cell lines. FISH confirmed the site-specific insertion ( Figure 3). The results of TNK-TPA activity measurement are shown in Table 1.
• HT1080 cell negative control TN-TPA activity was 0U / 10 6 cells / 24 hours
• TN-TPA activity after introduction was 408 U / 10 6 cells / 24 hours
• the expression level was 407 at 95 days after transformation.
• the expression is very stable, see table 1.
• the expression products have been confirmed by Western blot and amino acid sequencing ( Figure 4).
• TNK-TPA activity of TNK-TPA transformed positive cells ( ⁇ g / 10 6 cells /24 hours)
• Tl, T15 are two TNK-TPA positive cell lines
• TN-TPA / HT1080 cell line DMEM was cultured to 80% full bottom, the serum-free medium was changed, and the supernatant was collected after 24 hours, which contained the expression ⁇ - ⁇ ;

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• 2000
  • 2000-09-04 AU AU2000268170A patent/AU2000268170A1/en not_active Abandoned
  • 2000-09-04 WO PCT/CN2000/000260 patent/WO2002020802A1/zh not_active Ceased
• 2001
  • 2001-02-16 CN CN018151094AA patent/CN1216149C/zh not_active Expired - Fee Related
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  • 2001-02-16 JP JP2002527274A patent/JP2004508066A/ja active Pending
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