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Method for producing human tissue plasmin activator

A technology of plasminogen and activator, which is applied in the field of bioengineering to achieve the effect of high output and short development cycle

Inactive Publication Date: 2009-07-29
YANGZHOU UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] In order to overcome the deficiencies in the existing production of tPA, the invention provides a method for producing human tissue plasminogen activator, which effectively solves the problem

Method used

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  • Method for producing human tissue plasmin activator
  • Method for producing human tissue plasmin activator
  • Method for producing human tissue plasmin activator

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Experimental program
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Embodiment 1

[0026] 1. Experimental materials

[0027] Plasmids pBC1, pcDNA3.0, pcDNA3.1, and pCEP-4 were all from Invitrogen Company (Invitrogen LifeTechnologies, Catalog no.K270-01, V044-50, 8903026; Easy / vector TA cloning vector was from Gene Company (Gene Company, Shanghai ); PGEM vector was purchased from Promega. BnF95 is composed of PGEM plasmid inserted into the core promoter+NEO (from pcDNA3.1)+hLF.

[0028] The enzymes and kits used for molecular cloning come from domestic bioengineering companies.

[0029] DMEM / F 12 15.6mg / ml+NaHCO 3 1.2mg / ml+sodium acetate 5mM+transferrin 5μg / ml+ethanolamine 0.5mM+insulin 10μg / ml+hydrocortisone 5μg / ml+penicillin 100U / ml+streptomycin 100μg / ml+10%FBS (pH7.2~7.4)

[0030] G418 screening medium: DMEM / F 12 15.6mg / ml+NaHCO 3 1.2mg / ml+sodium acetate 5mM+transferrin 5μg / ml+ethanolamine 0.5mM+insulin 10μg / ml+hydrocortisone 5μg / ml+penicillin 100U / ml+streptomycin 100μg / ml+10%FCS+400μg / mlG418 (pH 7.2~7.4 )

[0031] Cell induction medium: DMEM / F ...

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Abstract

The invention belongs to the field of biological engineering, and particularly relates to a method for producing human tissue plasminogen activator (tPA) in large scale. The method is to use an expression vector composed of regulatory sequence containing a core promoter and cDNA and a BLG signal peptide to prepare a transgenic mouse; the lymphocyte or the mammary epithelial cell of the transgenic mouse is taken and fused with a tumour cell to obtain a transgenic hybridoma; a tPA transgenic hybridoma is induced by prolactin and is screened to obtain a high-expression cell strain; then the expression cell is transfected and screened again to obtain a high-expression hybridoma strain; the high-expression cell is inoculated into the abdominal cavity of the BALB / C mouse in a lactation period; then ascites is collected to obtain the recombinant human tissue plasminogen activator. The production cost thereof is remarkably reduced compared with a cell culture method; the product is restively safe in use; the production flow is simple; and complex instruments and devices are not needed.

Description

technical field [0001] The invention belongs to the field of bioengineering, and specifically relates to a method for large-scale production of human tissue plasminogen activator (tPA), the production cost of which is significantly lower than that of the cell culture method, the product is relatively safe to use, the production process is simple, and no complicated instruments are required equipment. Background technique [0002] Transgenic animal mammary gland bioreactor is to use animal mammary gland organs to produce biopharmaceuticals or biological products, which has high commercial application value, but the preparation efficiency of mammary gland bioreactor is very low, and the expression rate and expression level in transgenic animals are random and inconsistent. It was determined that most of the expression levels were low. At present, the general mammary gland-specific expression gene is composed of milk protein regulatory element and functional gene. Although thi...

Claims

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Application Information

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IPC IPC(8): C12N9/48C12N15/85C12N5/20A01K67/027
Inventor 成勇
Owner YANGZHOU UNIV
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