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Method for producing recombined endothelium chalone in bacillus colis and application

A technology of endostatin and Escherichia coli, which is applied in the field of genetic engineering biology, can solve the problems of expensive separation and purification, time-consuming production process, etc., and achieve the effects of convenient promotion, increased yield, and simple method

Inactive Publication Date: 2005-08-31
NANJING UNIV
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  • Summary
  • Abstract
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  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, the separation and purification of recombinant human endostatin mainly rely on heparin-affinity chromatography medium to obtain high-purity recombinant human endostatin. The production process is time-consuming and expensive for separation and purification.

Method used

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  • Method for producing recombined endothelium chalone in bacillus colis and application
  • Method for producing recombined endothelium chalone in bacillus colis and application
  • Method for producing recombined endothelium chalone in bacillus colis and application

Examples

Experimental program
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Effect test

Embodiment

[0017] Example: Cloning, expression, isolation, purification and characterization of recombinant human endostatin gene:

[0018] 1. Expression of recombinant human endostatin: recombinant human endostatin expression plasmid pET23a-hE and molecular chaperone expression plasmid pG-TF2 (co-expressing molecular chaperone TF and GroEL / ES) and pG-KJE8 (co-expressing molecular chaperone DnaK -DnaJ-GrpE and GroEL / ES) co-transform Escherichia coli BL21 (DE3) strain (Novagen). TF and GroEL / ES were induced by 20ng / ml tetracycline, DnaK-DnaJ-GrpE was induced by 0.3mg / ml L-arabinose, and 1mM IPTG was used to induce the expression of recombinant human endostatin. The expressed cells were ultrasonically disrupted and centrifuged at 8000 rpm for 10 min to collect the cells.

[0019] 2. Separation and purification of recombinant human endostatin: 1 liter of expressed bacteria was suspended in 50 ml of ultrasonic solution (20 mM Tris-Cl, pH 7.5, 2 mM EDTA), and ultrasonicated for 20 minutes in...

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Abstract

A process for producing soluble bioactive recombinant human endothelium inhibin in colibacillus and separating and purifying it is disclosed. The resultant recombinant human endothelium inhibin can specifically and dosage-dependently suppress the reproduction of endothelium cells and the generation of new blood vessel.

Description

1. Technical field: [0001] The invention belongs to the field of genetic engineering biotechnology. 2. Background technology: [0002] Angiogenesis is essential for the growth and metastasis of solid tumors. In order to promote angiogenesis, tumor tissue produces a series of angiogenic factors (such as bF6F), and many tumors also produce anti-angiogenic proteins, one of which is endostatin (endostatin), which was first discovered by Dr. Folkman's research group at Harvard University Isolated from the conditioned medium of a murine hemangioendothelioma cell line (O'Reilly, M.S., Boehm, T., Folkman, J. Cell 88:277-285, 1997). Endostatin is the C-terminal fragment of collagen XVIII with a molecular weight of 20kDa. It can specifically inhibit the proliferation and migration of endothelial cells, reduce glioblastoma blood vessels and blood flow, and effectively inhibit various primary tumors in mice. People try to produce biologically active endostatin, but the recombinant end...

Claims

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Application Information

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IPC IPC(8): A61K38/17A61P35/00C07K14/435C12N15/70C12P21/02
Inventor 华子春徐寒梅
Owner NANJING UNIV
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