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Method and use of producing soluble recombinant protein in colibacillus

A technology of Escherichia coli and protein, which is applied in the direction of recombinant DNA technology, bacteria, and the use of vectors to introduce foreign genetic materials, etc., can solve the problems of separation and purification and quality control difficulties, time-consuming and laborious production processes, and achieve inhibition of vascular endothelial cell proliferation, The effect of increased yield and simple method

Active Publication Date: 2005-05-11
NANJING JIRUIKANG BIOTECHNOLOGY RES INST CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, recombinant human endostatin in clinical research in China is mostly produced by in vitro refolding of E. coli inclusion body products. The production process is time-consuming, laborious, and difficult to separate, purify, and quality control.

Method used

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  • Method and use of producing soluble recombinant protein in colibacillus
  • Method and use of producing soluble recombinant protein in colibacillus
  • Method and use of producing soluble recombinant protein in colibacillus

Examples

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Embodiment 1

[0034]Example 1: Cloning, expression, isolation, purification and characterization of recombinant human endostatin gene:

[0035] 1. Cloning of human endostatin cDNA and construction of expression plasmid: Grind 50 mg of human liver tissue, dissolve it in 1 ml TRIZOL RNA Isolation Reagent (Gibco BRL), isolate RNA according to the reagent instructions, and use the isolated RNA as a template for One-step RT-RNA, amplified cDNA, the method was carried out according to the instructions of OneStep RT-RNA PCR Kit (TaKaRa), the forward primer used: 5'-ttc cat atg cacagc cac cgc gac ttc cag-3', which added Nde I enzyme Cutting site (see bold part); reverse primer 5'-ccg ctc gag cta ctt gga ggc agt cat g-3', a translation stop codon (CTA) was added behind the endostatin coding sequence, for the convenience of cloning, The restriction site XhoI (bold) was also added. The obtained PCR product was about 550bp, and the obtained PCR product and plasmid pET23a (Novagen Company) were digeste...

Embodiment 2

[0042] Example 2: Synthesis, cloning, expression, separation, purification and characterization of recombinant human angiostatin gene:

[0043] 1. Synthesis and cloning of human angiostatin gene and construction of its expression vector: referring to the codon preferred by Escherichia coli, ten oligonucleotides were chemically synthesized, the length of oligonucleotides was 28-35 bases, adjacent The oligonucleotides have 5-8 base pairing with each other, the sequence of the oligonucleotides and the overlap pattern between them are shown in Fig. First dilute all oligonucleotides to 100pmol / μl, and then follow the following steps to process sequentially: T4 polynucleotide kinase phosphorylation, annealing at 30°C, Klenow large fragment fills the gap, T4 DNA ligase ligates all gaps (nick), finally obtained a 183bp double-stranded DNA fragment encoding human calreticulin 120 to 180 amino acids. The fragment was amplified by PCR, the forward primer used was: 5'-CGGGATCCTGGACGACGAC...

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Abstract

The invention was involved in that target protein expression strain and molecular partner were expressed in E.coli.at the same time at low temperature to prevent the formation of target protein inclusion body effectively and increase yields of soluble production largely. Soluble recombinant human endostatin and human angiostatin were increased largely by the technique. It provided a new simple and cheap technique to produce soluble recombinant protein with biological activity. It was applied in bioengineering pharmaceutical factory, gene engineering, biochemistry and molecular biology with high efficiency, cheap price, wide application and strong promotion.

Description

1. Technical field: [0001] The invention belongs to the field of genetic engineering biotechnology. 2. Background technology: [0002] The Escherichia coli expression system has the characteristics of fast growth, clear genetic background, high expression level and low cost, and is an ideal foreign protein expression system. However, the biggest disadvantage of this expression system is that foreign proteins often cannot be folded correctly and exist in the form of inactive inclusion bodies, and the yield of inclusion body refolding is usually very low. Finding a simple, easy, and versatile method to improve or prevent the formation of inclusion bodies is a difficulty and a bottleneck in the current E. coli expression system. [0003] 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 bFGF), and many tumors also produce anti-angiogenic proteins, one of whic...

Claims

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Application Information

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IPC IPC(8): C12N1/21C12N15/63C12P21/02
Inventor 华子春徐寒梅孙启明
Owner NANJING JIRUIKANG BIOTECHNOLOGY RES INST CO LTD
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