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Preparation of recombinant protein of human vascular endothelial cell growth inhibition factor rhVEGI-192A

A technology of VEGI-192A and growth inhibitor, which is applied in the biological field, can solve the problems of low cost and lack of high efficiency, and achieve the effects of reducing production costs, optimizing expression conditions, and increasing production

Active Publication Date: 2009-08-12
SUN YAT SEN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, in the development of VEGI-192A as a tumor drug, there is still a lack of a high-efficiency, low-cost, stable and controllable large-scale production technology

Method used

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  • Preparation of recombinant protein of human vascular endothelial cell growth inhibition factor rhVEGI-192A
  • Preparation of recombinant protein of human vascular endothelial cell growth inhibition factor rhVEGI-192A
  • Preparation of recombinant protein of human vascular endothelial cell growth inhibition factor rhVEGI-192A

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0037] Embodiment 1: Construction of recombinant protein rhVEGI-192A expression vector

[0038]The rhVEGI-192A target gene was amplified from total RNA of human umbilical vein endothelial cells by PCR. The upstream primer of PCR is 5'-TTCCATATGCAACTCACAAAAGGGCCGTCT-3', and the downstream primer is: 5'-CGCGGATCCCTATAGTAAGAAGGCTCCAAAGAAGGTT-3'. Both upstream and downstream primers contain Nde I restriction enzyme cutting sites at the 5' end and BamH I restriction enzyme cutting sites at the 3' end. Then use the above two restriction endonucleases to cut out the target gene, and finally connect it to the expression vector pET-30a to construct the recombinant protein rhVEGI-192A expression vector. The 3' end of the insertion site contains a nucleotide sequence encoding 6 histidines. The accuracy and correct reading frame of the rhVEGI-192A recombinant were confirmed by sequence determination.

Embodiment 2

[0039] Example 2: IPTG-induced expression of rhVEGI-192A in Escherichia coli and screening of highly expressed clones.

[0040] 1. IPTG induction: Use the expression vector pET-30a-rhVEGI-192A to transform the competent cell BL21(DE3)pLysS, randomly pick 16 clones and colonize them in 3 mL LB medium, add kanamycin to a final concentration of 50 μg / ml , 37 ° C recovery culture overnight. The next day, measure the density of each tube of bacterial liquid. If the OD600 is between 0.6 and 1, add IPTG to a certain final concentration, and induce culture for 4 hours. The negative control is induced without adding IPTG. The recombinant expression was analyzed by SDS-PAGE. 183 clonal colonies were obtained.

[0041] 2. LB medium: 1% peptone, 0.5% yeast extract, 1% sodium chloride, pH7.0; carbenicillin: 50mg / ml; IPTG solution: 100mmol / L;

[0042] 3. Screening high-expression clones of rhVEGI-192A protein: Using high-throughput SDS-PAGE method, 16 single colonies with the highest exp...

Embodiment 3

[0044] Example 3: Condition optimization of IPTG-induced expression system

[0045] Different culture conditions affect the expression of the target protein, including temperature, IPTG concentration, density of engineered bacteria during induction (OD600 value), host bacteria, induction time and induction system. The optimal expression conditions are explored through preliminary experiments. The #3 clone colony was selected for the optimization experiment of protein induction expression conditions. figure 2 It is the condition optimization of the IPTG-induced expression system of the present invention, including the culture time before bacterial induction and the amount of IPTG added, and the host bacteria is BL21(DE3)pLysS. Each group of screening changes a parameter. When screening the best colonies, the host bacteria used is BL21(DE3)pLysS, and the induction conditions are IPTG: 1mM, 16h, temperature: 25°C; when screening the best IPTG concentration, the host bacteria use...

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Abstract

The invention provides a method for preparing recombinant protein of a human vascular endothelial cell-growth inhibiting factor rhVEGI-192A. The method comprises the steps of using a nucleotide sequence of VEGI-192A gene coding to construct an expression vector and inducing the expression vector to express in a host cell. Particularly, total RNA of human umbilical vein vascular endothelial cell line HUVEC is amplified to obtain a VEGI-192A gene fragment; a target gene is obtained through digestion by use of restriction endonuclease double enzyme; the target gene is connected to the expressionvector pET-30a to construct a recombinant protein expression vector; and the recombinant protein expression vector is transformed into escherichia coli host bacteria to induce expression so as to obtain purified protein. The method has the advantages of building a target-protein prokaryotic expression system with high efficiency, high yield, high activity and high purity on the basis of maintaining the natural spatial conformation of rhVEGI-192 recombinant protein, optimizing expression conditions and building a method for separating, purifying and renaturing target products.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a method for preparing a recombinant protein of human vascular endothelial cell growth inhibitory factor rhVEGI-192. Background technique [0002] Vascular endothelial cell growth inhibitory factor (VEGI) is a recently discovered angiogenesis inhibitory factor, mainly produced by vascular endothelial cells. In 1997, Tan et al. first discovered human VEGI by screening the cDNA library of human umbilical vein endothelial cells, and named it TLI (TNF-like ligand 1) at that time. Subsequent biological activity research found that VEGI can significantly inhibit the proliferation of endothelial cells, hence the name. The full-length VEGI gene is 17Kb, consisting of four exons I, II, III, and IV and three introns. Three kinds of mRNA were spliced ​​according to different splicing patterns, encoding VEGI-251, VEGI-192 and VEGI-174 isoforms composed of 251, 192 and 174 amino acid residues, ...

Claims

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Application Information

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IPC IPC(8): C12N15/63C12N15/70C12P21/02C12R1/19
Inventor 黎孟枫朱勋李鲁远袁洁吴珏珩何振健古明晖夏蕾贺海朋马剑达
Owner SUN YAT SEN UNIV
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