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

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 l) 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, r...

Examples

Embodiment 1

[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.

Embodiment 2

[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 expression efficiency were screened from 183 clonal colonies. figure 1 It is the SDS-PAGE results of the lysates of 16 colony colonies obtained from the screening of high-expression clones in the IPTG-induced expression system, in which 1 to 16 are high-expression clone colony groups, C is the clone colony group without IPTG induction, and M is protein Molecular weight standard, the host bacterium is BL21(DE3)pLysS.

[0043] While carrying out subsequent expression and purification procedures, the screened clones were preserved at low temperature - 80°C. After SDS-PAGE, gel scanning software was used to analyze that the expression of the target protein accounted for 44% of the total bacterial protein.

[...

Embodiment 3

[0046] According to the above results, we concluded that the optimal conditions for the expression of rhVEGI-192A protein in BL21(DE3)pLysS host bacteria were the addition of IPTG: 1mM, and the selected culture time: 16h.

[0047] Example 4: Auto-induced expression of rhVEGI-192A in Escherichia coli and screening of high-expression clones.

[0048] 1. Automatic induction: Use the expression vector pET-30a-rhVEGI-192A to transform the competent cell BL21(DE3)pLysS, randomly pick a certain number of clone colonies into 3mL LB medium, add kanamycin to a final concentration of 50μg / ml , 37 ° C recovery culture overnight. The bacterial cell pellet was collected by centrifugation the next day, and the recombinant expression was analyzed by SDS-PAGE.

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 l) 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, r...

Examples

Embodiment 1

[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.

Embodiment 2

[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 expression efficiency were screened from 183 clonal colonies. figure 1 It is the SDS-PAGE results of the lysates of 16 colony colonies obtained from the screening of high-expression clones in the IPTG-induced expression system, in which 1 to 16 are high-expression clone colony groups, C is the clone colony group without IPTG induction, and M is protein Molecular weight standard, the host bacterium is BL21(DE3)pLysS.

[0043] While carrying out subsequent expression and purification procedures, the screened clones were preserved at low temperature - 80°C. After SDS-PAGE, gel scanning software was used to analyze that the expression of the target protein accounted for 44% of the total bacterial protein.

[...

Embodiment 3

[0046] According to the above results, we concluded that the optimal conditions for the expression of rhVEGI-192A protein in BL21(DE3)pLysS host bacteria were the addition of IPTG: 1mM, and the selected culture time: 16h.

[0047] Example 4: Auto-induced expression of rhVEGI-192A in Escherichia coli and screening of high-expression clones.

[0048] 1. Automatic induction: Use the expression vector pET-30a-rhVEGI-192A to transform the competent cell BL21(DE3)pLysS, randomly pick a certain number of clone colonies into 3mL LB medium, add kanamycin to a final concentration of 50μg / ml , 37 ° C recovery culture overnight. The bacterial cell pellet was collected by centrifugation the next day, and the recombinant expression was analyzed by SDS-PAGE.

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