Expression, Purification Of Human Vascular Endothelial Growth Factor 165(VEGF₁₆₅) Fusion Protein

Objective: Malignant tumor has galactic detriment to people's health. And its attack rate is adscendent year by year. Tumor growth and metastasis largely depend on angiogenesis and the vascular endothelial growth factor (VEGF) play a critical rule in tumor-associated angiogenesis. So if inhibit the signal transduction of VEGF may inhibit angiogenesis, that may inhibit tumor growth and metastasis. We plan to construe the inducible His-VEGF165 recombinant prokaryotic expression vectors, and induce the target recombinant fusion proteinsto express in certain conditions. The study of next phase such as preparing and purifying the VEGF165 target proteins on a large scale, inoculating animals, obtainting a lot of monoclonal antibodies of VEGF165, and stydy the anti-tumor effect of monoclonal antibodies of VEGF165, would be built on these works.methods: The primers was designed according to the sequene characters of the VEGF165 cDNA and multiple cloning sites of pET-15b prolaryotic expression vector. The target gene fragment of VEGF, VEGF165 full-length fragment (576bp), was amplified by RT-PCR from the tissue total RNA of Colorectal Cancer. The target gene fragment of VEGF165 was recovered and purified from low melting point agarose gels. The pET-15b vector was transformed into the competent Escherichia coli DH5α, and then be prepared on a small scale by alkaline lysis method agter collecting cells from the culture products. The target gene fragment VEGF165 and the pET-15b vector fragment were recovered and purified from low melting point agarose gels after the restriction endonuclease digestion of the RT-PCR products and pET-15b vector by BamHI & NdeI. The recombinant expression plasmid pET-15b/VEGF165 was constructed through the ligation reaction between the VEGF165 target gene fragment and corresponding vector fragment under the catalyze of T4 DNA ligase. The ligation product was transformed into the competent E. coli DH5αand the recombinants was obtained by ampicillin screen. The positive clones namely were the engineering E. coli which contained the recombinant expression plasmids. The recombinant expression plasmid was prepared on a small scale by alkaline lysis method after the mutant was cultured in LB culture medium, then analyzed by restriction endonuclease digestion and sequencing to verify the taget gene sequences and the vector's reading frame right or not. The right recombinant expression plasmid above-mentioned was transformed into the competent E. coli BL21(DE3), which was an expression type E. coli. After being screened by ampicillin, the engineering E. coli BL21(DE3) which could express VEGF165 gene was obtained. The engineering E. coli was cultured in LB culture medium which contained ampicillin at 37℃until OD600 arrived 0.4~0.6, then added IPTG into the culture, taget fusion protein was induced to express for 1~8 hours. The induced product was analyzed by SDS-PAGE and Western blotting after being collected and lysed by 2×SDS loading buffer and supersonic. The condition of induced expression was be optimized after the fusion protein expression was confirmed. Then confirm which part was the fusion protein expression in the engineering E. coli. The fusion protein expressed with the manner of inclusion bodies was determined and purified by HisPrep FF 16/10 metal chelate affinity chromatography. A high quantity fusion protein was obtained.results: The restriction endonuclease digestion of recombinant expression plasmid demonstrated that the size of every fragments were in accord with their theoretic size completely. The further sequencing analysis showed that VEGF165 gene sequences and reading frames of the recombinant expression plasmid was correct tntirely. The prokaryotic recombinant expression plasmid of VEGF165 was constructed successfully. The SDS-PAGE analysis showed that there was indeed VEGF165 recombinant fusion protein in the induced products of the engineering E. coli. The expression level of VEGF165 recombinant fusion protein was high and the fusion protein expressed with the manner of inclusion bodies. The futher Western blotting analysis indicated that the VEGF165 recombinant fusion protein could react with the polyclonal antibodies of VEGF exclusively. It could be concluded that the VEGF165 recombinant fusion protein was the right target protein we wanted to express. The optimum condition of induced expression was that the engineering E. coli was cultured in LB culture medium which contained ampicillin at 37℃until OD600 arrived 0.4~0.6, then IPTG was added to its final concentration at 0.5mmol/L, taget fusion protein was induced to express for 4~5 hours. The fusion protein expressed was determined and purified by HisPrep FF 16/10 metal chelate affinity chromatography. A high quantity fusion protein was obtained.Conclusion: The recombinant expression plasmid which could expressed VEGF165 recombinant fusion protein was constructed and induced to express successfully. The expression level of VEGF165 recombinant fusion protein was satisfactory. A high quantity fusion protein was obtained. The success of construction and expression of pET-15b/VEGF165 prokaryotic expression plasmids would make it possible to obtain the VEGF165 target protein on a large scale, to manufacture VEGF monoclonal antibody, and to study and research its effect of antitumor.