Human endostatin vasostatin120-180 in E. coli expression purification and preliminary anti-angiogenic activity.docVIP

 PAGE \* MERGEFORMAT 22 Human endostatin vasostatin120-180 in E. coli expression purification and preliminary anti-angiogenic activity 【Abstract】 Objective: Cloning and expression in Escherichia coli of Human Endostatin vasostatin120-180 (VAS) gene, and were isolated and inhibit angiogenesis activity of identification. Methods: According to the E. coli codon preference, the application method of chemical synthesis and molecular cloning of human sources to be 120-180 endostatin fragment (ie VAS) encoding genes. By PCR and restriction enzyme digestion, the VAS encoding gene was cloned into the E. coli expression vector pET28a and BL21 (DE3) strain induced by IPTG and expressed with a His tag fusion recombinant protein His-VAS. VAS protein expression level of about 45% of the total bacterial proteins, most of the target protein to form inclusion bodies, inclusion bodies after in vitro denaturation, renaturation and purification, using SDSanalysis of identification, with the chick chorioallantoic membrane angiogenesis inhibition test for the biological activity of activity of the recombinant protein identification. Results: DNA sequence analysis identified recombinant plasmid pET28a-VAS was successfully constructed, IPTG induced by the fusion protein in E. coli highly expressed, Western blotting analysis and identified the molecular properties of VAS. By chick embryo chorioallantoic membrane test verification, re-VAS protein was able to inhibit angiogenesis in a good way. Conclusion: The access to high-level expression, high-purity VAS, the VAS for further functional analysis and inhibit angiogenesis activity of the foundation. 【Key Words】 preferred codon; endostatin 120-180; inclusion body; Renaturation; chick chorioallantoic membrane test Tumor growth and proliferation depend on angiogenesis - the formation of new blood vessels [1]. Anti-angiogenesis has become a very promising method of cancer treatment, namely, the development of fast-growi