重组人可溶性VEGI在大肠杆菌中的高效表达

目的:在大肠杆菌中高效表达重组人可溶性VEGI,纯化重组蛋白并分析其生物学活性.方法:采用PCR方法对编码人VEGI全基因进行修饰,获得编码成熟蛋白第29～174位氨基酸的C端胞外区基因片段,将PCR产物亚克隆到 pMD-18T中,经DNA测序证实后亚克隆入高表达载体pLOU-1,转化大肠杆菌BL21,获得高表达菌株.经IPTG诱导获得高表达,纯化表达产物并检测其对内皮细胞ECV304增殖的抑制活性 .结果:重组人可溶性VEGI在大肠杆菌中获得高效表达,约占细菌总蛋白的40％;纯化的重组蛋白经复性后具有直接抑制内皮细胞增殖的活性.结论: 重组人可溶性VEGI在大肠杆菌中获得高效表达,且表达的重组蛋白具有抑制内皮细胞生长作用,为研究其抗肿瘤作用打下了基础.

High level expression of recombinant human soluble VEGI in E.coli

Objective:To express recombinant human soluble VEGI in E. coli and to analyze its biological activity on endothelial cells. Methods:With PCR m ethod a VEGI gene fragment coding for mature protein from amino acid residue 2 9to 174 was obtained and cloned into p MD- 18T. The gene sequence was confirm ed by automatic DNA sequencing. The VEGI2 9- 1 74 gene fragm ent was subcloned into expression plasmid p L OU - 1. Recombinant plasmid p L OU - VEGI2 9- 1 74 was used to transfect E. coli BL 2 1. The recom binant VEGI2 9- 1 74 protein was purified and tested on endothelial cells ECV 30 4 . Results:The truncated VEGI2 9- 1 74 was obtained by PCR method and its sequence was confirmed. High level expression of recom binant hum an soluble VEGI2 9- 1 74 was achieved in BL 2 1,about 4 0 % of total bacterial proteins. The recom binant protein was purified and renatured and showed marked inhibitory effect on proliferation of endothelial cells ECV30 4 in vitro. Conclusion:A trancated VEGI2 9- 1 74 gene fragment is obtained,and the recombinantprotein VEGI2 9- 1 74 is highly expressed in E.coli and shows inhibitory activity on ECV30 4 proliferation.