重组人可溶性VEGI在大肠杆菌中的高效表达

High level expression of recombinanthuman soluble VEGI in E.coli

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 2 9～ 174位氨基酸的 C端胞外区基因片段 ,将 PCR产物亚克隆到 p MD-18T中 ,经 DNA测序证实后亚克隆入高表达载体 p L OU- 1,转化大肠杆菌 BL 2 1,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 ECV30 4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 4 0 % ;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 。

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. [