| 摘要: |
| 目的：制备纯化的新型血管生长抑制剂可溶性嵌合蛋白VEGI+，为进一步研究其活性奠定基础。方法：应用PCR方法制备血管内皮细胞生长抑制因子（VEGI）与寡肽CTTHWGFTLC相嵌合的融合基因VEGI+，将PCR产物克隆至pGEM-T载体中，经酶切及测序鉴定后，与原核表达载体pET30a(+)重组，构建重组表达载体pET30a-VEGI+，转化大肠杆菌BL21并诱导表达，纯化并鉴定表达产物。结果：成功扩增融合基因VEGI+，构建的重组质粒pET30a-VEGI+酶切鉴定结果与预期一致，诱导后表达产物以包涵体为主。SDS-PAGE电泳及Western印迹结果表明，复性纯化后的嵌合蛋白VEGI+相对分子质量约为23 000，纯度约为90%。结论：成功构建了重组表达载体，并在大肠杆菌诱导表达，亲和层析纯化后获得纯度较高的可溶性嵌合蛋白VEGI+。 |
| 关键词: 血管内皮生长抑制因子 寡肽类 基因融合 |
| DOI：10.3724/SP.J.1008.2007.01324 |
| 投稿时间：2007-06-26 |
| 基金项目:上海市科委专项基金（05nm05010）. |
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| Expression and purification of high purity soluble chimeric protein VEGI+ |
| DING Li-li，WEI Rui-li*，CAI Ji-ping，LI You |
| (Department of Ophthalmology，Changzheng Hospital，Second Military Medical University，Shanghai 200003，China) |
| Abstract: |
| Objective：To prepare a novel vascular endothelial growth inhibitor-soluble chimeric protein VEGI+ , so as to lay a basis for studying its biological activity. Methods: Chimeric molecule VEGI+ was constructed by grafting oligopeptide CTTHWGFTLC to extracellular region of VEGI (VEGI23-174). Before ligation into pET30a(+) expression vector, PCR product of the recombinant gene was cloned into pGEM-T vector and verified by restriction enzyme digestion and DNA sequencing, then pET30a-VEGI was used to transfect BL21 (modified E. coli strain). The chimeric protein was purified by metal affinity chromatography. Western blotting and coomassie blue staining were used for protein identification. Results: The chimeric molecule VEGI+ was confirmed by restriction enzyme digestion and DNA sequencing. The constructed pET30a-VEGI was confirmed by enzymatic digestion. The expression was mainly in the form of inclusion body. SDS-PAGE electrophoresis and Western blotting revealed a chimeric protein about 23 000, with a purity of about 90%. Conclusion: We have successfully constructed the recombinant plasmid pET30a-VEGI+ and expressed it in E. coli. And we have obtained high purity of soluble chimeric protein VEGI+ through affinity chromatography. |
| Key words: vascular endothelial growth inhibitor oligopeptides gene fusion |
