人血管内皮生长因子A(VEGFA)的原核载体构建、蛋白表达及鉴定  

Prokaryotic Vector Construction, Protein Expression and Identification of Human Vascular Endothelial Growth Factor A(VEGFA)

作　　者：赵瑜[1] 胡月新[2] 卢敏南[2] 焦扬[3] 赵洪波[3] 刘建军[3] 

机构地区：[1]昆明医科大学生物医学工程研究中心,云南昆明650500 [2]昆明医科大学科研实验中心,云南昆明650500 [3]昆明医科大学分子临床医学研究院,云南昆明650500

出　　处：《昆明医科大学学报》2015年第6期31-34,共4页Journal of Kunming Medical University

基　　金：云南省应用基础研究基金资助项目(2012FB152)

摘　　要：目的克隆人VEGFA基因,与原核载体融合表达、纯化及其鉴定,并将其应用于肿瘤血管生长研究过程中.方法采用PCR方法扩增人VEGFA序列,将其克隆入p GEX-KG原核载体,获得p GEX-KG-h VEGFA重组质粒.将该质粒转化入E.coli菌株BL21(DE3),经IPTG诱导其表达,以GSTrap亲和层析柱纯化融合蛋白,采用SDS-PAGE电泳,Western Blot方法鉴定融合蛋白的表达.结果 PCR扩增获得了h VEGFA基因片段,经测序证实与Gen Bank公布的序列一致,重组的质粒载体经过PCR、酶切鉴定,证明重组质粒中已成功的插入了h VEGFA基因片段.成功构建了该蛋白的原核表达载体p GEX-KG-h VEGFA.结论融合蛋白在IPTG终浓度为0.5 mmol/L、25℃条件下诱导表达,并获得纯化融合蛋白.采用Western Blot的方法可检测到目的融合蛋白GST-h VEGFA的表达.Objective To clone human VEGFA gene, induce its prokaryotic expression, purify and identify the fusion protein, which will be applied to study the process of tumor angiogenesis. Methods VEGFA segment was amplified by RT- PCR and cloned into expression vector p GEX- KG to construct the expression of plasmid p GEX- KG- h VEGFA. The recombinant plasmid was transfected to BL21（DE3）, and GST fusion protein was expressed by IPTG. The protein was purified by GST affinity chromatography. The methods of SDS- PAGE electrophoresis and Western Blot were used to identify the expressed fusion protein. Results h VEGFA gene segments were obtained by PCR amplification, and the sequence was consistent with that reported in Gen Bank. We have successfully constructed the prokaryotic expression vector ofp GEX- KG- h VEGFAby proving that the target gene h VEGFA had been inserted into the recombinant plasmid after PCR and Enzyme digestion. Conclusion The fusion protein could be induced under the condition when temperature was 25°C and the final concentration of the IPTG was 0.5mmol/L. After purified by GST affinity chromatography column, the expression of the fusion protein could be tested by Western Blot.

关 键 词：VEGFA 原核载体 融合蛋白 亲和纯化 

分 类 号：Q785[生物学—分子生物学]