小鼠VEGFR-2胞外片段重组蛋白的制备及其在检测抗VEGFR-2抗体中的初步应用  

Preparation of recombinant protein of extracellular domains of mouse VEGFR-2 and its applications in detecting VEGFR-2 antibody

作　　者：肖博宇[1] 付晓[1] 李春阳[1] 夏燕 唐淑君 杨文[1] 王艳林 

机构地区：[1]三峡大学医学院,湖北省宜昌市443002 [2]分子生物学研究所 [3]化学与生命科学学院

出　　处：《实用医学杂志》2013年第17期2793-2796,共4页The Journal of Practical Medicine

基　　金：湖北省高等学校优秀中青年创新团队计划项目(编号:T201203);三峡大学“求索”大学生创新基金项目

摘　　要：目的:原核表达和纯化小鼠血管内皮生长因子受体2(VEGFR-2)胞外区功能片段并用于抗VEGFR-2抗体的检测。方法:用PCR技术扩增编码VEGFR-2胞外区的DNA片段,将其克隆到原核表达载体pET28a(+)并转化大肠杆菌Rossetta(DE3)。在尿素变性条件下用Ni-NTA树脂亲和层析纯化IPTG诱导表达的基因融合蛋白,并用抗His抗体和抗VEGFR-2抗体分别对融合蛋白进行Western blotting鉴定。ELISA法用于分析VEGFR-2胞外区功能片段在检测抗VEGFR-2抗体中的应用价值。结果:克隆获得为小鼠VEGFR-2胞外功能区(Aa134-Aa417)编码的DNA片段并构建原核表达质粒。将上述质粒转化大肠杆菌后,高效诱导表达出VEGFR-2胞外区功能片段的融合蛋白。尿素变性条件下纯化的融合蛋白能特异性被抗His标签抗体和抗VEGFR-2抗体识别和结合。用上述原核表达的VEGFR融合蛋白建立的ELISA方法可检测样品中抗VEGFR-2抗体的含量。结论:本实验成功建立了VEGFR-2胞外区功能片段原核表达和纯化的方法,获得的VEGFR-2胞外区功能片段可用于抗VEGFR-2抗体效价的检测。Objective To prokaryotic express and purify the functional fragment of the extracellular domains of mouse vascular endothelial growth factor receptor-2 （VEGFR-2）, and to apply it in detecting VEGFR-2 antibody. Methods DNA fragment encoding the extracellular ligand binding domains of VEGFR-2 was amplified from mouse total RNA by RT-PCR technology, and was cloned into prokaryotic expressed vector pET28a （＋）. The resulted plasmid was used to transform E. coli Rossetta （DE3）. Recombinant protein was expressed by IPTG induction and purified under urea denatural condition. Western blotting assay was used to identify purified recombinant protein with His-tag antibodies and VEGFR-2 antibodies. ELISA was used to analyze the application value of recombinant protein in detecting the VEGFR-2 antibody. Results The DNA fragment encoding the extracellular ligand binding domains （Aa134-Aa417） of VEGFR-2 was acquired from mouse total RNA and was cloned into prokaryotic expressed vector. After transforming resulted plasmid into Escherichia coli, the expression of recombinant protein of extraceUular ligand binding domains of VEGFR-2 was induced efficiently. This recombinant protein purified in urea denatural condition could be recognized and bound specifically by His-tag antibody and VEGFR-2 antibody. The ELISA method established by this prokaryotic expressed VEGFR recombinant protein could be used to detect the content of VEGFR-2 antibodies in samples. Conclusion This experiment had successfully established a method for prokaryotic expression and purification of the extracellular ligand binding domains of VEGFR-2. The acquired recombinant protein can be used to detect the content of VEGFR-2 antibody.

关 键 词：血管内皮生长因子受体2 原核表达 蛋白纯化 小鼠 

分 类 号：R440[医药卫生—诊断学]