小鼠VEGFR2胞外1-4IgG样结构域的原核表达、纯化及活性鉴定  

Prokaryotic expression,purification and antigenicity identification of mouse VEGFR2 extracelluar 1-4 IgG-like domains

作　　者：王伟[1,2] 殷小涛[1,2] 李云奇[1,2] 田仁礼[1,2] 阎瑾琦[2] 高江平[1] 于继云[2] 

机构地区：[1]解放军总医院泌尿外科,北京100853 [2]军事医学科学院基础医学研究所,北京100850

出　　处：《南方医科大学学报》2013年第1期13-17,共5页Journal of Southern Medical University

基　　金：国家自然科学基金(30840094);国家高技术研究发展计划(863)项目(2007AA02Z451)~~

摘　　要：目的获得小鼠血管内皮生长因子Ⅱ型受体(mVEGFR2)胞外1-4IgG样结构域融合蛋白并验证其免疫原性及生物活性。方法利用RT-PCR法从孕龄14 d的Balb/c小鼠胚胎组织中扩增mVEGFR2D1-4基因,测序正确后将其克隆至原核表达载体pET-42a,构建重组质粒pET-42a-mVEGFR2D1-4。将其转化至大肠杆菌BL21(DE3)中IPTG诱导表达,经SDS-PAGE分析,目的蛋白用Western blotting鉴定后,亲和层析法纯化融合蛋白,ELISA法检测纯化蛋白的抗原活性,并通过体外细胞培养检测该融合蛋白对血管内皮生长因子(VEGF)促人脐静脉内皮细胞(HUVEC)增殖的阻断作用。结果测序结果证实成功扩增出目的基因mVEGFR2D1-4;酶切和测序结果证实pET-42a-mVEGFR2D1-4原核表达载体构建成功;转化后可以成功诱导并纯化出大小与预期一致的蛋白;Western blotting证实纯化的蛋白能与特异性抗体发生反应;ELISA检测显示纯化后的mVEGFR2D1-4融合蛋白具有免疫原性,并且体外细胞培养试验证实其能阻断VEGF对HUVEC的促增殖作用。结论成功获得mVEGFR2D1-4/GST融合蛋白,该蛋白具有良好的免疫原性及生物活性,为进一步研究以VEGFR2为靶点的抗肿瘤主动免疫治疗奠定了基础。Objective To obtain 1-4 IgG-like domains of mouse vascular endothelial growth factor receptor 2 （VEGFR2） fusion protein （mVEGFR2D1-4/GST） and identify its antiginicity and biological activity. Methods The gene of mVEGFR2D1-4 was amplified by RT-PCR from 14-days embryos of Balb/c mice. The PCR product was cloned into pET-42a prokaryotic expression vector to construct the recombinant plasmid pET-42a-mVEGFR2D1-4, which was transformed into E. coli BL21 （DE3） strain for mVEGFR2D1-4/GST expression. The fusion protein was identified by SDS-PAGE and Western blotting, and the antigenicity of the protein purified by affinity chromatography was characterized by ELISA. The VEGF blocking effect of the purified protein in human umbilical vein endothelial cells （HUVECs） were evaluated in in vitro cell cultures. Results The mVEGFR2D1-4 gene was obtained, which had an identical sequence to that retrieved in GenBank. The prokaryofic expression vector for mVEGFR2D1-4 was successfully constructed as confirmed by enzyme digestion and DNA sequencing. Both Western blotting and ELISA demonstrated the anfigenicity of the purified mVEGFR2D1-4 fusion protein, which obviously blocked the effect of VEGF in promoting HUVEC proliferation in vitro. Conclusion The mVEGFR2D1-4/GST fusion protein obtained shows a strong antigenicity and biological activity to facilitate further study of active anti-tumor immunotherapy targeting VEGFR2.

关 键 词：血管内皮生长因子Ⅱ型受体 1-4IgG样结构 原核表达 蛋白纯化 

分 类 号：R730.5[医药卫生—肿瘤]