小鼠VEGFR2胞外1-4IgG样结构域的原核表达、纯化及活性鉴定

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。

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

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 prokaryotic expression vector for
mVEGFR2D1-4 was successfully constructed as confirmed by enzyme digestion and DNA sequencing. Both Western blotting
and ELISA demonstrated the antigenicity 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.