摘要
目的分析人VEGF165和小鼠VEGF164的共同抗原表位,为血管内皮生长因子(Vascular endothelial growth factor,VEGF)表位疫苗的设计和验证奠定基础。方法分析人VEGF165和小鼠VEGF164蛋白与KDR结合的关键位点,确定其共同表位。将共同表位插入到VEGF骨架蛋白中,扩增含共同表位的骨架蛋白基因,与原核表达载体pET-24a连接,构建重组表达质粒pET-24a-FV,转化大肠杆菌BL21(DE3),IPTG诱导表达,表达产物经Sephacryl S-100凝胶柱进行纯化,纯化的蛋白进行SDS-PAGE分析。以纯化的表位展示蛋白免疫小鼠,获得免疫血清,ELISA法测定血清效价,Western blot和间接免疫荧光法检测免疫小鼠血清对小鼠VEGF164和VEGF165的识别。结果人VEGF165和小鼠VEGF164共同抗原表位为KDR 40s loop结合区的EYPDEIEYIFKP;重组表达质粒经双酶切及测序证明构建正确;表位展示蛋白主要以包涵体形式表达,表达量占菌体总蛋白的20%,纯度可达92%;小鼠VEGF164和人VEGF165均能与小鼠免疫血清发生反应,血清效价达到1∶104;免疫血清不仅能识别表位展示蛋白,还能识别小鼠VEGF164和人VEGF165单体和二聚体,还可与人HeLa、小鼠B16肿瘤细胞之间存在阳性反应,表明表位展示蛋白能展示VFGF的共同抗原表位。结论骨架蛋白展示的VEGF164和VEGF165共同表位免疫小鼠后,能产生针对这两个蛋白的特异免疫反应,证实EYPDEIEYIFKP表位是VEGF164和VEGF165的共同抗原表位。
Objective To analyze the common antigenic epitope of mouse vascular endothelial growth factor（VEGF）164 and human VEGF165 and lay a foundation of design and verification of VEGF epitope vaccine.Methods The candidate common epitope of VEGF164 and VEGF165,EYPDEIEYIFKP,was determined by analyzing the interaction of VEGF and its receptor KDR.The common epitope was inserted into skeleton protein.The skeleton protein gene containing the common epitope was amplified and inserted into prokaryotic expression vector pET-24a.The recombinant plasmid pET-24a-FV was transformed to E.coli BL21（DE3）for expression under induction of IPTG.The expressed product was purified by Sephacryl S-100 chromatography,then analyzed by SDSPAGE.BALB / c mice were immunized with the purified epitope display protein and determined for serum antibody titer by ELISA,and for recognition to mouse VEGF164 and VEGF165 by Western blot and IFA.Results The common antigenic epitope was the 40S Loop of KDR binding region.Restriction analysis and sequencing proved that recombinant plasmid pET-24a-FV was constructed correctly.Epitope display protein was mainly expression in a form of inclusion body,which contained 20% of total somatic protein and reached a purity of 92%.Both moue VEGF164 and human VEGF165 showed reactions with immune sera of mice,with a titer of 1 ∶ 104.The immune sera recognized not only epitope display protein,but also the monomers and dimmers of mouse VEGF164 and human VEGF165,and showed reactions with human HeLa and mouse B16 cells,indicating that the epitope display protein could display the common antigen epitope of VEGF.Conclusion The common epitope of VEGF164 and VEGF165 was displayed by skeleton protein and induced the corresponding specific immune responses in mice,suggesting that EYPDEIEYIFKP was the common epitope of VEGF164 and VEGF165.
出处
《中国生物制品学杂志》
CAS
CSCD
2012年第8期989-994,共6页
Chinese Journal of Biologicals
关键词
血管内皮生长因子
抗原表位
免疫
Vascular endothelial growth factor； Antigenic epitope； Immunization
