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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 epitopes of human VEGF165 and mouse VEGF164 and lay the foundation for the design and verification of Vascular endothelial growth factor (VEGF) epitope vaccine. Methods The key sites for the binding of human VEGF165 and mouse VEGF164 proteins to KDR were determined and their common epitopes were determined. The common epitope was inserted into the VEGF backbone protein. The backbone protein gene containing the common epitope was amplified and ligated with the prokaryotic expression vector pET-24a to construct the recombinant expression plasmid pET-24a-FV. The recombinant plasmid was transformed into E. coli BL21 (DE3), IPTG The expressed product was purified by Sephacryl S-100 gel column and the purified protein was analyzed by SDS-PAGE. Immunized mice were immunized with the purified epitope-displaying protein. Serum titer was determined by ELISA. Western blot and indirect immunofluorescence assay were used to detect the expression of VEGF164 and VEGF165. Results The co-antigenic epitope of human VEGF165 and mouse VEGF164 was EYPDEIEYIFKP in the KDR 40s loop binding region. The recombinant plasmid was confirmed by double enzyme digestion and sequencing. The expressed protein was mainly expressed in inclusion bodies, 20% of protein and 92% of purity. Both mouse VEGF164 and human VEGF165 reacted with mouse serum, the serum titer reached 1:104. The immune serum not only recognized epitope display protein but also recognized mouse VEGF164 and human VEGF165 monomers and dimers, but also positive reaction with human HeLa and mouse B16 tumor cells, indicating that the epitope-displaying protein can display the common antigenic epitope of VFGF. Conclusion Skeletal protein-immunoprecipitation of VEGF164 and VEGF165 co-epitopes can produce specific immune responses against these two proteins and confirm that the EYPDEIEYIFKP epitope is a common epitope of VEGF164 and VEGF165.