bFGF与VEGF抗原表位筛选及复合肽的表达鉴定

Screening the antigen epitopes of bFGF/VEGF and expressing and identifying their complex peptide

目的筛选bFGF与VEGF抗原表位,构建复合肽进行原核表达,并对复合肽免疫原性及抑瘤效果进行研究。方法生物信息学方法分别预测bFGF和VEGF抗原表位,筛选优势表位,通过柔性Linker连接各表位构建复合肽,合成引物,重叠延伸PCR(gene splicing by overlap extension PCR,SOE PCR)方法合成复合肽cDNA序列,克隆入原核表达载体pET32a进行表达,表达产物进行SDS-PAGE和Western-Blot鉴定,Ni-NTA柱纯化复合肽并免疫小鼠,间接ELISA法测定抗bFGF抗体和抗VEGF抗体滴度。构建黑色素移植瘤C57BL/6小鼠模型,研究复合肽免疫后肿瘤生长情况。结果生物信息学方法筛选获得3个bFGF表位和2个VEGF表位连同一个噬菌体多肽库筛选表位构建成复合肽,SOEPCR方法成功合成复合肽cDNA序列,构建原核表达载体表达bFGF与VEGF复合肽,纯化的复合肽免疫小鼠产生抗bFGF抗体和抗VEGF抗体;并且体内能有效抑制黑色素瘤生长。结论生物信息学方法筛选bFGF和VEGF获得的抗原表位构建的复合肽在体内同时激发抗bFGF抗体和抗VEGF抗体,并且能抑制肿瘤的生长,为研究bFGF与VEGF表位的抗肿瘤作用奠定基础。

Our study aims to screen the bFGF and VEGF epitopes and express their complex epitope peptides in E.coli.By using of the biotic software and network servers,five strong antigen epitopes of bFGF and VEGF were selected.Then the cDNA of the antigen epitopes were amplified by SOE PCR and subcloned into the expression vector pET32a to generate multi-epitopes recombinant complex peptide plasmids.The immunoresponse of the recombinant fusion proteins were analyzed by SDS-PAGE and Western blot.The recombinant complex peptide was purified by affinity chromatography.The immunogenicity of the recombinant complex peptide was analyzed in C57BL/6J mice.Three bFGF epitopes and three VEGF epitopes were combined together with the soft linkers and the cDNA was constructed to the expression vector pET32a.The recombinant complex peptide was expressed in E.coli and then purified.The complex peptide can induce high titer anti-bFGF and anti-VEGF antibodies,and remarkably reduces tumor burdens in the mouse melanoma model.So we concluded the complex peptide could be used as a vaccine in tumor inhibition.