O型口蹄疫病毒VP1表位重组蛋白疫苗的构建、表达和纯化

Construction,expression,and purification of a recombinant epitope vaccine for type O foot-and-mouth disease virus

目的为了克服灭活口蹄疫病毒疫苗可能存在的传播病毒的潜在危险,构建一种能预防O型口蹄疫病毒感染的VP1表位重组蛋白疫苗。方法采用O型口蹄疫病毒(FMDV)表面VP1蛋白上的B细胞表位肽和T细胞表位肽,以PCR扩增和克隆连接等方法构建VP1表位六聚体重组蛋白(VP1epi)基因,并在大肠杆菌中进行诱导表达,镍亲和层析纯化。用ELISA法检测VP1epi免疫豚鼠血清中抗FMDV抗体的水平。结果构建了一种由FMDV表面VP1中B细胞表位肽重复六次的蛋白质多肽,采用pET28原核表达系统,在大肠杆菌BL21(DE3)中获得了高效表达,表达产量约占菌体蛋白的30%左右,经镍亲和层析后获得纯度高于90%的VP1表位重组蛋白。VP1epi免疫的豚鼠血清中含有一定量的抗FMDV抗体。结论VP1epi重组蛋白能诱导机体产生抗O型FMDV的抗体,说明这种重组蛋白可能成为预防O型FMDV感染的基因工程蛋白质疫苗。

Objective To developed a recombinant protein vaccine with epitopes on VPI protein for prevention of foot-and-mouth disvirus （FMDV） spreading by inactive-FMD vaccine. Methods B cell epitopes and T cell epitopes on VPI protein of type O FDMV were used to construct the gene encoding Vial epitope-recombinant protein （VP1epi） by PCR and DNA cloning. The VP1epi protein was expressed in E. coli with pET28 prokaryotie expression system and purified by nickel affinity chromatography. The anti-FMDV antibody in sera of guinea pig immunized with VPI epi protein was detected by ELISA method. Results Vial epi protein constructed by epitopes on VPI protein of type O FMDV was highly expressed in BL21 （DE3） and the expression yield was about 30% of total bacterial protein. The purity of VP1epi protein purified by nickel affinity chromatography was more than 90%. The anti-FMDV antibody was found in sera of VP1epi-immunized guinea pig. Conelusion The recombinant VPI epi protein ean induce animal to produce specific anti-FMDV antibody and may be used as a novel genetic protein vaccine for prevention of type O FMDV infection.