摘要
在分析脑心肌炎病毒VP1蛋白抗原表位基础上人工合成一段含VP1抗原表位双链DNA序列(150 bp),克隆于原核表达载体pGEX-6p-1,经酶切鉴定后转化大肠杆菌(E.coli)BL21感受态细胞,以异丙基硫代-β-D-半乳糖苷(IPTG)诱导表达融合蛋白。经过GSTrap FF亲和层析柱获得纯化蛋白,经SDS-PAGE电泳显示相对分子质量为30 800。以该纯化蛋白免疫家兔,获得效价分别为1∶25 600和1∶1 600的抗血清。W estern-b lot结果显示该融合蛋白与经过空载体pGEX-6p-1表达的GST蛋白孵育的抗血清反应后有明显的特异性条带。结果表明:脑心肌炎病毒VP1蛋白抗原表位区150 bp编码基因在E.coli中获得正确表达,纯化后的蛋白具有抗原性。
A 150-base-pair （bp） double strands DNA of the epitope of the VP1 coding gene obtained from GenBank of EMCV was synthesized artificially, which had BglⅡ and EcoR Ⅰrestriction enzymes site on the 5＇ ends and 3＇ ends, respectively. And it was inserted into plasmid pGEX-6p-1 to construct the recombinant prokaryotic expression vector. After transformating the recombinant plasmid into E. coli competent cells BL21 （DE3）, the fusion protein was expressd by isopropyl-β-D-thiogalactoside （IPTG）, and purified through GSTrap FF column. It was visualized on 12% of sodium dodecyl sulfate-polyacrylamide gel electrophoresis （SDS-PAGE） with relative molecular weigh of 30 800. To evaluate the antigeneity of the the recombinant protein, it was injected into two rabbits, and the serum antibody against the protein was made and estimated with indirect ELISA and Western-blot. The results showed that the titer of two rabbits serum were 1 ： 25 600 and 1 ： 1 600, and the fusion protein could react with the serum incubated by GST protein. Conclusion： The 150 bp gene of the epitope of VP1 protein of EMCV was correctly expressed in E. coli BL21 cells, and the purified protein has antigenicity.
出处
《南京农业大学学报》
CAS
CSCD
北大核心
2005年第4期100-103,共4页
Journal of Nanjing Agricultural University
基金
教育部重点项目(104101)
关键词
脑心肌炎病毒
VP1蛋白抗原表位
原核表达
抗原性
encephalomyoearditis virus （EMCV）
epitope of VP1 protein
prokaryotie expression
antigenieity
