【摘要】 为制备Asia 1型口蹄疫病毒(FMDV)特异性诊断抗原,根据国内Asia 1型FMDV流行毒株的序列,合成了2个FMDV流行毒株VP1基因的5个抗原表位。采用InsightⅡ软件进行蛋白空间构象模拟,证实设计的多表位分子结构符合要求。将合成的多表位基因按设计的酶切位点进行酶切连接,构建了Asia 1型FMDV VP1双拷贝基因重组质粒(pMD18-dVP1 Asia)。从该重组质粒获得dVP1 Asia基因,与毕赤酵母表达载体pPIC9K连接,构建了重组表达质粒pPIC9K-dVP1 Asia。用BglⅡ将重组表达质粒线性化后,转化GS115酵母菌。经筛选、鉴定,成功获得了表达FMDV多表位VP1蛋白的阳性重组酵母菌。该阳性重组酵母菌经甲醇诱导,在培养液上清中可检测到目的蛋白。Western-blot结果显示,该蛋白能特异识别Asia 1型FMDV抗体,且具有很好的反应原性。更多还原

【Abstract】 In order to prepare the specific diagnostic antigen of foot-and-mouth disease virus(FMDV) type Asia 1,according to sequences of FMDV type Asia 1 isolates in China,five epitopes from two isolates of FMDV type Asia 1 were chosen and synthesized.Their conformation was stimulated with software Insight Ⅱ,and the structure of multi-epitope was consistent with that of the design model.The multi-epitope gene was doubled and inserted into pMD18-T,and the recombinant plasmid pMD18-dVP1Asia was constructed.Then,the dVP1Asia gene was cloned into Pichia pastoris expression vector pPIC9K to construct the recombinant plasmid pPIC9K-dVP1Asia,which was linearized with BglⅡ,and transformed into GS115 cells by electroporation.The positive clones were selected by MD/MM plates and confirmed by PCR.SDS-PAGE analysis showed that expression product could secreted into the supernatant,and the recombinant dVP1Asia proteins could specifically react with the bovine positive sera against FMDV type Asia 1 in Western-blotting test.更多还原