Asia1型口蹄疫病毒多表位基因的设计及其在毕赤酵母中的表达  

Molecular design of multi-epitope VP1 genes of foot-and-mouth disease virus type Asia 1 and their expression in Pichia pastoris

作　　者：鲁会军[1] 金宁一[1] 郑敏[1] 韩松[1] 霍晓伟[1,2] 胡博[1,3] 田明尧[1,3] 金扩世[1] 

机构地区：[1]中国人民解放军军事医学科学院军事兽医研究所,吉林长春130062 [2]内蒙古民族大学动物科技学院,内蒙古通辽028000 [3]吉林大学畜牧兽医学院,吉林长春130062

出　　处：《中国兽医科学》2009年第1期11-15,共5页Chinese Veterinary Science

基　　金：国家高技术研究发展计划(863)项目(2006AA10A204);国家“十一五”科技支撑计划项目(2007BAD55B05)

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。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 In- sight 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 pPICgK to construct the recombinant plasmid pPlC9K-dVP1Asia,which was linearized with Bgl Ⅱ ,and transformed into GSl15 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 dVPlAsia proteins could specifically react with the Western blotting test. bovine positive sera against FMDV type Asia 1 in Western blotting test.

关 键 词：ASIA1型口蹄疫病毒 多表位基因 分子设计 毕赤酵母 表达 

分 类 号：S852.659.6[农业科学—基础兽医学] Q786[农业科学—兽医学]