口蹄疫病毒表位嵌合蛋白在枯草芽胞杆菌中的分泌表达

Secretive expression of epitope-chimeria of foot-and-mouth disease virus by Bacillus subtilis

为了将口蹄疫病毒(FMDV)中和表位展示在病毒样颗粒(VLP)表面,以猪圆环病毒2型(PCV2)衣壳蛋白为骨架,用FMDV O/Mya-98/2010毒株G-H环替换PCV2自身中和表位,构建嵌合衣壳蛋白CaO。为分泌表达CaO蛋白,在芽胞杆菌穿梭质粒pHCMC05的多克隆位点中顺次插入枯草芽胞杆菌P43启动子和中性蛋白酶B分泌信号肽(nprBsp),构建分泌型大肠杆菌-枯草芽胞杆菌穿梭质粒p7257-P43。然后将合成的CaO基因插入p7257-P43质粒nprBsp的下游,构建分泌表达质粒p7257-P43-CaO。将该质粒转化枯草芽胞杆菌WB800,氯霉素诱导表达。上清经SDS-PAGE检测,嵌合蛋白CaO在枯草芽胞杆菌中得到表达。Western-blot证实,该蛋白具有反应原性。免疫电镜可观察到直径约15nm±2nm的VLP,表明嵌合蛋白在体外能有效组装。

The capsid protein of porcine circovirus type 2（PCV2） was used as a protein skeleton,and its neutralization epitope was replaced by the G-H loop of VP1 of foot-and-mouth disease virus strain O/ Mya-98/2010,which was used to constitute a chimeric capsid protein designated as CaO. Subsequently, based on plasmid pHCMC05, an Escherichia coli-Bacillus subtilis shuttle plasmid p7257-P43 was construc- ted by inserting of p43 promoter（P43） and neutral protease B signal peptide（nprBsp） of B. subtilis orderly into its multiple cloning sites（MCS）. The CaO gene was synthesized artificially,and was induced into MCS following the nprBsp to construct the secretary expression plasmid p7257-P43-CaO. The expression plas- raids were transformed into the B. subtilis strain WB800 for induction expression by chloromycetin. SDS- PAGE analysis showed that CaO could be expressed by B. subtilis WB800 strain secretively,and the Wes- tern-blot analysis demonstrated that the chimeric protein had reactinogenicity. By electron microscopy , these chimeric capsid proteins were found to self-assemble to virus-like particles in vitro,which was about 15 nm±2 nm in diameter.