猪流行性腹泻病毒M基因及其片段原核表达效果分析

将猪流行性腹泻病毒的M基因及编码M蛋白膜外区M基因片段（M’）分别亚克隆到原核表达载体pJLA605和pGEX-6p-1中，构建了重组质粒pJLA605-M、pGEX-6p-M-GST、pGEX-6p-M和pGEX-6p-M’，转化大肠杆菌后。探索了M蛋白在N端和C端不带有谷胱苷肽巯基转移酶（GST）、N端和C端均带有GST、只在N端带有GST以及只含有M蛋白的膜外区M’蛋白的表达情况。SDS-PAGE分析结果表明，N端和C端带有GST的pGEX-6p-M-GST和只含有膜外区M’基因的重组质粒pGEX-6p-M’获得了高效表达，经Bandscan5．0软件分析，其表达产物分别以融合蛋白GST-M和GST-M’形式存在，表达量分别为20％和45％。在对重组质粒pJLA605-M转化的大肠杆菌诱导表达过程中发现菌体生长的抑制现象，说明M蛋白对宿主菌有一定的毒性，进一步提取包涵体进行SDS-PAGE发现M蛋白以包涵体的形式得以表达。猪流行性腹泻病毒M蛋白的毒性作用可能与M蛋白具有的出芽功能有关，因而表现出对大肠杆菌的细胞壁的破坏作用。

Prokaryotic expression analysis of recombinant M gene and its truncated M' fragment of porcine epidemic diarrhea virus

The whole M gene of porcine epidemic diarrhea virus (PEDV) and its truncated fragment M' gene which encodes the exterior of the viral envelope, were subcloned into pJLA605 and pGEX-6p-1, respectively. The recombinant plasmids pJLA605-M, pGEX-6p-M-GST, pGEX-6p-M and pGEX-6p-M' were constructed and transformed into E.coli for expression. SDS-PAGE analysis showed recombinant M protein and truncated M' protein were highly expressed by pGEX-6p-M-GST and pGEX-6p-M', and the expression products as fusion protein GST-M and GST-M' account reached 20 % and 45 % in the whole bacteria protein, respectively. With the analysis of software Bandscan5.0 and SDS-PAGE, the extract of inclusion body (IB) confirmed M proteins were expressed in the form of IBs. In addition, we found the phenomenon of growth inhibition of E.coli anchored plasmid pJLA605-M after induced,which suggest that M proteins were toxic for host bacteria, and the toxic effect may be contributed to the sprout function of M proteins and causes damage to the cell wall of E.coli.