猪瘟病毒保护抗原E2和猪IgG重链基因的合成及其表达蛋白

Construction and Expression of the E2 of CSFV and Swine IgG Heavy Chain Fused Gene

根据已发表文献及DNAstar软件分析 ,选取双拷贝Shimen株猪瘟病毒E2基因A ,D区和猪IgG重链中可与SPA非特异性结合的区域作为目的基因进行串联表达。根据GenBank发表的序列 ,Primer5 0软件的辅助下分别设计三对引物 ,并在扩增第二条和第三条片段的上游引物 5′端分别设计一个长 5个氨基酸的Linker,将扩增的三片段串联克隆入原核表达载体pET 32a中 ,构建成重组质粒pET 2eh ,对重组质粒诱导表达 ,表达蛋白经纯化后标记于CowanⅠ株金黄色葡萄球菌SPA上 ,与收集的 80份待检血清进行平板凝集试验 ,结果与现有诊断试剂盒检测结果具有较好的符合性。该方法具有简单 ,快速 ,敏感 ,易于操作的特点。

According to the published data concerned,the A,D domains of the CSFV Shimen strain E2 gene and the relevant region of swine IgG heavy chain gene,which codes the peptide suitable for conjugating SPA,were selected as target fragments with the aids of DNAstar software.3 pairs of primers were designed with the help of Primer 5.0 software,and a linker of 5 amino acids-coding sequence was designed at the 5′terminal of the sense primers of the second and the third pairs of primers,respectively.Then the three types of PCR products were cloned tandemly one by one into the prokaryotic expressing vector pET-32a to get the recombinant plasmed pET-2eh.The inducing expressed protein were purified and labelled by the SPA of staphylococcus to inspect the sera antibody of CSF.The indirect Co-agglutination Test of SPA results showed that the method established here is in good agreement with the present indirect haemagglutination detective kit,and with the advantage of simple ,quick and sensitive.;