猪瘟兔化弱毒株E2基因的原核表达及间接ELISA的初步建立

猪瘟病毒E2蛋白C端含有一段30多个疏水性氨基酸组成的跨膜区域(Transmembraneregion,TMR),用RTPCR和巢式PCR分别扩增了含不同长度TMR的猪瘟兔化弱毒E2基因,并克隆入pGEX4T1的MCS中,构建了原核表达载体pGEXTE2339(无TMR)、pGEXTE2355(1TMR)、pGEXTE2375(3TMR)。因E2基因含有大量大肠杆菌稀有密码子,选择了BL21CodonPlus(DE3)RP作为表达受体菌,结果表明pGEXTE2339、pGEXTE2355以包涵体的形式正确表达了目的蛋白,而pGEXTE2375没有明显表达。并利用pGEXTE2339、pGEXTE2355表达的融合蛋白初步建立了间接ELISA方法。

Expression of Hog Cholera Lapinised Virus E2 Gene in E.coli and the Development of Indirect ELISA with Recombinant Protein

The envelope glycoprotein E2 of classical swine fever virus(CSFV) was an important protective antigen with more than thirty hydrophobic amino acids at the carboxyl terminal. E2 genes with different lengths of transmembrane region(TMR) of Hog cholera lapinised virus(HCLV) were amplified by RT-PCR,and cloned into pGEX-4T-1 to construct recombinant plasmid pGEXTE2-339, pGEXTE2-355, pGEXTE2-375, respectively. In view of codon usage in prokaryocyte, these plasmids were transfered into E.coli BL21-CodonPlus(DE3)-RP. After induction by IPTG, pGEXTE2-339, pGEXTE2-355 were successfully expressed as inclusion bodies, but pGEXTE2-375 was obviously not expressed. It was showed that TMR could decrease expression ability of HCLV E2 gene in E.coli.Furthermore,after inclusion bodies being washed and denatured, the fusion protein was used as antigen to detect antibody of CSFV and indirect ELISA was developed.