传染性脓疱病毒112基因的原核表达及亚细胞定位

Prokaryotic expression and subcellular localization of orf virus 112 gene

对本实验室分离的传染性脓疱病毒(ORFV)AH-F10株的112基因进行克隆、原核表达及亚细胞定位。通过PCR方法获得其112基因,并分别构建原核表达重组质粒pET-32a-112及真核表达重组质粒pEGFP-C3-112。pET-32a-112经IPTG诱导表达,获得112蛋白并制备超免疫血清。同时将pEGFP-C3-112转染BHK21细胞,观察病毒112蛋白的亚细胞定位。结果显示,pET-32a-112在大肠杆菌中主要以包涵体形式表达,大小约为60 ku;Western-blot结果表明,重组112蛋白具有良好的反应原性;112蛋白主要定位于细胞质中。本试验为进一步研究112蛋白的功能奠定了一定的理论基础。

This study was designed to prokaryotically express and subcellularly localize the 112 gene of the ORFV AH-F10 strain.At first,112 gene was amplified by PCR.Then, the prokaryotic and eukaryotic expression recombinant plasmids named pET-32 a-112 and pEGFP-C3-112 were successfully constructed.Subsequently,112 recombinant proteins were acquired by induction with IPTG.The expressed protein was purified and immunized with BALB/c mice to prepare for antiserum.The pEGFP-C3-112 was transfected into BHK-21 cells by Lipofectamine 2 000.Then,the subcellular localization of the expressed 112 protein was observed by inverted fluorescence microscopy.The results showed that pET-32 a-112 was successfully expressed in the form of inclusion bodies in E.coli with a size of about 60 ku.The results of Western-blot demonstrated that the expressed 112 protein had good reactionogenicity with the antiserum.The results of subcellular localization showed that the 112 protein was mainly located in the cytoplasm.The present study laid a theoretical foundation for further study of thefunction of 112 protein.