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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。
Clone, prokaryotic expression and subcellular localization of the 112 gene of infectious buccal virus (ORFV) AH-F10 isolated in this laboratory. The 112 gene was obtained by PCR and the prokaryotic expression plasmid pET-32a-112 and eukaryotic expression plasmid pEGFP-C3-112 were constructed respectively. pET-32a-112 was induced by IPTG expression to obtain 112 protein and prepare hyperimmune serum. Meanwhile, pEGFP-C3-112 was transfected into BHK21 cells to observe the subcellular localization of the virus 112 protein. The results showed that pET-32a-112 was mainly expressed in inclusion bodies in Escherichia coli and its size was about 60 ku. The result of Western-blot showed that the recombinant 112 protein had good reactivity and the 112 protein mainly localized in the cytoplasm. This experiment lays a certain theoretical foundation for the further study of the function of 112 protein.