宿主细胞CyPB基因的克隆及表达分析

Cloning and expression analysis of CyPB gene of host cell

CyPB作为亲环蛋白(cyclophilins,CyPs)家族成员之一,是环孢菌素A、K506、雷帕霉素等免疫抑制药物在细胞内的主要结合蛋白。本课题组前期利用抑制性消减杂交技术对羊传染性脓疱病病毒(orf virus,ORFV)感染MDBK前后宿主细胞差异基因进行筛选时发现,正向文库中CyPB显著上调表达,但其在ORFV侵染过程中的作用尚不清楚。为了进一步明确CyPB对ORFV侵染复制的影响,根据从GenBank中获取的牛源CyPB基因编码区全长651bp基因序列,设计1对针对牛源CyPB基因的特异性引物,利用RT-PCR方法扩增获得CyPB基因全长基因序列。将获得的CyPB基因连接至PET-32a载体,转化到BL21菌株中,经IPTG诱导表达,SDS-PAGE分析可见在25 000处有与预期大小一致的蛋白条带。上述结果表明,本研究成功诱导表达纯化出His-CyPB融合蛋白,该结果将为针对牛源CyPB蛋白功能的研究提供重要的物质基础。

CyPB is a member of cyclophilins(CyPs)family,which is a binding protein of the immunosuppressive drugs including cyclosporin A,K506 and rapamycin.The suppression subtractive hybridization cDNA library of MDBK cells in the early phase of ORFV infection had been constructed.Among of the differentially expressed genes selected from the cDNA library,the CyPB gene was significantly up-regulated expression in the forward library.However,the role of CyPB gene in the process of ORFV infection was still not clear.In order to further clarify the role of CyPB in Orf virus(ORFV)early infection,the full length of the CyPB was 651 bp,which was amplified by PT-PCR using the specific primers based on the CyPB gene sequence from Genbank database.The amplications were inserted into PET-32 avector,and the recombinant plasids were transformed into E.coli BL21(DE3)and induced by IPTG.The 25 000 protein band was observed by SDS-PAGE electrophoresis,which was consistent with His-CyPB fusion protein.The above results will provide important material basis for the study of CyPB function.