伪狂犬病病毒Ea株UL18基因的克隆、序列分析及表达  

Expression of UL18 gene of pseudorabies virus strain Ea

作　　者：薛念波[1] 肖少波[1] 江云波[1] 常天明[1] 刘曼莉[1] 赵骞[1] 罗锐[1] 陈焕春[1] 方六荣[1] 

机构地区：[1]华中农业大学动物医学学院动物病毒实验室

出　　处：《中国预防兽医学报》2008年第4期255-259,共5页Chinese Journal of Preventive Veterinary Medicine

基　　金：国家自然科学基金(30400322)

摘　　要：本研究以伪狂犬病病毒Ea株基因组DNA为模板,PCR扩增含UL18全长基因的片段并克隆至pMD18-T载体,采用双脱氧末端终止法测序。序列分析显示UL18全长891bp,可编码297个氨基酸。将该基因亚克隆到原核表达载体pQE-82L的6×His标签下游,获得原核表达质粒pQE-UL18,转化大肠杆菌DH5α,经IPTG诱导在大肠杆菌中成功表达,表达的融合蛋白6×His-UL18的分子量约为33ku。Western blot证实,表达的融合蛋白能与抗6×His的单抗发生特异性反应。进一步将UL18基因插入真核表达载体pEGFP-N1中EGFP基因的5'端,获得与EGFP融合表达的真核表达质粒pEGFP-UL18,转染HeLa细胞,通过激光共聚焦显微镜观察发现,转染后24h融合蛋白EGFP-UL18主要定位于细胞浆,仅有部分定位于细胞核,而48h时则主要定位在细胞核,但对照载体pEGFP-N1转染细胞的荧光一直呈弥散型分布于整个细胞中。In this study, the UL 18 gene of pseudorabies virus （PRV） Strain Ea was amplified by PCR and cloned into pMD 18-T vector. Sequence analysis showed that the UL18 gene was 891 base pairs in length encoding 297 amino acids （aa）. The full-length UL18 fragment was subcloned into downstream of hexahistidine sequence of prokaryotic expression vector pQE-82L and transformed into E.coli DH5 α. Expression of a fusion protein （6 x His-UL18） with 33 ku molecular mass was detected by Western blot. The UL18 gene was inserted into eurokaryotic expression vector pEGFP-N1 and transfected into HeLa cells. Protein expression was indicated by fluorescence. The results showed that the protein was mainly located in the cytoplasm 24 h post-tmnsfection, and relocated to cell nucleolus 48 h post-transfection. However the fluorescence in pEGFP-Nl-transfected cells was found uniformly distributed throughout the cytoplasm of the cell.

关 键 词：伪狂犬病病毒 UL18基因 序列分析 表达 

分 类 号：S852.65[农业科学—基础兽医学]