伪狂犬病病毒Ea株UL14基因的克隆、序列分析及表达  

Cloning,sequence analysis and expression of UL14 gene of pseudorabies virus Ea strain

作　　者：张辉[1] 肖少波[1] 方六荣[1] 牛传双[1] 陈焕春[1] 

机构地区：[1]华中农业大学动物医学院,湖北武汉430070

出　　处：《中国兽医学报》2008年第3期264-267,共4页Chinese Journal of Veterinary Science

基　　金：国家自然科学基金资助项目(30400322);霍英东青年教师基金资助项目(91029)

摘　　要：从伪狂犬病病毒Ea株基因组DNA中克隆含UL14基因的BamHⅠ第3片段并进行序列分析。根据测定的序列设计1对能扩增UL14基因完整编码区的引物,PCR扩增UL14基因并将其插入原核表达载体pGEX-KG,获得原核表达质粒pKG-UL14,转化BL21(DE3),在IPTG诱导下,GST-UL14融合蛋白获得高效表达,表达产物的相对分子质量为44 000,并以包涵体形式存在。纯化的表达产物免疫兔,获得了针对UL14蛋白的高效价多抗血清。进一步将UL14基因克隆到真核表达载体pEGFP-C1中EGFP基因的3′端,获得与EGFP融合表达的真核表达质粒pEGFP-UL14,转染Hela细胞,通过荧光显微镜观察发现,转染后24、48 h的融合蛋白EGFP-UL14主要定位在胞浆,但转染后72 h的融合蛋白主要定位在细胞核。上述研究结果为进一步阐明UL14的结构与功能奠定了基础。BamHⅠ-3 fragment containing UL14 gene was cloned from pseudorabies virus genome DNA and sequenced.According to result of sequencing,UL14 gene was amplified and inserted into prokaryotic expressing vector pGEX-KG.After transformed into E.coli BL21（DE3）,the fusion protein GST-UL14 was expressed under induction with IPTG.The results of SDS-PAGE analysis showed that GST-UL14 was 44 000 in size and presented as inclusion body.Anti-UL14 polyclonal antisera were produced in rabbits with the purified fusion protein GST-UL14.Eukanyotic expressing plasmid pEGFP-UL14 expressing EGFP-UL14 fusion protein was constructed,transfected into Hela cells,and detected at 24,48,72 h post transfection.Fluorescence assay of Hela cells transfected with pEGFP-UL14 indicated that most of UL14 consist in cytoplasm at 24,48 h post transfection and translocated to nucleolus 72 h post transfection.The above results lay foundation for further studying the structure and functions of UL14 gene.

关 键 词：伪狂犬病病毒 UL14 序列分析 表达 

分 类 号：S852.65[农业科学—基础兽医学] R535[农业科学—兽医学]