小反刍兽疫病毒P蛋白及非结构蛋白V表达特性研究  

Expression of P/V Gene of Peste des Petits Ruminants Virus and the Studies of the Biological Characteristics

作　　者：严欢[1,2] 马玉馨 信爱国 高华峰[1] YAN Huan MA Yu-xin XIN Ai-guo GAO Hua-feng(Yunnan Tropical and Subtropical Animal Viral Disease Laboratory, Kunming 650224, China College of Veterinary Medicine,Yunnan Agricultural University, Kunming 650201, China)

机构地区：[1]云南省热带亚热带动物病毒病重点实验室,云南昆明650024 [2]云南农业大学动物医学院,云南昆明650201

出　　处：《中国兽医杂志》2017年第8期3-6,共4页Chinese Journal of Veterinary Medicine

基　　金：国家自然科学基金项目(31160499)

摘　　要：从小反刍兽疫病毒全长c DNA中对磷酸化蛋白P及其编码的非结构蛋白V基因进行特异扩增,PCR产物回收后分别连接于T载体酶切及测序分析后,将其亚克隆至真核载体pEGFP-N2及原核表达载体pET32a,获得的真核表达质料命名为pEGFP-P、pEGFP-RV与空载体对照转染Vero细胞,经浓度为800μg/μL G418筛选后获得均一表达,荧光定位观察表明P蛋白与V蛋白的表达并不相同,P蛋白严格定位于细胞浆,而V蛋白则能在胞浆及胞核内观察到;获得原核表达质粒pET32a-P和pET32a-RV在37℃通过条件优化后以1.0 mmol/L IPTG诱导后分别收获细胞上清及沉淀用于SDS-PAGE及Western Blot对重组P、V蛋白的检测,结果表明,重组P蛋白为90 ku,重组V蛋白为55 ku的可溶性融合蛋白,可溶性表达产物经500 mmol/L咪唑两次纯化后获得80%以上纯度的表达,纯化得到的蛋白免疫兔子后能产生特异性抗体。The P/V gene fragment of peste des petits ruminants vaccine strain N75/1 was amplified by PCR from c DNA library,and the purified products were cloned into pMD19-T vector. Confirmed with enzyme digestion and sequence analysis, P and V genewas subcloned into vector pEGFP-N2 and pET32 a. The eukaryotic expression vector with p/v gene was constructured and finallynamed pEGFP-P,and pEGFP-RV, respectively. The recombinant plasmid with P/V gene and the empty vector control was trans-fected into Vero cells and the cells were selected with G418. The PPRV P gene was expressed only in cytoplasm. Prokaryotic Expres-sion vector of pET32 a with P/V gene was constructed and named as pET32a-P and pET32a-RV respectively. The fusion proteinwas induced by IPTG with the final titer 1. 0 mmol/L at 37℃. Fusion protein mainly expressed in the supernatant was purified with NI-NTA spin with 500 mmol/L imidazole twice and used to immunize the rabbits to prepare polyclonal antibodies. The results showedthat the molecular weight of recombinant protein was about 90 ku and 55 ku. The antiserum of rabbits analyzed by western blotting showedthat the bands at 90 ku and 55 ku molecular weights were detected. It shows that the two purified proteins have biological functions.

关 键 词：小反刍兽疫病毒 P/V蛋白 细胞定位 原核表达 

分 类 号：S855.3[农业科学—临床兽医学]