应用痘苗病毒载体表达猴轮状病毒VP4抗原基因  

Expression of Simian Rotavirus Vp4 by a Vaccinia Virus Vector

作　　者：姜惠英[1] 杨安道[1] 周静仪[1] 张惠芳 洪涛[1] 

机构地区：[1]中国预防医学科学院病毒学研究所

出　　处：《中华实验和临床病毒学杂志》1995年第3期254-257,共4页Chinese Journal of Experimental and Clinical Virology

基　　金：国家"863"高科技生物技术领域资助

摘　　要：把编码猴轮状病毒（Ｒｈｅｓｕｓｒｏｔａｖｉｒｕｓ，ＲＲＶ）Ｖｐ４抗原的第４基因片段插入到痘苗病毒表达载体ｐＪＳＡ１１７５的Ｐ７．５启动子下游，构建成在痘苗病毒Ｐ７．５启动子调控下表达猴轮状病毒Ｖｐ４抗原基因的重组质粒ＰＪＳＡ１１７５－ＶＰ４。应用磷酸钙沉淀技术将ＰＪＳＡ１１７５－ＶＰ４ＤＮＡ转入ＴＫ－１４３细胞，在ＢＵＤＲ和Ｘ－ｇａｌ存在下筛选蓝色蚀斑。经３代以上纯化和病毒增殖，获重组病毒Ｒ－ＶＪＳＡ１１７５－Ｖｐ４。蚀斑滴定其满度达到１５×１０１１ＰＦＵ／Ｌ。经核酸杂交试验证明所获得的重组痘苗病毒带有猴轮状病毒Ｖｐ４抗原基因。用重组病毒感染ＴＫ－１４３细胞（或Ｖｅｒｏ细胞），在感染后４８ｈ，用酶免疫法（ＥＩＡ）检测受染细胞上清液和细胞裂解液中表达的猴轮状病毒Ｖｐ４抗原基因均呈阳性反应。本试验为本研究室轮状病毒基因工程疫苗的一部分，为深入了解轮状病毒基因结构及其功能在方法学上奠定了必要的基础。Rhesus rotavirus (RRV) gene 4 that encodes Vp4 protein of the virus was cloned and inserted into the downstream of promotor 7.5 of a Chinese strain of vaccinia virus vector. The constructed vaccina-RRV Vp4 recombinant(PJSA1175-Vp4)plasmid was transfected into TK-143 cells with wild type vaccinia DNA. The recombinant virus were obtained with the xgal as resulting gene by selection of the blue plaques.Then the recombinant virus infected newly prepared TK-143 cells. After 4-7 days, the cell lysates were detected by enzyme immunoassay and low level of RRV-Vp4. 1.5 × 10 PFU / L was expressed in the TK-143 cells. The present experiment has laid a basis for further characterization of the major gene product,Vp4.

关 键 词：轮状病毒 痘苗病毒 基因表达 抗原 基因 

分 类 号：R373.2[医药卫生—病原生物学] R373.12[医药卫生—基础医学]