腺病毒介导HAV结构基因表达及其产物的免疫学性质检测  

Expression of structural gene of HAV mediated by replication-defective adenovirus and testing of its immunological properties

作　　者：吴 娟[1] 李嘉琦[2] 孙茂盛[2] 戴长柏[2] 

机构地区：[1]云南烟草科学研究院生物技术重点实验室,云南昆明650106 [2]中国医学科学院协和医科大学医学生物学研究所,云南昆明650118

出　　处：《免疫学杂志》2002年第3期192-200,共9页Immunological Journal

摘　　要：目的 探讨重组腺病毒免疫实验动物后的抗体反应。方法 利用RT-PCR方法,从HAV-L8株的RNA中扩增出结构蛋白vp3+vpl基因,克隆到穿梭质粒pXCX2 NotI上。采用磷酸钙-DNA共沉淀技术,将E1缺失的复制缺陷型腺病毒载体pJM17与pXCX2-CMV-HAV共转染293细胞。结果 通过胞内同源重组,经RT-PCR、免疫荧光染色和蛋白印迹鉴定表明,获得了复制缺陷型重组腺病毒rAdHAV。纯化后的rAdHAV滴度为1×109.0TCID50/mL。昆明种小白鼠口服rAdHAV后,诱导产生了抗HAV IgG。结论 复制缺陷型腺病毒可望成为发展口服病毒疫苗的有效载体系统。ve To discuss the immune reaction in the animals inoculated with the reeombinant adenovirus. Methods The structural gene (vp3 + vpl) of HAV-L8 was amplified from its RNA by RT-PCR and was cloned into Ad shuttle plasmid (pXCX2 Not I ). A reeombinant replication-defective adenovirus was rescued in 293 packaging cells via homologous recombination in vivo of both plasmid pXCX2-CMV-HAV and pJM17 containing Ad5 genome with deletions of El region. Results A series of methods such as RT-PCR, immunofluorescence and western blot were employed to identify the generated reeombinant adenovirus (rAdHAV). The titer of stocks of rAdHAV was up to 1× 109.0 TCID50/mL. After rAdHAV was delivered to Kunmimg mice by oral administration, anti-HAV IgC was tested in the mice. Conclusions A reeombinant replication-defective adenovirus will become an efficient vector system to develop an oral vims vaccine.

关 键 词：重组腺病毒 HAV vp3+vp1 免疫反应 基因表达 病毒疫苗 

分 类 号：R392[医药卫生—免疫学]