表达HAV结构基因的重组腺病毒鉴定及细胞病变分析  

Identification and CPE Analysis of Recombinant Adenovirus Expressed Structural Gene of HAV

作　　者：李嘉琦[1] 吴娟[1] 孙茂盛[1] 戴长柏[1] 

机构地区：[1]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：《动物医学进展》2003年第2期68-71,共4页Progress In Veterinary Medicine

摘　　要：利用RT-PCR方法,从HAV-L8株的RNA中扩增出结构基因(vp3+vp1),克隆到穿梭质粒pXCX2NotI上。采用磷酸钙-DNA共沉淀技术,将腺病毒载体pJM17与pXCX2-CMV-HAV共转染293细胞。在光学显微镜下观察到明显的细胞病变(CPE);经RT-PCR、免疫荧光染色和蛋白印迹鉴定证明HAV的结构基因已插入到腺病毒基因组中;在电子显微镜下观察发现有二十面体的病毒颗粒。以上结果表明获得了重组腺病毒rAdHAV,纯化后的rAdHAV滴度为1×109.0TCID50/mL。The structural gene (vp3+vp1) of HAV L8 strain was amplified from its RNA by RT PCR and was cloned into adenovirus(Ad) shuttle plasmid (pXCX2NotI). A recombinant adenovirus was rescued in 293 packaging cells via homologous recombination in vivo of both plasmid pXCX2 CMV HAV and pJM17. Cytopathic effects(CPEs)were observed with the optical microscope;a series of methods such as RT PCR,immunofluorescence and western blot showed that the structural gene of HAV L8 strain was inserted into the Ad genome; and icosahedrous particles were seen under the electron microscope. These results indicated that we had obtained the recombinant adenovirus (rAdHAV). The titer of stocks of rAdHAV was up to 1×10 9.0 TCID 50 /mL.

关 键 词：重组腺病毒 HAV结构基因 鉴定 细胞病变 甲型肝炎病毒 基因工程病毒疫苗 转移载体 

分 类 号：R512.61[医药卫生—内科学]