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

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.