狂犬病毒3aG株糖蛋白基因在人5型重组腺病毒中的表达研究  

Expression of glycoprotein gene of the rabies virus 3aG strain by E-3 deleted adenovirus recombinant

作　　者：张新梅[1] 张云[1] 李文辉[1] 翟文静 王树蕙[1] 

机构地区：[1]中国医学科学院基础医学研究所中国协和医科大学基础研究所病原学室,北京100005

出　　处：《中华实验和临床病毒学杂志》2000年第3期281-284,共4页Chinese Journal of Experimental and Clinical Virology

基　　金：国家"8 6 3"基金资助 !(86 3-10 1-0 5 -0 3-0 4)

摘　　要：目的　构建表达狂犬病毒糖蛋白基因 (GP)的重组人腺病毒。方法　克隆狂犬病毒疫苗株GP基因并测序 ,将其插入E3区缺失的Ad5载体 ,置于E3区早期启动子的控制下 ,通过同源重组 ,蚀斑纯化 ,筛选表达GP的重组人腺病毒。用ELISA法检测表达的糖蛋白 ,快速荧光灶抑制试验 (RFFIT)法测定此重组病毒免疫小鼠后产生的中和抗体。结果　获得的 3aG株基因的开放读码框为 15 75个核苷酸 ,编码 5 2 4个氨基酸 ;经同源重组和蚀斑纯化获得了E3区表达狂犬病毒GP基因的重组人腺病毒 ;其表达产物能特异性地被抗狂犬病毒人免疫血清所识别 ;免疫小鼠后能产生一定水平的中和抗体。结论　成功构建了E3区表达狂犬病毒GP基因的重组人腺病毒 。Objective To analysis Chinese rabies virus vaccine strain 3aG glycoprotein(GP) gene and further produce GP by E3-deleted human adenovirus recombinant. Methods Chinese rabies virus vaccine strain 3aG glycoprotein gene was cloned by RT-PCR and its sequence was determined by DNA sequencing. Cotransfection was performed to obtain adenovirus recombinant. The expressed glycoprotein was examined by ELISA and its immunogenicity was evaluated by testing neutralizing antibody level of mice inoculated with the recombinant virus. Results DNA sequencing showed that the open reading frame of GP gene contains 1 575 nucleotides and five of the deduced amino acids are different from the previous report. The recombinant adenovirus containing GP gene in E3 region was obtained by cotransfecting 293 cells and rounds of plaque purification. ELISA assay demonstrated that the GP gene can be efficiently expressed and rapid fluorescent focus inhibition test (RFFIT) showed it can also elicit GP specific neuralizing antibody. Conclusion Chinese rabies virus vaccine strain 3aG glycoprotein was successfully expressed by E3-deleted human adenovirus recombinant and specific neutralizing antibody can be elicited in mice after immunization by the recombinant.

关 键 词：狂犬病病毒 糖蛋白 序列分析 腺病毒 中和抗体 

分 类 号：R373[医药卫生—病原生物学]