肠道病毒71型微复制子的构建  

Construction of the minireplicon of enterovirus 71

作　　者：马英伟[1] 孙乐乐[1] 李静[1] 乔乔[1] 庄志超[1] 赵丽[1] 于学杰[1] 王志玉[1] 温红玲[1] 孙妍琳[1] 

机构地区：[1]山东大学公共卫生学院病毒学实验室,山东济南250012

出　　处：《山东大学学报（医学版）》2015年第9期35-40,共6页Journal of Shandong University:Health Sciences

基　　金：国家自然科学基金(81371833);山东省医药卫生科技发展计划(2013WS0211)

摘　　要：目的 构建EV71非结构蛋白及EV71 3C蛋白酶微复制子体系,研究微复制子的复制及表达情况。方法聚合酶链式反应扩增绿色荧光蛋白（EGFP）,利用特定酶切位点及连接酶,将目的基因片段连接至p MD19-T载体,分别构建EV71非结构蛋白及EV71 3C蛋白酶微复制子体系。荧光显微镜下观察微复制子表达情况,实时荧光定量PCR观察微复制子复制情况。结果 EV71非结构蛋白微复制子及EV71 3C蛋白酶微复制子表达后,均可观察到特异性绿色荧光,荧光强度有明显差异;转染后12~72 h取样所测RNA含量几乎不变。结论 非结构蛋白全长及3C蛋白酶启动微复制子转录强度不同,所构建微复制子不具备复制功能,提示结构蛋白编码区对病毒RNA复制有重要意义。Objective To construct the minireplicon of non-structural protein and 3C protease of EV71 and study the replication and expression of the minireplicon. Methods The genes of the enhanced green fluorescent protein（ EGFP）, amplified by PCR, and the genes of non-structural protein and 3C protease were inserted into pMD19-T vector through specific restriction sites and ligase. The expression and replication of the minireplicon were detected by fluorescence mi- croscope and checked by real-time t＇CR respectively. Results Specific green fluorescence was detected after in vitro transcription and transfection of the plasmid containing the genes of EV71 non-structural proteins and the 3C protease, and there were significant differences in the intensity of the fluorescence among different groups. The content of the RNA remained same between 12 h to 72 h after transfection. Conclusion The abilities of transcriptions of the full- length non-structural protein and the 3C protease were different. The replication of the minireplicon could not be detec- ted in RD cells, which suggested the importance of the structural protein to the replication of the virus.

关 键 词：肠道病毒71型 微复制子 报告基因 非结构蛋白 

分 类 号：R373.2[医药卫生—病原生物学]