肠道病毒71型的全长cDNA感染性克隆的构建与鉴定

基础医学与临床 ›› 2013, Vol. 33 ›› Issue (7): 840-844.

肠道病毒71型的全长cDNA感染性克隆的构建与鉴定

王冠洲¹,叶飞²,赵阿妮³,韩为⁴,阴彬³,彭小忠⁴

1. 中国医学科学院基础医学研究所北京协和医学院基础学院
2. 中国医学科学院基础医学研究所
3. 中国医学科学院基础医学研究所北京协和医学院基础学院
4. 中国医学科学院基础医学研究所北京协和医学院基础学院医学分子生物学国家重点实验室

收稿日期:2013-05-17 修回日期:2013-05-19 出版日期:2013-07-05 发布日期:2013-06-26
通讯作者: 彭小忠 E-mail:pengxiaozhong@pumc.edu.cn
基金资助:
自发性高血压大鼠心、脑、肾和肠系膜微动脉缝隙连接特性的比较研究》国家自然科学基金（国家自然科学基金）

Construction of an infectious clone of enterovirus 71

Received:2013-05-17 Revised:2013-05-19 Online:2013-07-05 Published:2013-06-26
Contact: Xiao-zhong PENG E-mail:pengxiaozhong@pumc.edu.cn

摘要/Abstract

摘要： 目的构建一株肠道病毒71型的全长cDNA克隆并验证感染性。方法用长片段高保真RT-PCR方法扩增肠道病毒71型87-2008 Xi’an株的cDNA，装入pBR322载体。将该克隆线性化，体外转录后转染RD细胞后，通过观察CPE及RT-PCR验证其感染性。随后利用空斑法测定拯救病毒的一步生长曲线。结果成功构建了肠道病毒71型87-2008 Xi’an株的全长cDNA克隆；体外转录及转染RD细胞60h后可观察到明显的CPE现象；RT-PCR在转染RD细胞中检测到病毒的存在；成功利用空斑法测定了拯救病毒的一步生长曲线，最大滴度达到2×107 pfu/ml。结论成功构建了肠道病毒71型87-2008 Xi’an株的全长cDNA克隆并验证了其感染性，同时测定了拯救病毒的一步生长曲线，与野生型病毒相近。

关键词: 关键词：肠道病毒71型, 感染性克隆, 一步生长曲线

Abstract: Objective To construct full length infectious cDNA clone of enterovirus 71. Methods A cDNA fragment with a T7 promoter at the 5’ end and a poly (A) tail at the 3’ end was amplified by RT-PCR from the genome of the EV71/87-2008 Xi’an strain directly. This fragment was then ligated to pBR322 via SalI and BspEI site. Then the in vitro synthesized RNA transcripts were transfected into RD cells to produce the rescued virus. The negative-strand viral RNA was detected from the RD cells lysates by RT-PCR. The one step growth curve of recovered EV71 was measured by plaque assay. Results The full length cDNA clone of EV71/87-2008 Xi’an was constructed and the infectious of this clone was validated successfully. The EV71 recovered virus was proved functionally identical to its template. Conclusion Results suggest that the infectious clone of EV71/87-2008 Xi’an strain was constructed successfully.

Key words: Key words: EV71, infectious clone, one step growth curve

中图分类号:

Q939.47

王冠洲叶飞赵阿妮韩为阴彬彭小忠. 肠道病毒71型的全长cDNA感染性克隆的构建与鉴定[J]. 基础医学与临床, 2013, 33(7): 840-844.