肠道病毒71型（EV71）AH3株感染性克隆的构建与鉴定

Construction and identification of infectious clone of enterovirus 71 AH3

目的 构建肠道病毒71型（Enterovirus71,EV71） AH3株的全基因序列感染性克隆.方法 通过分段扩增和酶切连接将EV71全基因组cDNA片段逐步克隆入PWSK29载体,构建含全病毒基因组序列的重组质粒.转染Vero细胞后,获得拯救病毒.经RT-PCR、酶切、测序、间接免疫荧光（IFA）等方法进行鉴定.结果 酶切鉴定结果与预期一致,序列分析显示为EV71基因；其转染Vero细胞后,可观察到典型的细胞病变；所获得的拯救子代病毒滴度（TCID50）为107.5；IFA检测可见感染细胞出现绿色荧光.结论 成功构建EV71全基因序列感染性克隆,为病毒致病机理及减毒疫苗的研究奠定基础.

Objective To construct an infection clone of enterovirus 71 AH3.Methods Through segmented amplification and enzyme ligation,the fragments of EV71 were gradually directional cloned into PWSK29 vector,and the complete genome plasmid was constructed.The rescued virus of EV71 AH3 plasmid was obtained after transfecting vero cells and identified by RT-PCR,sequencing and IFA.Results The full-length clone of EV71 was constructed successfully,and the enzyme-cutting identification result was in line with the expectation.The sequence analysis indicates it is EV71 gene.The typical CPE was observed after transfecting vero cells.The TCID50 of rescued progeny virus is 10-75.And the infected cells can be detected green fluorescence by IFA.Conclusion The infectious clone of the complete genome of EV71 is successfully constructed,which will serve as the foundation for further study on gene function of the virus.