登革2型病毒全长基因组的长链RT-PCR法扩增

贡树基; 赵卫; 曹虹; 张文炳; 周浩; 陈丽丹

doi:10.11847/zgggws2006-22-04-29

登革2型病毒全长基因组的长链RT-PCR法扩增

Amplification of complete genome of dengue 2 virus by long RT-PCR

摘要

摘要:
目的采用长链RT-PCR技术扩增登革2型病毒新几内亚株(NGC)全长基因组。
方法根据登革2型病毒NGC株基因组全序列设计引物, 在上游引物中加入Sp6 RNA聚合酶启动子核心序列, 下游引物中加入ClaⅠ内切酶位点。从病毒感染的乳鼠脑中提取RNA, 采用长链RT-PCR技术进行扩增。以获得的PCR产物为模板分别扩增3个NGC株特异的基因片段。
结果长链RT-PCR法扩增出约11 kb基因片段, 经PCR法证实为登革2型病毒NGC株特异序列。
结论通过长链RT-PCR技术, 获得了登革2型病毒NGC株全长基因组, 为进一步构建感染性克隆打下基础。

Abstract:
Objective To establish the long reverse transcription PCR for amplification of the complete genome of dengue 2 virus.
Methods The primers were designed according to the published nucleotide sequence of DEN2NGC strain.Sp6 was added to 5.terminal and restriction endonuclease site for Cla ⅳ was added to 3' terminal.After virus RNA was extracted from the brains of the infected new-born mice, the co mplete genome of DEN2NGC strains was amplified by the long RT-PCR.Three fragments of specific lengths were re-amplified from PCR products for identification.
Results The 11kb full-length genome of dengue 2 virus was amplified successfully by the long RT-PCR.
Conclusion The complete genome of dengue 2 virus was successful to be amplified using the long RT-PCR, and it will be advantageous for further constructing infectious clone.

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