登革2型病毒NGC株基因组亚克隆的构建及其鉴定

Construction and identification of genomic cDNA subclones of dengue 2 virus NGC strain

目的构建登革2型病毒NGC株基因组全序列的亚cDNA克隆,为进一步构建全长感染性cDNA克隆奠定基础。方法根据登革2型病毒NGC株基因组全序列中的酶切位点设计2对引物,从病毒感染的乳鼠脑中提取RNA,采用长链RT-PCR技术扩增出覆盖病毒基因组全长的2个cDNA片段,并克隆至pCR-XL-TOPO载体后测序。结果经酶切鉴定和序列测定表明,获得的cDNA克隆为登革2型病毒NGC株特异的。结论已成功构建出登革2型病毒NGC株基因组的2个cDNA亚克隆。

Objective To construct the eDNA subclones spanning the entire genome of dengue 2 virus NGC strain for further construction of full-length infectious viral eDNA clone. Methods Two pairs of primers were designed according to the restriction endonuelease sites in the viral genome of dengue 2 virus NGC strain. After viral RNA extraction from the brain of infected new-born mice, two parts of full-length viral eDNA were amplified by long RT-PCR and cloned into the vector pCR-XL-TOPO. The partial sequence of the recombinant plasmid was determined. Results and Conclusion Sequence analysis and digestion with restriction enzymes demonstrated that the two eDNA subelones were specific for dengue 2 virus NGC strain, suggesting the successful construction of the two eDNA subelones of dengue 2 virus NGC strain.