登革病毒NS3全基因的扩增与克隆

登革热(ＤＦ)、登革出血热及登革休克综合征(ＤＨＦ/ＤＳＳ)是由登革病毒所致的两种不同临床类型的急性传染病,广泛流行于全球热带及亚热带地区。ＤＨＦ/ＤＳＳ以高热、出血、休克、高病死率为主要特征,近年来其发病率有迅速增加的趋势,已成为严重影响人类健康的公共卫生问题。迄今,ＤＨＦ/ＤＳＳ的发病机制仍不清楚,亦无有效的特异性预防方法1]。登革病毒属于黄病毒科的黄病毒属,有Ⅰ、Ⅱ、Ⅲ、Ⅳ四个血清型,基因组为单股正链ＲＮＡ,全长约11ｋｂ,编码三种结构蛋白和七种非结构蛋白。基因组顺序为5′ＣＰｒＭＥＮＳ1ＮＳ2ａＮＳ2ｂＮＳ3Ｎ…

Amplification and Cloning of NS3 Gene of Dengue Virus

The RNA of dengue 2 virus (New Guinea C strain, NGC) was extracted from small amount of dengue 2 virus culture supernatant. The large NS3 gene fragment was amplified from RNA of dengue 2 virus with RT PCR method. The amplified NS3 gene fragment was cloned directly into the pUC18 plasmid vector. The recombinant plasmid was identified by agarose gel electrophoresis analysis after digestion with restriction endonuclease and nested PCR method. The results showed that the amplified NS3 gene fragment was about 1978 bp in length; the recombinant plasmid contained NS3 gene of dengue 2 virus. The method of amplified large fragment of dengue virus from small amount of dengue virus culture supernatant was established.