采用逆转录-聚合酶链式反应(RT-PCR)技术扩增了我国登革2型病毒E基因, 大小为1.29kb. 将扩增的E基因片段首先克隆到T载体(pBluescript ⅡKS+)中, 用PCR和限制性酶切分析鉴定出阳性重组子. 然后利用M13通用引物直接对插入的E基因片段进行序列分析. 结果表明, 经限制性内切酶鉴定和DNA序列分析证明所克隆的序列与已报道的E基因序列是一致的.
The E gene fragment was amplified from RNA isolated the Chinese dengue-2 virus by RT-PCR method. It was 1.29 kb in length. This unmodified E fragment was directly inserted into the EcoRV-cut T-tailed pBluescript ⅡKS+ vector DNA. The positive recombinant colonies were identified by the PCR and the restriction endonuclease digesting method. Then nucleotide sequence analysis of the inserted E gene fragment was conducted directly by using the common M13 primer. The result of the restriction endonuclease and sequence analysis confirmed that the 120 by of 5’-teminal nucleotides sequence of the amplified E gene fragment was the same sequence as reported.
于曼,秦鄂德,杨佩英,孙蕾,徐品芳,司炳银,闫国珍.登革2型病毒E基因的克隆及碱基序列测定[J].生物化学与生物物理进展,1996,23(5):457-459
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