登革2型43株病毒包膜蛋白E基因的聚合酶链反应扩增

Polymerase Chain Reaction Amplification of Envelope Protein E Gene of Dengue-2 Virus 43 Strain

以我国登革２型４３株病毒ＲＮＡ为模板，采用逆转录－聚合酶链反应技术（ＲＴ－ＰＣＲ）扩增Ｅ基因片段。拟扩增的Ｅ基因片段始于登革病毒编码序列５＇端的１０３６ｂｐ，止于３＇端的２３０４ｂｐ，其间含有１个ＨｉｎｄⅢ酶切位点，３＇端有１个ＳｍａⅠ位点，５＇端有１个ＥｃｏＲⅠ位点。建立了扩增大片段的实验条件并制备出了大于１２００ｂｐ以上的片段。该片段与标准分子量对比为１２９２ｂｐ左右，经ＨｉｎｄⅢ酶切，出现７８１ｂｐ和５１１ｂｐ２个片段，用Ｓｏｕｔｈｅｒｎｂｌｏｔ和光敏生物素标记核酸探针证实制备出的１２９２ｂｐ片段为Ｅ基因片段，此片段可直接用于克隆表达。

This paper describes the direct amplification of E gene fragmants, using reverse transcription (RT) PCR technique with China Dengue-2 virus 43 strain RNA as template. We assume that the location of the target gene fragments in the coding sequence begins at 1 036bp terminates site at 5’-end, and a EcoR Ⅰ site at 3’-end. We designed a amplification method for big fragments and get a required one, about 1 290bp in length compared with MW marker. and confirm it with Hind Ⅲ. Two fragments of 781bp and 511bp come out respectively. Southern blot and photobiotin labelled nuclear acid probe also proved that this fragment was indeed E gene.The preparation of this fragment constructs a basis for E gene cloning and expression, and for further, study of the relationship between its structure and function, which may contribute to the antigenic diagnosis, research and the application as dengue fever subunit vaccine.