基于DNA和RNA的双功能Semliki森林病毒复制子载体的构建

Construction of DNA and RNA Based on Bifunctional Replicon Vector Derived from Semliki Forest Virus

以semliki森林病毒衍生的复制子载体pSFV1和辅助载体pSFVhelper2为骨架,用CMVIE和T7启动子替换SP6启动子并在3′UTR下游插入BGH转录终止子,构建了基于DNA和RNA的复制子表达载体pSMCTA和辅助载体pSHCTA。在DNA和RNA二种递送方式上证实该表达载体可高水平表达外源基因,与辅助载体共转染可制备具有感染能力并能表达外源基因的重组病毒颗粒。构建的基于DNA和RNA的双功能复制子载体显著地提高SFV载体应用范围,在体外可用于高水平表达外源基因及大规模制备重组病毒颗粒,在体内也可用于研制复制子疫苗和基因治疗载体。

DNA-based replicon expression vector pSMCTA and helper vector pSHCTA were constructed by replacing the SP6 promoter used in the original system pSFVI and pSFV-helper2 derived from Semliki Forest virus （SFV） with the RNA polymerase Ⅱ -dependent cytomegalovirus immediate early （CMV IE） enhancer/promoter and T7 promoter, and inserting BGH transcription termination and polyadenylation signal downstream 3＇-untranslated region （UTR）. The RNA polymerase Ⅱ-dependent cytomegalovirus immediate early （CMV IE） enhancer/promoter and T7 promoter in pSMCTA and pSHCTA could drive transcription to produce replicon RNA in vivo and ex vivo. High level expression of foreign genes （GFP and LacZ） could be demonstrated by transfecting BHK21 cells with the new replicon expression vectors based on both DNA and RNA, and recombinant virus particles （RVP） be prepared by cotranfecting the expression vectors with the helper vectors. Foreign genes were also highly expressed in cells （ BHK21 ） which were infected with RVP activated by α-chymotrypsin. The bifunctional replicon vectors can be used in highly efficient expression of foreign genes and preparation of RVP ex vivo, also in development of replicon vaccines and gene therapy vectors in vivo.