登革2型病毒PrM基因的重组甲病毒RNA介导的抗病毒作用的研究

将扩增的登革 2型病毒株PrM基因导入pSFV载体的SP6启动子下游 ,筛选出含该基因正、反向插入的重组质粒DNA。用SpeI酶分别将重组的和辅助的质粒DNA线性化 ,并将其体外转录成 5′末端含帽子结构的RNA。再将这两种RNA共转染BHK细胞。然后将转染的宿主细胞用登革 2型病毒株攻击 ,并分别观察含正、反义PrM基因的重组甲病毒RNA介导的抗病毒效果。通过碱基序列测定 ,筛选出含PrM基因正、反向插入的pSFV PrM重组质粒。并获得了经重组RNA与辅助RNA共转染细胞而产生的重组病毒颗粒。含有反义PrM基因的重组病毒RNA ,在宿主细胞中具有抗登革 2型病毒复制的作用 ,而且强于含正义PrM基因的重组病毒RNA。

STUDIES OF THE RESISTANCE OF THE RECOMBINANT ALPHAVIRUS RNAs CONTAINING DENGUE-2 PrM GENE TO VIRUS INFECTION

The amplified PrM gene of dengue-2 virus was cloned into the downstream SP6 promoter-pSFV vector and the recombinant plasmid (pSF.rM2) DNA which contained sense- or antisense-PrM gene, was selected. pSF.rM2 DNA and helper DNA linearized by the enzyme SpeI digestion were both transcribed in vitro into recombinant RNAs which contained the capping analog on the 5'-end and contaansfected into BHK cells by electroportation. The the transfected host cells were challenged with dengue-2 virus and the resistant efficiency of recombinant virus RNAs containing sense- or antisense-PrM gene to virus infection were observed, respectively. The recombinant plasmids (pSFV-PrM) containing sense- or antisense-PrM gene were selected with determination of the nucleotide sequence. The recombinant virus particles were obtained with recombinant RNA and helper RNA co-transfected into BHK cells. Host cells transfected with antisense-PrM RNA derived complete resistance to dengue-2 virus replication and the efficiency was higher than that of the recombinant virus RNA containing sense-PrM gene.