【摘要】 本研究根据编码A亚型人呼吸道合胞病毒(HumanRespiratorySyncytialVirus,HRSV)核壳体蛋白(Nucleocapsidprotein,N)和磷蛋白(phosphoprotein,P)的基因序列,各设计一对特异性的引物,应用RT-PCR技术,从感染HRSV的HEp-2细胞中扩增获得n和p的基因片断,克隆至真核表达载体pcDNA3.1(+)。获得的重组质粒通过脂质体Lipofectamine2000转染COS-7细胞,72h后再用Westernblot鉴定蛋白的表达。结果显示真核表达载体pcDNA3.1(+)/N和pcDNA3.1(+)/P的限制性内切酶分析结果与预期一致,基因序列分析显示没有发生无义突变,利用蛋白印记方法也检测到了N和P的特异性条带。于是我们认为成功构建了含有HRSVN和P编码基因的真核表达载体,在真核细胞内能顺利表达。为进一步开展HRSV反向遗传学等研究奠定了基础。更多还原

【Abstract】 The n and p genes from subgroup A human respiratory syncytial virus (HRSV), were amplified by RT-PCR. The n product was cloned into pGEM-T easy vector and that of p into pcDNAII vector and the two genes were subcloned into the eukaryotic expression vector pcDNA3.1(+). The resulting recombinant plasmids pcDNA3.1(+)/N and pcDNA3.1(+)/P were checked by restriction enzymes and DNA sequencing and were tranfected into COS-7 cells with the aid of Lipofectamine 2000. Expression of N and P were detected by western blots at 72 hours post transfections. Expressed N and P in COS-7 cells was confirmed by western blot. The constructed expression vectors will be used for the further reverse genetics experimentations on HRSV.更多还原