人呼吸道合胞病毒微型基因组拯救系统的建立及功能鉴定

Establishment and functional identification of minigenome rescue system of human respiratory syncytial virus

目的建立人呼吸道合胞病毒(human respiratory syncytial virus,hRSV)微型基因组拯救系统,并鉴定其功能。方法利用包含T7启动子、锤头状核酶、hRSV的前导序列、基因起始信号、非编码区、多克隆位点、基因终止信号、尾随序列、丁肝核酶、T7终止子的基本载体pRSV1和pRSV2,与增强型绿色荧光蛋白(enhanced green fluorescent protein,eGFP)基因构建微型基因组质粒pRSV1-eGFP和微型反基因组质粒pRSV2-eGFP。以pcDNA3.1(+)为表达载体,与优化后的RSV的核蛋白(N)、磷蛋白(P)、聚合酶大片段(L)和转录延长/终止抑制因子(M2-1)基因序列构建辅助质粒pcDNA-N、pcDNA-P、pcDNA-L和pcDNA-M2-1。将微型基因组质粒或微型反基因组质粒通过单转染、与RSV共感染或与辅助质粒共转染至表达T7 RNA聚合酶(T7 RNP)的BSR-T7/5细胞,荧光显微镜或流式细胞仪观察eGFP的表达,并鉴定微型基因组的功能及辅助蛋白的生物学活性。结果微型基因组质粒、微型反基因组质粒和4个辅助质粒经双酶切及测序证明构建正确;微型基因组质粒通过先感染后转染或与辅助质粒共转染,观察到eGFP的表达,证实了微型基因组的功能活性;缺乏N、P或L蛋白,检测不到eGFP的表达;缺乏M2-1蛋白,eGFP的表达量降低。结论成功建立了RSV微型基因组拯救系统,并证实了其拯救功能,为利用反向遗传学手段拯救重组RSV,研发RSV减毒活疫苗奠定了基础。

Objective To establish the minigenome rescue system of human respiratory syncytial virus(hRSV)and identify its function.Methods The basic vectors pRSV1 and pRSV2 were available,which harbored T7 promoter,hammerhead ribozyme,leader sequence of h RSV,gene start signal,non-coding sequence,multiple clone site(MCS),gene end signal,trailer sequence,hepatitis virus ribozyme and T7 terminator.Minigenome plasmid and mini-antigenome plasmid were constructed by ligating pRSV1 or pRSV2 to enhanced green fluorescence protein(eGFP)gene.Helper plasmids pcDNA-N,pcDNA-P,pcDNA-L and pcDNA-m2-1 were constructed by using vector pcDNA3.1(+)and the optimized nucleoprotein(N),phosphoprotein(P),polymerase large fragment(L)and transcription elongation/termination inhibition factor(M2-1)respectively.BSR-T7/5 cells expressing T7 RNA polymerase(T7 RNP)were transfected with minigenome plasmid or mini-antigenome plasmid alone,after infection with RSV and in combination with helper plasmids respectively,and observed for expression of eGFP by fluorescent microscopy and flow cytometry to confirm the function of minigenome and biological activity of helper proteins.Results Restriction analysis and sequencing proved that the minigenome plasmid,mini-antigenome plasmid and four helper plasmids were constructed correctly.The expression of eGFP was observed in the cells transfected with minigenome plasmid after RSV infection or in combination with helper plasmids,which proved the functional activity of minigenome rescue system.No eGFP expression was detected in absence of N,P or L protein,while the expression level decreased in absence of M2-1 protein.Conclusion The minigenome rescue system was established successfully,of which the rescue function was confirmed.It laid a foundation of rescue of recombinant RSV and development of live attenuated RSV vaccine by reverse genetic technique.