表达绿色荧光蛋白的重组人偏肺病毒的构建及其中和抗体检测方法的应用

摘要

Human metapneumovirus (hMPV) is a newly identified pathogens, which remains poorly characterized and has been reported to replicate inefficiently in vitro. To construct and recovery recombinant hMPV (rhMPV-NL-EGFP) expressing green flurescent protein (GFP), BHK-21 cells were transfected with the full-length cDNA plasmid pPS-hMPV-EGFP, togethering with 4 major helper recombinant plasmids of CITE-N, pCITE-L, pCITE-P and pCITE-M2.1. The results demonstrated that infectious rhMPV was rescued by reverse genetics technique. rhMPV replicated in vero E6 cells as efficiently as the parental hMPV. The EGFP expression of rhMPV was stable for at least 10 passages in vero E6 cells. The ability to monitor EGFP expression in living cells greatly facilitated the detection of neutralization antibody using this recombinant virus, and the antibody titeration results were identical to that of indirect immunofluorescence assay (p>0.05). It should be feasible to construct the rhMPV vaccine virusexpressing foreign antigens. The results provides the basis for developing a live attenuated, multivalent hMPV vaccine.%人偏肺病毒(hMPV)是最新发现的与呼吸道疾病有关的病原体.为构建表达增强型绿色荧光蛋白(EGFP)的B型重组hMPV,本研究利用反向遗传学技术,以表达EGFP的hMPV NL/1/99株全长cDNA质粒重组pPS-hMPV-EGFP,及其4种辅助重组质粒pCITE-N、pCITE-P、pCITE-L、pCITE-M2.1为基础,共转染BHK-21细胞进行重组病毒的拯救.鉴定结果表明拯救的重组病毒rhMPV-NL-EGFP高效表达外源基因EGFP.rhMPV-NL-EGFP与亲本病毒株具有相似的生物学特性,生长滴度可达106.7 TCID50/mL.rhMPV-NL-EGFP在细胞中连续传代10代仍然维持外源基因的高效和稳定表达,病毒滴度于室温条件下可维持长时间稳定.分别利用亲本病毒株和重组病毒rhMPV-NL-EGFP进行免疫小鼠血清中和抗体检测,结果显示两种方法具有较好的相似性(p＞0.05).本研究为病毒中和抗体的检测提供了更为快捷的新方法,同时为hMPV感染发病机制及疫苗研究奠定了基础.