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  

Construction of recombinant human metapneumovirus expressing green fluorescent protein for the detection of virus neutralization antibodies

作　　者：李晓艳[1,2] 葛金英[3] 哈斯苏荣[1] 王琪[3] 焦丽红[3] 王永[2] 步志高[3] 

机构地区：[1]内蒙古农业大学兽医学院,内蒙古呼和浩特010018 [2]哈尔滨医科大学公共卫生学院,黑龙江哈尔滨150081 [3]中国农业科学院哈尔滨兽医研究所兽医生物技术国家重点实验室/农业部兽医公共卫生重点开放实验室,黑龙江哈尔滨150001

出　　处：《中国预防兽医学报》2011年第9期665-670,共6页Chinese Journal of Preventive Veterinary Medicine

基　　金：国家自然科学基金(青年科学基金项目:30800984)

摘　　要：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。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 virus expressing foreign antigens.The results provides the basis for developing a live attenuated,multivalent hMPV vaccine.

关 键 词：人偏肺病毒 反向遗传技术 病毒拯救 绿色荧光蛋白 

分 类 号：S852.65[农业科学—基础兽医学]