狂犬病病毒GX074株P-M^(S20F)突变体构建及其生长特性鉴定  

Construction of rabies virus GX074 P-M^(S20F) mutant and identification of its growth characteristics

作　　者：彭璟 李文芳 陆丽莹 韦显凯[1,2] 李晓宁 罗廷荣[1,2] PENG Jing;LI Wen-fang;LU Li-ying;WEI Xian-kai;LI Xiao-ning;LUO Ting-rong(College of Animal Science and Technology,Guangxi University,Nanning,Guangxi 530004,China;State Key Laboratory for Conservation and Utilization of Subtropical Agro-bioresources,Nanning,Guangxi 530004,China)

机构地区：[1]广西大学动物科学技术学院,广西南宁530004 [2]亚热带农业生物资源保护与利用国家重点实验室,广西南宁530004

出　　处：《南方农业学报》2024年第1期253-262,共10页Journal of Southern Agriculture

基　　金：国家自然科学基金项目(32070161);广西自然科学基金项目(2020GXNSFAA297212)。

摘　　要：【目的】明确狂犬病病毒(RABV)强毒株GX074的P蛋白和M蛋白第20位苯丙氨酸(Phe)替换至弱毒株r RC-HL的重组突变体在宿主细胞内的生长特性,为P蛋白和M蛋白转录复制机理研究提供理论依据。【方法】运用反向遗传技术以强毒株GX074的M蛋白第20位Phe替换弱毒株r RC-HL(GX074P)的M蛋白第20位丝氨酸(Ser),构建r RC-HL(GX074P-M^(S20F))突变体感染性c DNA克隆并拯救病毒。以重组突变体感染BSR/T7-9细胞,进行多步生长曲线测定,比较重组突变体与亲本毒株的生长能力,采用Western blotting检测N蛋白、P蛋白和M蛋白相对表达水平,利用实时荧光定量PCR检测N基因、P基因和M基因的相对表达量。【结果】经RT-PCR和测序鉴定,拯救的突变体r RC-HL(GX074P-M^(S20F))M蛋白第20位Ser成功替换为Phe。多步生长曲线测定结果显示,构建的重组突变体在感染24、48、72和96 h后,病毒滴度均高于亲本弱毒株r RC-HL和对照弱毒株r RC-HL(GX074PM1),平均约为亲本弱毒株r RC-HL的10倍。在蛋白表达水平上,r RC-HL(GX074P-M^(S20F))毒株在感染48 h后,N蛋白和M蛋白相对表达水平均极显著高于亲本弱毒株r RC-HL(P<0.01),说明强毒株GX074的P蛋白联合M蛋白第20位Phe替换至弱毒株r RC-HL后,增加了突变体N蛋白和M蛋白表达。感染24和48 h后,r RC-HL(GX074P-M^(S20F))毒株N基因、P基因和M基因的相对表达量均高于亲本弱毒株r RC-HL和对照弱毒株r RC-HL(GX074PM1)。【结论】强毒株GX074的M蛋白第20位Phe替换可提高病毒的增殖能力,同时增强病毒的复制与转录能力,M蛋白第20位Phe可能是影响病毒复制和转录的关键位点。【Objective】To clarify the growth characteristics of the recombinant mutant with the replacement of P pro-tein and the 20th phenylalanine(Phe)of M protein of the strong strain of rabies virus(RABV)strain GX074 to the attenua-ted strain rRC-HL in host cells,and to provide theoretical basis for the study of the transcription and replication mecha-nism of the P and M proteins.【Method】The 20th serine(Ser)of M protein of attenuated strain rRC-HL(GX074P)was re-placed by the 20th Phe of M protein of strong strain GX074 by reverse genetic technique to construct the infectious cDNA clone of rRC-HL(GX074P-M^(S20F))mutant and rescue the virus.BSR/T7-9 cell was infected by recombinant mutant,and multi-step growth curve determination was performed to compare the growth ability of the recombinant mutant and that of the parent strain.The relative expression levels of N,P and M proteins were detected by Western blotting,and the rela-tive expression levels of N,P and M genes were detected by real-time fluorescence quantitative PCR.【Result】RT-PCR and sequencing confirmed that the 20th Ser of the M protein of the rescued mutant rRC-HL(GX074P-M^(S20F))was success-fully replaced by the 20th Phe.The results of the multiple-step growth curve demonstrated that the viral titer of the recombi-nant mutant was higher than that of both the parental attenuated strain rRC-HL and the control attenuated strain rRC-HL(GX074PM1)at 24,48,72 and 96 h after infection,with 10 times of the parent attenuated rRC-HL.In terms of protein expression,the relative expression levels of N protein and M protein of rRC-HL(GX074P-M^(S20F))strain were extremely significantly higher than those of the parental attenuated strain rRC-HL at 48 h after infection(P<0.01).These results indi-cated that the substitution of the P protein of strong strain GX074 with the 20th Phe of M protein to the attenuated strain rRC-HL increased the expression of N and M proteins in the mutant.The relative expression levels of N,P and M genes of rRC-HL(GX074P-M^(S20F))strain wer

关 键 词：狂犬病病毒(RABV) P蛋白 M蛋白 生长特性 反向遗传 

分 类 号：S852.659.5[农业科学—基础兽医学]