型内嵌合口蹄疫病毒全长感染性cDNA克隆的构建

【目的】近年来,O型口蹄疫的不断暴发严重危害了我国畜牧业的发展,其病原——O型口蹄疫病毒已演化出3种谱系:中国型猪毒系、泛亚系和缅甸98系。其中中国型猪毒系病毒高度嗜猪,对养猪业危害最大。目前应用的疫苗已不能有效保护中国型猪毒系变异株的流行,这给我国猪口蹄疫的防控带来了极大的困难。为了进一步发展免疫原性好、抗原谱广的猪O型口蹄疫疫苗候选株,本研究以O/HN/93现用疫苗毒株的感染性克隆为骨架,用流行的新猪毒系病毒的部分VP3和VP1基因(主要是替换VP1蛋白上的B-C环和G-H环)替换疫苗毒株的相应部分,构建了嵌合的FMDV全长cDNA克隆。【方法】线化的嵌合全长质粒和表达T7 RNA聚合酶的真核质粒pcDNAT7P共转染BHK-21细胞,体内转录拯救嵌合病毒。【结果】嵌合全长质粒转染BHK-21细胞36h后,出现明显的FMDV致细胞病变效应。对收获的病毒分别用RT-PCR、间接免疫荧光、电子显微镜观察结果证实成功拯救到嵌合的FMDV。拯救的病毒乳鼠致病性试验结果表明该拯救病毒对乳鼠的致病力减弱。该嵌合病毒的成功拯救为研制口蹄疫新型疫苗等奠定了基础。

Construction of a full-length infectious cDNA clone of inter-genotypic chimeric foot-and-mouth disease virus

[Objective]A series of type O foot-and-mouth disease(FMD) outbreaks occurred in China seriously affect development of Chinese husbandry.Its causative agent—type O foot-and-mouth disease virus(FMDV) has been evolved three topotypes: Cathay,Middle East-South Asia and Southeast Asia,specifically,the viruses of Cathay topotype are highly adapted to pig,representing the biggest threat on Chinese pig industry.The available FMD vaccine in China provides insufficient protection against some arising viruses of Chathay topotype,which exert important obstacle to control porcinophilic FMD epidemics in China.To develop vaccine candidate with characteristics of good immunogenicity and broad spectrotype,a full-length infectious cDNA clone of inter-genotypic chimeric FMDV was constructed,which replaced part VP3 and VP1 gene of O/HN/93 strain with the corresponding to the variants of Cathay topotype(mainly replaced the B-C and G-H loop of structure protein VP1).[Methods] Linearized recombinant plasmid and plasmid expressing T7 RNA polymerase were cotransfected into BHK-21 cells to rescue the chimeric virus in vivo.[Result] The transfected cells showed apparent cytopathic effects(CPE) after 36 h post-transfection.The rescued virus was checked by RT-PCR,indirect immunofluorescence,electron microscope.Results show that chimeric FMDV was successfully rescued in vivo.The study of sulk mice pathogenicity show chimeric FMDV has attenuated pathogenicity for suckling mice compared with its parental virus.[Conclusion] The construction of inter-genotypic chimeric FMDV will lay the basis for developing novel vaccine against FMD.