马传染性贫血病毒疫苗株S2基因不同变异位点逆向突变感染性克隆的体外复制特性分析  

In vitro replication analysis of infectious clones of the Chinese attenuated infectious anemia virus vaccine strain reverse-mutated in the S2 gene

作　　者：高旭[1,2] 姜成刚[1] 高华[1] 林跃智[1] 赵立平[1] 相文华[1] 周建华[1] 

机构地区：[1]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大动物病研究室,黑龙江哈尔滨15000 [2]延边大学农学院动物医学系,吉林延吉133002

出　　处：《中国预防兽医学报》2009年第12期919-924,共6页Chinese Journal of Preventive Veterinary Medicine

基　　金：国家自然科学基金(30771994);十一五重大传染病专项(2008ZX1001-1010);哈尔滨市科技攻关计划项目(2006AA3AS040)

摘　　要：为研究EIAV弱毒疫苗株(EIAVFDDV15)S2基因发生的稳定性突变对疫苗株特性的作用以及S2基因的功能,以EIAVFDDV15感染性克隆质粒pFDDV3-8为模板,根据疫苗研制过程中S2基因发生的4个主要稳定性变异位点,构建及拯救出S2基因不同差异位点逆向突变为强毒株相应氨基酸的6株感染性克隆衍生毒株。经实时定量PCR、逆转录酶活性和western blot等检测表明,疫苗株S2基因4个稳定性变异位点全部逆向突变的感染性克隆衍生毒vpFDDVS2r1-3-4-5在体外培养靶细胞中的复制水平低于亲本疫苗株感染性克隆衍生病毒和其他组合的逆向突变的感染性克隆衍生病毒,提示EIAV疫苗株S2蛋白4个稳定突变位点的综合作用可能是决定疫苗株和强毒株特性差异的因素之一。以上结果为体内感染S2基因逆向突变感染性克隆衍生病毒,进一步揭示S2基因在EIAV弱毒疫苗致弱过程中的作用提供了依据。Our previous study identified four stable mutation sites in the S2 of Chinese EIAV attenuated vaccine strains occurred during the attenuation process. To investigate the effects of these stable mutations on the attenuated virulence of EIAV vaccine strains and the function of S2, the four stable mutation sites in this gene of pFDDV3-8, an infectious clone of the EIAV vaccine strain EIAVFDDV15, were reverse-mutated. Six S2 reverse-mutated infectious clones were constructed, containing different combinations of reverse-mutated sites. The in vitro replication properties of viruses rescued from these infectious clones were detected by real-time PCR, activity of reverse transcriptase and western blot. Results revealed that of these six viruses, only the virus vpFDDVS2r1-3-4-5, which contains all the four reverse-mutated sites, demonstrated a delayed dynamic replication rate when compared with the parental virus pFDDV3-8, This result indicates that the synergetic effect of all the four stable-mutated sites in the S2 protein of the EIAV vaccine strains may contribute partially for the attenuated virulence of the vaccine. Additionally, this study provides necessary information for in vivo investigation on these S2 reverse-mutated viruses for the intensive study of the S2 gene.

关 键 词：马传染性贫血病毒 S2基因 逆向突变 感染性克隆 复制特性 

分 类 号：S852.65[农业科学—基础兽医学]