CAJ | 학술논문

为研究中国马传染性贫血病毒(EIAV)弱毒疫苗致弱过程中基因组的进化特征,作者对EIAV_(FDDV)前病毒的囊膜基因env进行了分析.使用PCR方法体外扩增EIAV_(FDDV)前病毒DNA的env,并随机挑取PCR的阳性克隆子进行测序和序列比对分析.结果表明,随机挑取的PCR阳性克隆中,29/30的克隆子在第2128-2130位核苷酸处存在TGG→TGA(翻译中止密码)的突变.该基因对应的EIAV穿膜蛋白gp45的氨基酸数在突变株中较未截短株中减少154,变成259个aa.对EIAV_(FDDV)进行Western blot分析时发现,EIAV_(DLV)45 ku的gp45条带被约35 ku的条带取代.由此推测,EIAV_(FDDV)疫苗株绝大多数病毒颗粒的gp45是截短型.对该代次疫苗株免疫马匹第15和40天体内EIAV前病毒DNA和基因组RNA的序列进行分析,结果表明该截短毒株具有在体内和体外进行复制的能力.由于已有研究表明细胞嗜性改变与gp45的截短有关,因此,推断EIAV_(FDDV)的gp45的截短是其适应在体外培养的驴胎皮肤细胞上复制传代的结果.综上所述,本研究发现EIAV_(FDDV)的gp45存在高比例截短突变,证明该截短突变毒株具有在体内和体外进行复制的能力.但该截短突变是否可通过改变EIAV_(FDDV)在免疫马体内的细胞嗜性,进而影响其毒力和免疫原性,还有待进一步验证.
위연구중국마전염성빈혈병독(EIAV)약독역묘치약과정중기인조적진화특정,작자대EIAV_(FDDV)전병독적낭막기인env진행료분석.사용PCR방법체외확증EIAV_(FDDV)전병독DNA적env,병수궤도취PCR적양성극륭자진행측서화서렬비대분석.결과표명,수궤도취적PCR양성극륭중,29/30적극륭자재제2128-2130위핵감산처존재TGG→TGA(번역중지밀마)적돌변.해기인대응적EIAV천막단백gp45적안기산수재돌변주중교미절단주중감소154,변성259개aa.대EIAV_(FDDV)진행Western blot분석시발현,EIAV_(DLV)45 ku적gp45조대피약35 ku적조대취대.유차추측,EIAV_(FDDV)역묘주절대다수병독과립적gp45시절단형.대해대차역묘주면역마필제15화40천체내EIAV전병독DNA화기인조RNA적서렬진행분석,결과표명해절단독주구유재체내화체외진행복제적능력.유우이유연구표명세포기성개변여gp45적절단유관,인차,추단EIAV_(FDDV)적gp45적절단시기괄응재체외배양적려태피부세포상복제전대적결과.종상소술,본연구발현EIAV_(FDDV)적gp45존재고비례절단돌변,증명해절단돌변독주구유재체내화체외진행복제적능력.단해절단돌변시부가통과개변EIAV_(FDDV)재면역마체내적세포기성,진이영향기독력화면역원성,환유대진일보험증.
To investigate the genomic evolution of EIAV vaccine strains during attenuation,we analyzed the sequence of env in provirus of EIAV_(FDDV).After amplifying env in provirus DNA of EIAV_(FDDV) by using PCR,some positive T-A clones were selected randomly to sequence and analyze.A mutation from TGG to TGA,a premature stop codon,was detected at the position of 2128-2130 nt of the env in 29 of 30 randomly selected clones of env.This mutation causes a truncated transmembrane protein (TM,also termed as gp45) at the 262th aa residue that resulted this mu-tated glycoprotein 154 residues shorter than the wild gp45.Indeed,we can see the band of predicted truncated gp45 when EIAV_(FDDV) was analyzed by Western blot.To investigate the status of EIAV with the truncated gp45 in vivo ,the env sequence of peripheral blood mononuclear cells(PBMC) associated provirus DNA and circulating virions in EIAV_(FDDV)-vaccinated horses were analyzed.TGG to TGA mutation were both found in integrated provirus DNA and the genome of the circulating virions.The above results indicate that the virion with the truncated gp45 owns the ability to infect the target cell and replicate in vivo.Extend studies are needed to understand the contribution of the truncated gp45 to immunogenicity and virulence of attenuated EIAV vaccine strains.