表达非洲猪瘟病毒CD2v与P12蛋白的重组伪狂犬病毒的构建

Construction of Recombinant Pseudorabies Virus Expressing CD2v and P12 Proteins of African Swine Fever Virus

非洲猪瘟(African swine fever,ASF)与伪狂犬病(pseudorabies,PR)分别由非洲猪瘟病毒(African swine fever virus,ASFV)与伪狂犬病毒感染(Pseudorabies virus,PRV)引起的猪(或部分猪)高致死、传染性疾病。目前非洲猪瘟无商业化疫苗,两种疾病均给养猪业造成了巨大的经济损失。本研究以PRV-Fa经典毒株为载体通过CRISPR/Cas9(clustered regularly interspaced short palindromic repeats/Cas9)基因编辑技术将ASFV(Pig/HLJ/18)CD2v(EP402R)与p12(O61R)基因分别插入PRV-Fa株TK(UL23)及gI(US7)基因位点中,成功构建TK及gI缺失且重组表达ASFV CD2v与P12的重组弱毒株PRV-∆gI(P12)-∆TK(CD2v)。通过基因测序、蛋白质免疫印迹(Western blot)与免疫荧光表明重组毒株遗传性状稳定且CD2v与P12在Vero细胞中稳定表达。通过一步生长曲线、噬斑生长测定、小鼠毒力测试及小鼠病理学评估表明与野生型毒株PRV-Fa相比重组毒株毒力明显减弱,对小鼠不具致死能力,该重组毒株为研究预防PR及ASF的新型疫苗提供了新的选择。

ASF(African swine fever)and PR(pseudorabies)both are highly lethal and infectious diseases for pigs infected by African swine fever virus(ASFV)and pseudorabies virus(PRV),respectively.Both diseases have caused huge economic losses to the pig breeding industry and no commercial vaccine is available for preventing African swine fever.In this study,we inserted the ASFV(Pig/HLJ/18)CD2v(EP402R)and p12(O61R)genes into the PRV-Fa TK(UL23)and gI(US7)gene loci,respectively by CRISPR/Cas9 technology and classical viral strain PRV-Fa,and successfully constructed a recombinant attenuated strain PRV-∆gI-(P12)-∆TK-(CD2v)with deletion of TK and gI as well as expression of CD2v and P12.Gene sequencing,Western blotting and immunofluorescence analyses showed that the genetic traits of the recombinant strain were stable,and the CD2v and P12 were stably expressed in Vero cells.The one-step growth curve,plaque growth assay,mouse virulence test and pathological evaluation indicated that the virulence of the recombinant strain was significantly attenuated and not lethal to mice compared with the wild-type strain PRV-Fa.Therefore,the recombinant strain will become a new selection for researching of vaccine to prevent PR and ASF.