表达兔出血症病毒VP60蛋白的重组兔粘液瘤病毒的构建与初步评价

Generation and evaluation of a recombinant myxomavirus expressing the VP60 protein of rabbit haemorrhagic disease virus

兔出血症病毒(Rabbit hemorrhagic disease virus,RHDV)及兔粘液瘤病毒(Myxoma virus,MYXV)分别引起兔出血症(兔瘟)和兔粘液瘤病,是两种严重危害家兔养殖业以及导致原产地欧洲野兔-穴兔(Oryctolagus cuniculus)种群近濒危的重要病原。VP60为构成RHDV衣壳的主要抗原蛋白。为研制能同时免疫预防该两种疫病的重组二联疫苗,本研究分别以MYXV和其复制非必需基因——胸腺激酶(Thymidine kinase,TK)基因为重组载体和同源重组靶基因,构建穿梭载体p7.5-VP60-GFP。将p7.5-VP60-GFP载体转染被MYXV感染的兔肾细胞株RK13,经同源重组后,在荧光显微镜下筛选出表达GFP的重组病毒,并将其命名为rMV-VP60-GFP。通过PCR和Western blotting进行重组病毒vp60基因特异性插入和表达验证结果显示,vp60和gfp基因成功插入MYXV基因组中并且可成功表达,表明成功构建了表达RHDV衣壳蛋白基因vp60的重组MYXV。动物攻毒保护试验表明,制备的重组病毒能保护家兔抵抗MYXV的致死性攻击,这为后续疫苗的研发奠定了基础。

Rabbit haemorrhagic disease virus(RHDV)and myxoma virus(MYXV),are two pathogens that have harmful effect on rabbit breeding and population decline of European rabbits in their native range,causing rabbit haemorrhagic disease(rabbit fever)and myxomatosis,respectively.The capsid protein VP60 of the RHDV represents the major antigenic protein.To develop a recombinant bivalent vaccine candidate that can simultaneously prevent these two diseases,we used the nonessential gene TK(thymidine kinase)of MYXV as the insertion site to construct a recombinant shuttle vector p7.5-VP60-GFP expressing the RHDV major capsid protein(VP60)and the selectable marker GFP.Then the shuttle vector p7.5-VP60-GFP was transfected into rabbit kidney cell line RK13 which was previously infected with MYXV.After homologous recombination,the recombinant virus expressing GFP was screened under a fluorescence microscope and named as rMV-VP60-GFP.Finally,the specific gene-knock in and expression verification of the vp60 and gfp genes of the recombinant virus was confirmed by PCR and Western blotting.The results showed that these two genes were readily knocked into the MYXV genome and also successfully expressed,indicating that the recombinant MYXV expressing the vp60 of RHDV was generated.Protection against MYXV challenge showed that the recombinant virus induced detectable antibodies against MYXV which would shed light on development of the effective vaccine.