共表达PRRSV修饰的GP5与M蛋白的重组改良型痘苗病毒安卡拉株的构建及其生物学特性

将猪繁殖与呼吸综合征病毒(Porcine reproductive and respiratory syndrome virus, PRRSV)修饰的ORF5(MORF5)与ORF6基因分别克隆入笔者构建的改良型痘苗病毒安卡拉株(Modified vaccinia virus ankara, MVA) 转移载体pLR-gpt的两个多克隆位点中, 构建重组转移载体pLR-MORF5/ORF6。经脂质体介导, 将构建好的pLR-MORF5/ORF6 转染已感染亲本MVA 2h的BHK-21细胞单层, 用药物选择性培养基(MXHAT)在24孔板上进行连续筛选纯化, 得到带有筛选标记的重组病毒rMVAgpt-MGP5/M。将rMVAgpt-MGP5/M 感染表达Cre重组酶的BHK-21细胞(BHK-Cre), 获得删除筛选标记的重组病毒rMVA-MGP5/M。PCR证明MORF5与ORF6成功插入MVA基因组中; Western blotting与间接免疫荧光证明重组病毒能表达MGP5与M蛋白; 重组病毒的生长特性表明与亲本MVA出现病变的时间及病毒滴度基本一致。结果表明, 本研究成功构建了共表达PRRSV MGP5与M蛋白的重组MVA, 并且表达产物具有免疫原性; 外源基因的插入不影响MVA复制, 具较好的稳定性, 从而为PRRSV新型基因工程疫苗的研制奠定了良好的基础。

Construction and Biological Characteristic for the Recombinant Modified Vaccinia Virus Ankara Co-expressing Modified GP5 and M Protein of Porcine Reproductive and Respiratory Syndrome Virus

Modified ORF5 (MORF5) and ORF6 gene of PRRSV were cloned into two multiple cloning sites of MVA transfer vector pLR-gpt to construct the recombinant plasmid pLR-MORF5/ORF6. Homologous recombination between pLR-MORF5/ORF6 and the wtMVA on BHK-21 cell line was mediated with liposome by infecting the cell with 0.01 MOI wtMVA two hours before transfecting the recombinant plasmid into the cell. When the cytopathic effect (CPE) was obvious, virus was collected from the cell plate and the recombinant virus was selected with drug selecting medium (2% MXHAT). After 12 cycles of selection, rMVA with a selection marker Eco gpt was obtained and named as rMVAgpt-MGP5/M. By infecting BHK-Cre expressing Cre recombinant enzyme, the Eco gpt marker in rMVAgpt-MGP5/M was deleted and this rMVA was named as rMVA-MGP5/M. The insertion of MORF5 and ORF6 into the MVA genome was confirmed with PCR analysis and the expression of MGP5 and M protein was identified with Western blot and IFA. Through biological study on the recombinant MVA, no obvious difference was observed between rMVA-MGP5/M and the wtMVA regarding to the CPE and growth curve. The recombinant MVA constructed in this study could coexpress the modified GP5 and M protein and the expressed product had good immunocompetence. Furthermore, the insertion of the MORF5 and ORF6 into MVA genome had no obvious effect on the replication and biological characteristics of this virus.