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为了构建改良型痘苗病毒安卡拉株表达系统可删除筛选标记的双表达穿梭载体,利用Cre/LoxP DNA重组系统以及本实验室表达Cre酶的BHK-21细胞系(BHK-Cre),以大肠杆菌黄嘌呤-鸟嘌呤磷酸核糖转移酶(Eco gpt)为筛选标记构建可删除筛选标记的双表达穿梭载体pLR-gpt。将Eco gpt基因以及调控其表达的启动子基因置于2个同向的LoxP位点之间,2个独立的多克隆位点位于2个LoxP位点之外,最终获得的重组病毒可以在BHK-Cre细胞系上删除筛选标记Eco gpt。为了验证系统的有效性,将PRRSV NJ-a株ORF5与ORF6基因分别克隆入pLR-gpt的2个多克隆位点中,构建重组转移载体pLRgpt-ORF5/ORF6。经脂质体介导,将pLRgpt-ORF5/ORF6转染已感染亲本MVA 2 h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-GP5/M。将rMVAgpt-GP5/M感染BHK-Cre,获得删除筛选标记的重组病毒rMVA-GP5/M,Western blotting与间接免疫荧光证明重组病毒能表达GP5与M蛋白,经PCR和病毒生长曲线鉴定,获得删除筛选标记的重组病毒rMVA-GP5/M已经完全删除筛选标记gpt。结果表明成功构建了痘苗病毒表达系统可删除筛选标记的双表达穿梭载体,并且外源基因的插入不影响亲本病毒的复制与外源基因的表达,具有较好的稳定性。
To construct an improved vaccinia virus Ankara strain expression system, a double-tagged shuttle vector was deleted. The Cre / LoxP DNA recombination system and BHK-Cre cell line (BHK-Cre) expressing Cre enzyme in our laboratory were used. Purine-guanine phosphoribosyltransferase (Eco gpt) A double-tagged shuttle vector, pLR-gpt, was constructed as a selectable marker for deletion of the selectable marker. The Eco gpt gene and the promoter gene regulating its expression are placed between two LoxP sites in the same orientation. Two independent multiple cloning sites are located outside the two LoxP sites. The resulting recombinant virus can be expressed in BHK Remove the selection marker Eco gpt on the -Cre cell line. To verify the effectiveness of the system, ORF5 and ORF6 genes of PRRSV NJ-a strain were cloned into two multiple cloning sites of pLR-gpt to construct recombinant transfer vector pLRgpt-ORF5 / ORF6. After being transfected with pLRgpt-ORF5 / ORF6 by liposome, the monolayer of BHK-21 cells that had been infected with the parental MVA for 2 hours was transfected with 24-well plates with a selective medium (MXHAT) for continuous screening and purification Screening of recombinant virus rMVAgpt-GP5 / M. The rMVAgpt-GP5 / M was infected with BHK-Cre, and the recombinant virus rMVA-GP5 / M with deletion marker was obtained. Western blotting and indirect immunofluorescence showed that the recombinant virus could express GP5 and M protein and identified by PCR and virus growth curve. The screened recombinant virus rMVA-GP5 / M has completely removed the selection marker gpt. The results showed that the vaccinia virus expression system could successfully delete the double-tagged shuttle vector, and the insertion of the exogenous gene did not affect the replication of the parental virus and the expression of the exogenous gene, and had a good stability.