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

A novel double expression shuttle vector to get marker-free recombinant modified vaccinia virus Ankara

摘要:

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

Abstract:

A novel double expression shuttle vector named pLR-gpt was constructed for marker-free recombinant modified vaccinia virus Ankara generation. A delectable Eco gpt marker was adopted with Cre/LoxP DNA recombination system and a BHK-21 cell line that can express Cre enzyme. Eco gpt gene controlled by P7.5 promoter from Vaccinia virus was cloned between two LoxP sites in the same direction. Additionally, two multiple cloning site under control of other two Vaccinia virus promoters were constructed outside LoxP sites. With this new transfer vector, Eco gpt marker in rMVA can be deleted on BHK-Cre with interaction between Cre enzyme and LoxP sequence. In order to verify the efficacy of this system, ORF5 and ORF6 gene of Porcine reproductive and respiratory syndrome virus (PRRSV) NJ-a strain were cloned into two multiple cloning sites of pLR-gpt to construct recombinant plasmid pLR-ORF5/ORF6. Homologous recombination between pLR-ORF5/ORF6 and wtMVA on BHK-21 cell was mediated by liposome by infecting cells with 0.01 MOI wtMVA two hours before transfection. After twelve cycles of selection, recombinant MVA with selecting marker Eco gpt was obtained and named as rMVAgpt-GP5/M. By infecting BHK-Cre, the Eco gpt marker in rMVAgpt-GP5/M was deleted and this rMVA was named as rMVA-GP5/M. Expression of GP5 and M protein was identified with Western blotting and IFA. Results from PCR and biological study for rMVA indicated that Eco gpt marker was completely deleted. Conclusions: double expression transfer vector for marker-free recombinant Modified vaccinia virus Ankara generation was successfully constructed, and works well in MVA expression system.