共表达口蹄疫病毒O型P1-2A-IL-18基因和Asia I型P1-2A-3C基因重组鸡痘病毒的构建

目的构建共表达口蹄疫病毒(FWDV)O型P1-2A-IL-18基因和AsiaI型P1-2A-3C基因的重组鸡痘病毒。方法将酶切得到的FWDVO型P1-2A-IL-18基因和AsiaI型P1-2A-3C基因克隆至鸡痘病毒表达载体pUTAL上,构建重组鸡痘病毒转移质粒pUTAL-P1-2A-3C-P1-2A-IL-18,与鸡痘病毒(FPV)282E4株共转染鸡胚成纤维细胞(CEF),通过BrdU3次加压筛选,挑选出单克隆重组病毒株,进行RT-PCR及间接免疫荧光(IFA)鉴定。结果重组鸡痘病毒转移质粒pUTAL-P1-2A-3C-P1-2A-IL-18经双酶切鉴定证明构建正确。经RT-PCR和IFA鉴定,证明所筛选的重组鸡痘病毒在CEF中能正确表达P1-2A-3C-P1-2A-IL-18基因盒。结论已成功构建了共表达FMDVO型P1-2A-IL-18基因和AsiaI型P1-2A-3C基因的重组鸡痘病毒。

Construction of Recombinant Fowlpox Virus for Co-expressing P1-2A-IL-18 Gene of Foot-and-mouth Disease Virus FMDV Type O and P1-2A-3C Gene of FMDV Type Asia I

Objective To construct the recombinant fowlpox virus (rFPV) for co-expressing the P1-2A-IL-18 gene of foot-and-mouth disease virus (FMDV) type O and P1-2A-3C gene of FMDV type Asia I. Methods Digest recombinant plasmids T-P1-2A-IL-18 and T-P1-2A-3C with restriction endonuclease, and clone the obtained P1-2A-IL-18 and P1-2A-3C genes into expression vector pUTAL. Chick embryo fibroblast(CEF) was co-transfected with the constructed recombinant plasmid pUTAL-P1-2A-3C-P1-2A-IL-18 and FPV, then monoclonal recombinant virus strains were selected by 3 cycles of BrdU pressure screening and identified by RT-PCR and indirect IFA. Results Restriction analysis proved that recombinant plasmid pUTAL-P1-2A-3C-P1-2A-IL-18 was constructed correctly. RT-PCR and indirect IFA showed that P1-2A-3C-P1-2A-IL-18 gene cassette was successfully expressed in CEF transfected with rFPV. Conclusion The rFPV for co-expressing the P1-2A-IL-18 gene of FMDV type O and P1-2A-3C gene of FMDV type Asia I was successfully constructed.