共表达O型口蹄疫病毒P1-2A基因和猪白细胞介素18的重组鸡痘病毒的构建

目的:为获得能有效预防O型口蹄疫病毒的重组鸡痘病毒活载体疫苗奠定基础。方法：在O型口蹄疫病毒P1-2A基因上游引入Kozak序列，下游通过Linker与细胞因子IL-18联结，获得P1-2A基因与猪IL-18基因融合表达基因盒P1-2A-IL-18，将该表达基因盒克隆至鸡痘病毒中间转移载体pUTAL-3C中，构建重组鸡痘病毒转移载体质粒pUTAL-3C- P1-2A-IL-18。通过脂质体转染法，将pUTAL-3C- P1-2A-IL-18与鸡痘病毒282E4株共转染鸡胚成纤维细胞（CEF），通过BrdU三次加压筛选，挑选出单克隆重组病毒株。结果：经RT-PCR和间接免疫荧光法鉴定，证明所筛选的1株重组鸡痘病毒在CEF中能正确表达P1-2A-IL-18基因盒。结论：成功获得了一株共表达O型口蹄疫病毒P1-2A基因和猪白细胞介素18基因的重组鸡痘毒疫苗候选株rFPV-3C-P1-2A-IL-18。

Generation of a recombinant fowlpox virus co-expressing Foot-and-Mouth Disease virus P1-2A gene and porcine interleukin 18

Objective:In order to prevent the O-FMDV, a recombinant FPV which can co-expressing foot-and-mouth disease virus P1-2A gene and porcine interleukin -18 as the vaccine was constructed and screened. Methods:Two primers for amplifying the O-FMDV P1-2A gene were desigined:the Kozak sequence was inserted into the upstream primer;the linker was inserted into the downstream primer. Then pIL-18 gene was connected to P1-2A gene in order to construct the expression gene box, which named as P1-2A-IL-18. The gene box was inserted into the FPV transfer vector pUTA-16-LacZ-3C to construct recombinant transfer vector plasmid pUTAL-3C-P1-2A-IL-18.The recombinant transfer vector plasmid was cotransfected with 282E4 fowlpox viruses into the CEF. The recombinant fowlpox viruses-3C-P1-2A-IL-18 (rFPV-3C- P1-2A-IL-18) was selected 3 passages by culturing in CEF with MEM medirum containing BrdU. The selected viruses were plaque-purified in CEF without BrdU. After RT-PCR and IFA analysis, the recombinant FPVs expressing gene box P1-2A-IL-18 was obtained. Results:The rFPV-3C-P1-2A-IL-18 strains could express 3C-P1-2A gene and pIL-18 gene correctly. Conclusion:It indicated that a recombinant fowlpox viruses rFPV-3C-P1-2A-IL-18 which can co-expressing foot-and-mouth disease virus P1-2A gene and porcine interleukin 18 was successfully obtained.