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  

Generation of a Recombinant Fowlpox Virus Co-expressing Foot-and-Mouth Disease Virus P1-2A Gene and Porcine Interleukin -18

作　　者：刘慧娟[1] 金宁一[2] 马鸣潇[1] 金明兰[1] 张林[1] 李旭[1] 计越[1] 鲁会军[2] 金扩世[1] 

机构地区：[1]吉林大学畜牧兽医学院,长春130062 [2]中国人民解放军军事医学科学院军事兽医研究所全军基因工程重点实验室,长春130062

出　　处：《中国生物工程杂志》2006年第9期20-23,共4页China Biotechnology

基　　金：国家"863"计划资助项目(2001AA213071);吉林省科技发展计划资助项目(20040202-1)

摘　　要：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联接,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。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-3 C-P1-2A-IL-18 strains could express 3 C-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.

关 键 词：口蹄疫病毒 鸡痘病毒活载体 P1-2A基因 IL-18基因 3C基因 

分 类 号：Q786[生物学—分子生物学]