共表达Asia1口蹄疫病毒P1-2A-3C基因和猪IL-18基因重组鸡痘病毒构建及表达  

CONSTRUCTION AND EXPRESSION OF RECOMBINANT FOWLPOX VIURS CONTAINING P1-2A-3C GENE OF FOOT-AND-MOUTH DISEASE VIRUS TYPE ASIA1 AND SWINE IL-18 GENE

作　　者：鲁会军[1] 霍晓伟[1,2,3] 任静强[1,2] 常巧呈[1,2] 金扩世[1,2] 金宁一[1,2] 

机构地区：[1]军事医学科学院军事兽医研究所吉林省人兽共患病预防与控制重点实验室,长春130062 [2]吉林大学畜牧兽医学院,长春130062 [3]内蒙古民族大学动物科技学院,通辽028000

出　　处：《中国动物传染病学报》2011年第6期30-35,共6页Chinese Journal of Animal Infectious Diseases

基　　金：吉林省科技支撑重点项目(20090235)

摘　　要：为构建Asia1型口蹄疫重组鸡痘病毒疫苗,采集口蹄疫发病牛的水泡液及水泡皮,用RT-PCR法扩增出Asia 1型口蹄疫病毒的前体蛋白基因P1-2A片段和蛋白酶基因3C片段,分别克隆至pMD18-T载体上,通过酶切连接获得质粒pMD18-T-P1-2A-3C。再将P1-2A-3C片段与pUTAL-IL18片段连接起来,构建鸡痘病毒中间转移质粒pUTAL-P1-2A-3C-IL18。通过脂质体转染法,将pUTAL-P1-2A-3C-IL18与鸡痘病毒282E4株共感染染鸡胚成纤维细胞(chicken embryo fibroblasts,CEF),通过BrdU三次加压筛选,筛选出重组鸡病毒株vUTAL-P1-2A-3C-IL18。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-3C基因。本研究为研制安全、高效的亚洲Ⅰ型FMD重组鸡痘病毒疫苗奠定了基础。In order to construct the recombinant fowlpox virus vaccine against Foot-and-mouth disease virus （FMDV） type Asial, the structural protein precursor P1-2A gene and nonstructural protein 3C （proteinase） gene fragments of FMDV type Asial were amplified by RT-PCR from vesicular fluid of cattle. The P1-2A and 3C genes were inserted to vector pMD 18-T, respectively. The plasmid pMD18-T-P1-2A-3C was obtained by digestion with restriction endonuclease. Then, the fragment P1-2A-3C was inserted into the fowlpox virus shuttle plasmid pUTAL-IL18 under promoter ATI-P7.5 downstream to construct fowlpox virus shuttle plasmid pUTAL-P1-2A-3C-IL18, which was used to co-transfect into CEF with 282E4 fowlpox virus. The recombinant fowlpox virus P1-2A-3C-IL18 （rFPV-P1-2A-3C-IL18） was cloned 3 times in CEF with BrdU. The expression of P1-2A-3C-IL18 in the recombinant fowlpox virus was confirmed in RT-PCR and IFA. This study provided useful information for development of a safe and effective recombinant FPV vaccine against FMDV type Asia i.

关 键 词：Asia1口蹄疫病毒 鸡痘病毒载体 P1-2A基因 3C基因 IL-18基因 表达 

分 类 号：S852.659.6[农业科学—基础兽医学] Q7[农业科学—兽医学]