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.
出处
《中国动物传染病学报》
CAS
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)
