CAJ | 학술논문

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。
위구건Asia1형구제역중조계두병독역묘,채집구제역발병우적수포액급수포피,용RT-PCR법확증출Asia 1형구제역병독적전체단백기인P1-2A편단화단백매기인3C편단,분별극륭지pMD18-T재체상,통과매절련접획득질립pMD18-T-P1-2A-3C。재장P1-2A-3C편단여pUTAL-IL18편단련접기래,구건계두병독중간전이질립pUTAL-P1-2A-3C-IL18。통과지질체전염법,장pUTAL-P1-2A-3C-IL18여계두병독282E4주공감염염계배성섬유세포（chicken embryo fibroblasts,CEF）,통과BrdU삼차가압사선,사선출중조계병독주vUTAL-P1-2A-3C-IL18。경RT-PCR화간접면역형광법감정,증명소사선적1주중조계두병독재CEF중능정학표체P1-2A-3C기인。본연구위연제안전、고효적아주Ⅰ형FMD중조계두병독역묘전정료기출。
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.