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当前位置: 首页 > 文章 > 牛α-IFN及FMDV P12A3C双表达载体的构建及其在BHK-21细胞中的表达 生物技术通报 2012,0 (2) 80-84
Position: Home > Articles > Construction of a Coexpression Vector Containing Genes of P12A3C of FMDV and α-IFN and Its Expression in vitro Biotechnology Bulletin 2012,0 (2) 80-84

牛α-IFN及FMDV P12A3C双表达载体的构建及其在BHK-21细胞中的表达

作  者：
杨彬;兰喜;李宝玉;殷相平;李学瑞;柳纪省
单  位：
中国农业科学院兰州兽医研究所家畜疫病病原生物学国家重点实验室农业部畜禽病毒学重点开放实验室
关键词：
口蹄疫病毒;P12A基因;3C基因;α-干扰素;pBudCE4.1载体
摘  要：
从口蹄疫病毒Asia I/Jiangsu毒株的细胞毒中提取总RNA,通过RT-PCR方法分别获得FMDV的P12A及3C基因;同时以pMD18-T-α-IFN质粒为模板,PCR扩增得到α-IFN基因。将α-IFN基因及FMDV P12A及3C基因连接至双启动子表达载体pBudCE4.1上,构建成双效表达质粒pBudCE4.1-α-IFN-P12A3C,经电泳、PCR、双酶切和DNA测序鉴定表明双效质粒载体构建成功。用此重组质粒转染BHK-21细胞后并对其表达情况进行检测,表明该双表达质粒在BHK-21细胞中能够成功表达。以此重组质粒免疫乳鼠后12 h,按100 TCID50/0.1 mL的量进行攻毒,结果发现该质粒能够抑制病毒的增殖,对乳鼠有一定的保护作用。结果表明成功构建了牛α-IFN及FMDV P12A3C组合基因双表达载体,为进一步研究口蹄疫基因疫苗提供前期基础。
译  名：
Construction of a Coexpression Vector Containing Genes of P12A3C of FMDV and α-IFN and Its Expression in vitro
作  者：
Yang Bin Lan Xi Li Baoyu Yin Xiangping Li Xuerui Liu Jixing(Key Laboratory of Animal Virology of Ministry of Agriculture,State Key Laboratory of Veterinary Etiological Biology, Lanzhou Veterinary Research Institute,Chinese Academy of Agricultural Sciences,Lanzhou 730046)
关键词：
FMDV P12A3C gene α-IFN pBudCE4.1 vector
摘  要：
The gene cassette-P1-2A,3C of FMDV was acquired by RT-PCR,and the fragment of IFN-α was amplified by PCR.Genes encoding the bovine α-IFN and the P12A-3C of FMDV Asia I/Jiangsu 2005 and were cloned into the MCS of pBudCE4.1 expression vector under CMV promoter and EF1a promoter,respectively.The bi-functional recombinant plasmid was identified by restriction analysis and PCR.It was proved by DNA sequencing that the acquired recombinant pBudCE4.1-α-IFN-P12A3C contains the bovine α-IFN and the P12A-3C of FMDV.The recombinant plasmid was transfected into the BHK-21 cell using lipoid 2000,and the analysis revealed the recombinant plasmid could expressed in BHK-21 cells.The recombinant plasmids were administered to mice intramuscular injection.All of mice were challenged with 100 TCID50/0.1 mL FMDV Asia I/Jiangsu 2005 after injection 12 h.Vaccination of mice with recombinated expression plasmids were protected against the challenge of a virulent virus.This study demonstrates that the delivery of FMDV antigens via bicistronic vectors is feasible.Further experimentation with bicistronic delivery systems is required for the optimization and refinement of DNA vaccines to effectively prime protective immune responses against foot-and-mouth disease(FMD).

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