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当前位置: 首页 > 文章 > 含O型FMDVP1-2A、3C基因和EGFP基因表达盒的构建 甘肃农业大学学报 2009,44 (1) 15-20
Position: Home > Articles > The construction and identification of P1-2A、3C and EGFP genes cassette of type O foot-and-mouth disease virus Journal of Gansu Agricultural University 2009,44 (1) 15-20

含O型FMDVP1-2A、3C基因和EGFP基因表达盒的构建

作  者：
王建科;吴国华;叶奕优;颜新敏;朱海霞;李健;朱彩珠;张强;王雯慧
单  位：
深圳出入境检验检疫局动植物检验检疫技术中心;甘肃农业大学动物医学院;中国农业科学院兰州兽医研究所家畜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：
FMDV;启动子P7.5;P1-2A基因;3C基因
摘  要：
采用RT-PCR方法分别扩增口蹄疫病毒O/China99毒株的P1-2A和3C基因,将P1-2A基因连接到pUC119载体,3C基因连接到pMD18-T载体,分别得到重组载体pUC119-P1-2A和pMD18-T-3C;将重组载体pUC119-P1-2A用HindⅢ、BamHⅠ酶切,重组载体pMD18-T-3C用BamHⅠ、NheⅠ酶切;利用酶切所得到的基因片段P1-2A、3C有共同的BamHⅠ酶切位点,实现基因P1-2A、3C的连接,构建重组载体pMD18-T-P1-2A-3C.将基因P1-2A-3C与启动EGFP表达的双向串连痘苗病毒启动子P7.5相连,构建载体p-EGFP-N1-P7.5-P1-2A-3C,并进行PCR扩增、酶切鉴定及序列测定.结果表明:试验成功构建了一侧启动表达P1-2A-3C基因,一侧启动表达标记基因EGFP的表达盒p-EGFP-N1-P7.5-P1-2A-3C.
译  名：
The construction and identification of P1-2A、3C and EGFP genes cassette of type O foot-and-mouth disease virus
作  者：
WANG Jian-ke1,2,WU Guo-hua2,YE Yi-you3,YAN Xin-min2,ZHU Hai-xia2,LI Jian2,ZHU Cai-zhu2,ZHANG Qiang2,WANG Wen-hui1(1.College of Veterinary Medicine,Gansu Agricultural University,Lanzhou 730070,China; 2.Key Laboratory of Animal Virology of Ministry of Agriculture,State Key Laboratory of Veterinary Etiological Biology,Lanzhou Veterinary Research Institute,Chinese Academy of Agriculture Sciences,Lanzhou 730046,China;3.Technical Center of Animal and Plant Inspection and Quarantine,Shenzhen Exit & Entry Inspection and Quarantine Bureau,Shenzhen 518001,China)
关键词：
FMDV;promotor P7.5;P1-2A gene;3C gene
摘  要：
The P1-2A gene and the 3C gene of O/China 99 strain of foot-and-mouth disease virus were amplified by RT-PCR.The P1-2A gene fragment was inserted into pUC119 vector and the 3C gene was inserted into pMD18-T vector,then the recombinant plasmid pUC119-P1-2A and pMD18-T-3C were constructed.The recombinant plasmid pUC119-P1-2A was digested by Hind Ⅲ and BamH I and the recombinant plasmid pMD18-T-3C was digested by BamH I and Nhe I,and the ligated P1-2A gene and 3C gene were achieved.Due to the two gene fragments P1-2A,3C had the same BamH I enzyme site,the transfer vector pMD18-T-P1-2A-3C was constructed.At last the two directions poxvirus p7.5 gene controlled en hanced green fluorescent protein gene and P1-2A-3C gene was ligated.The vector was named p-EGFP-N1-P7.5-P1-2A-3C.The positive vector was analyzed by restriction enzyme,PCR and nucleic acid sequencing.The results showed that the gene expression cassette pEGFP-N1-P7.5-P1-2A-3C was constructed successfully,the P7.5 promoter controlled P1-2A-3C gene in one direction and in the opposite oriention controlled EGFP.

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