含O型FMDVP1-2A、3C基因和EGFP基因表达盒的构建  

The construction and identification of P1-2A、3C and EGFP genes cassette of type O foot-and-mouth disease virus

作　　者：王建科[1,2] 吴国华[2] 叶奕优[3] 颜新敏[2] 朱海霞[2] 李健[2] 朱彩珠[2] 张强[2] 王雯慧[1] 

机构地区：[1]甘肃农业大学动物医学院,甘肃兰州730070 [2]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,甘肃兰州730046 [3]深圳出入境检验检疫局动植物检验检疫技术中心,广东深圳518001

出　　处：《甘肃农业大学学报》2009年第1期15-20,共6页Journal of Gansu Agricultural University

基　　金：国家重点基础研究发展计划(973)项目(973-2005CB523201);国家科技基础条件平台项目(2005DKA21205-3)

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.The PI-2A gene and the 3C gene of O/China 99 strain of foot-and-mouth disease virus were amplified by RT-PCR. TheP1-2A gene fragment was inserted into pUC119 vector and the3C 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 HI and BamH I and the recombinant plasmid pMD18-T-3C was digested by BamH I and Nhe were achieved . Due to the two gene fragments P1-2A,3C had I, and the ligated P1-2A gene and 3C gene the same BarnH I enzyme site , the transfer vector pMD18-T-PI-ZA-3C was constructed. At last the two directions poxvirusp5 5 gene controlled enhanced green fluorescent protein gene andP1-2A-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-PT. 5-P1-2A-3C was constructed successfully, thePT. 5 promoter eontrolledP1-2A-3C gene in one direction and in the opposite oriention controlled EGFP.

关 键 词：FMDV 启动子P7 5 P1—2A基因 3C基因 

分 类 号：S852.65[农业科学—基础兽医学]