霍乱弧菌毒素协同调节茵毛基因tcpA的克隆及序列分析

目的以新疆分离0139霍乱弧菌XJ93006株的DNA为模板，自行设计引物克隆毒素协同调节菌毛亚单位A(tcpA)基因(包括侧序列)并构建重组载体。方法以0139霍乱弧菌新疆分离株XJ93006基因组DNA作为模板；根据0l群ElTor型霍乱弧菌N16961全基因组序列设计tcpA PCR引物，高保真酶扩增tcpA片段：限制性内切酶切PCR产物和pNEB193空质粒，T4DNA连接酶连接酶切产物，重组质粒转化大肠什菌TB1工程菌，蓝白斑菌落试验筛选阳性克隆；提取质粒经酶切鉴定后测序并利用生物信息数据库对该序列进行序列分析，比较.结果DNA限制性内切酶可从重组质粒pNEB193-tcpA的EcoRI和Sal I位点之间切出一个1037bp的DNA片段，测序结果与01群ETTor型霍乱弧菌N16961、O139霍乱弧菌标准株M045的tcpA同源性均达到99.9％以上。结论成功构建0139霍乱弧菌新疆分离株XJ93006毒素协同调节菌毛亚单位A(tcpA)基因的重组质粒pNEB193-tcpA；序列分析表明霍乱弧菌毒素协同调节菌毛亚单位A(tcpA)基因住01群ETTor型霍乱弧菌和0139霍乱弧菌中是一类高度保守的原核基因，可作为霍乱弧菌疫苗的候选基因。另外为研究0139霍乱弧菌的来源奠定基础。

Cloning and sequence analysis of toxin-coregulated pilus subunit (tcpA)gene of vibrio cholerae

Objective To clone toxin_coregulated pilus subunit A (tcpA) gene of vibrio cholerae O139 strain XJ93006 and construct the recombinant cloning vector of pNEB 193_tcpA.Methods Prepare genomics DNA from vibrio cholerae O139 strain XJ93006 as PCR template, design primers according to the genomics sequemce of vibrio cholerae O1 biovar E1Tor strain N16961 published on Genbank, tcpA gene was amplified by PCR using Pyrobest DNA polymerase. PCR production of tcpA and clone vector pNEB 193 were digested by the same restiction endonuclease and linked by T 4 DNA ligase. The recombined plasmid was transformed into E.coli TB 1.The positive clones were detected by blue_white screening, then identified by restriction endonuclease digesting and DNA fragment sequencing.Results A 1055bp DNA fragment has been inserted into pNEB 193 vector betweenEcoR I and Sal I restriction sites. Compared with vibrio cholerae O1 biovar ElTor strain N16961 and vibrio cholerae O139 strain MO45 and other vibrio cholerae O1 biovar E1Tor strains and vibrio cholerae O139 strains,the homology is more than 99.9%. Conclusion pNEB193_tcpA, the recombined cloning vector of tocxin_coregulated pilus subunit A (tcpA) gene has been successfully constructed, and sequence analysis indicate that the tapA gene of other vibrio cholerae O1 biovar E1Tor and vibrio cholerae O139 is a highly conserved prokaryotic gene and a potential candidate for vibrio cholerae vaccine development. Besides, the result can be abase to study the source of vibrio cholerae O139.