阴道毛滴虫氢化酶体腺苷酸激酶基因真核表达载体的构建与鉴定  

Construction of Eukaryotic Exression Vector for Hydrogenosomal Adenynate Kinase(AK) of Trichomonas Vaginalis

作　　者：帖超男[1] 王雅静[1] 谢辉[2] 毕世樑[3] 刘佩娜[1] 廖琳[1] 

机构地区：[1]四川大学华西基础医学与法医学院寄生虫学教研室,四川成都610041 [2]郑州市第五人民医院检验科 [3]四川大学华西第二医院妇产科

出　　处：《预防医学情报杂志》2008年第11期862-865,共4页Journal of Preventive Medicine Information

基　　金：国家自然科学基金(No.39970667)

摘　　要：目的构建阴道毛滴虫氢化酶体腺苷酸激酶(adenynate kinase,AK)基因真核表达载体并予以鉴定。方法根据AK基因已知序列设计合成一对引物,应用PCR技术从阴道毛滴虫基因组DNA中扩增出AK基因,并将其克隆入pMD18-T Simple载体。阳性克隆的重组质粒经酶切及PCR鉴定后,用双脱氧链末端终止法进行基因序列测定。应用BLAST软件辅助分析所测基因与Genbank中阴道毛滴虫氢化酶体AK序列的同源性。克隆载体PMD-18T-AK经限制性内切酶BamHI和XbaI双酶切,得到含这2个酶切位点之AK DNA片段,T4连接酶连接到含有这2个酶切位点的真核表达载体pcDNA3.1(+),构建出重组真核表达载体pcDNA3.1(+)-AK,经凝胶电泳鉴定、酶切鉴定、PCR鉴定证实DNA片段大小的正确性。结果经凝胶电泳鉴定、酶切鉴定、PCR鉴定证实含大小正确的AK DNA片段。结论成功地构建了基因真核表达载体。Objective To construct and identify eukaryotic expression vector of the hydrogenosomal adenynate kinase of Trichomonas vaginalis. Method A pair of primers was designed according to the known sequence of AK gene . The AK gene fragment was amplified by PCR, and ligated to a sequenceing vector, pMD18-T simple vector. Positive recombinants were detected by PCR, digested by restriction enzyme and sequenced with dideoxy nucleotide chain termination method. The cloning vectors（ containing full length of AK DNA and two restriction sites ： BamHI and XbaⅠ）were first cut by two restriction andonuclease： BamHI and XbaI, and the same as the eukaryotic expression vector; then, the AK DNA and the digested vector were ligated by T4 DNA ligase, and rcombinant rukaryotic expression vector was formed. Its length was certificated by agarose gel eleetorphoresis analysis, digestion with BamHI and XbaⅠ, and PCR. Result The results of agarose gel electrophoresis analysis, digestion, and PCR confirmed the right length of inserted DNA, which was the same as the AK DNA. Conclusion peDNA3.1 -AK, a eukaryotic expression vector, is successfully constructed.

关 键 词：阴道毛滴虫 氢化酶体 腺苷酸激酶 基因 克隆 序列分析 真核表达载体 

分 类 号：R382.21[医药卫生—医学寄生虫学]