PCR产物靶向克隆法构建原核表达载体pET15b-CAT  

Construction of prokaryotic expression vector pET15b-CAT with PCR targeting cloning

作　　者：王家宁[1] 郭凌郧[1] 孔霞[1] 郑飞[1] 谭艳[1] 黄永章[1] 

机构地区：[1]郧阳医学院附属人民医院临床医学研究所,湖北十堰442000

出　　处：《第四军医大学学报》2008年第20期1851-1854,共4页Journal of the Fourth Military Medical University

基　　金：湖北省高等学校优秀中青年科技创新团队计划(T200811);十堰市重大科技项目(2006030Z)

摘　　要：目的:将人过氧化氢酶(Catalase,CAT)cDNA的PCR扩增产物定向克隆到pET15b载体上以构建重组质粒pET15b-CAT.方法:扩增目的基因的PCR引物的5′端加上与线性化载体同源的碱基序列,以使PCR的扩增产物两端分别带上15个和线性化载体两端同源的碱基序列.以pZeoSV2(+)-CAT为模板进行靶向克隆所需的人CATcDNA扩增.将纯化的人CATcDNAPCR产物与经XhoⅠ和BamHⅠ酶切的线性化载体pET15b以6∶1摩尔数之比混合于含重组酶的反应液中,25℃反应30min,将PCR产物定向克隆于目标载体pET15b上,获得重组质粒pET15b-CAT.重组质粒经PCR,XhoI酶切和DNA测序鉴定.结果:人CATcDNA的PCR扩增产物与经XhoⅠ和BamHⅠ酶切的线性化载体pET15b发生了同源重组反应,经鉴定采用PCR产物靶向克隆法成功构建了pET15b-CAT.结论:PCR产物靶向克隆法是一种简便、高效地将PCR产物定向克隆于目标载体的方法,而无需对PCR产物进行酶切、末端抹平、载体去磷酸化及连接反应,适用于任何载体、任何插入片段和任意酶切位点.AIM： To clone human catalase CAT） cDNA PCR product into pET15b-CAT to generate recombinant plasmid pET15b-CAT. METHODS： A pair of PCR primers having 15 bases of homology with sequences flanking the desired site of insertion in the cloning vector were designed. The plasmid pZeoSV2 （ ＋ ）-CAT was used as the template for amplification of human CAT cDNA for PCR cloning. The purified human CAT cDNA and the linearized pET15b with Xho I and BamH I digestion was mixed together at a molar ratio of 6：1 within the tube containing recombinant enzyme and incubated at 25℃ for 30min, then transformed the reaction mixture into competent DHSα. The prokaryotic expression vector was identified with PCR, Xho I digestion and DNA sequencing. RESULTS： Homologous recombination occurred between PCR product of human CAT cDNA and linearized pET15b. Human CAT cDNA was successfully inserted into pET15b to generate pET15b-CAT. CONCLUSION： PCR targeting cloning is a simple, efficient method for cloning of PCR products without the need for restriction enzymes, ligase, or blunt-end polishing and dephosphorylation of the vector. This method rapidly generates precise constructs with inserts in the correct orientation and it is universal - it works with any inser - t and any vector at any restriction site.

关 键 词：过氧化氢酶 聚合酶链反应 靶向克隆 

分 类 号：Q78[生物学—分子生物学]