构建T载体用于PCR克隆  

Construction of T-vector for PCR cloning

作　　者：马琪[1] 周莉[1] 马立新[1] 

机构地区：[1]湖北大学生命科学学院,湖北武汉430062

出　　处：《湖北大学学报（自然科学版）》2005年第2期162-165,共4页Journal of Hubei University：Natural Science

基　　金：国家"十五"科技攻关项目(2001BA708B04-05)资助

摘　　要：通过克隆甘露聚糖酶基因到pMD-18T载体构建了质粒pMD-18Tman2XcmI,克隆的甘露聚糖酶基因两侧各引进了一个特定的XcmI酶切位点;用XcmI酶切质粒pMD-18Tman2XcmI可以制备T载体.因为甘露聚糖酶基因作为填充片段和筛选标记受半乳糖苷酶基因启动子的控制,甘露聚糖酶能够表达,从而水解甘露聚糖产生水解圈,指示转化了部分酶切质粒的背景菌落.此外,在甘露聚糖酶基因中存在第3个不同序列的XcmI酶切位点,因此残余的甘露聚糖酶基因引起的背景可以进一步降低.抽提大量质粒贮存,随时制备T载体使用,增加了克隆的稳定性.该T载体克服了一般T载体质量不稳定,费钱,克隆效率低,假阳性高的缺点.使用构建的T载体完成了10个基因的克隆,结果表明重组效率达到了90%～100%.A plasmid ,pMD-18Tman2XcmI is developed,the plasmid is constructed by cloning an Xcm?I cassette with mannanase gene (man 1?101?bp long) between two Xcm?I restriction sites into pMD-18T.The XcmI cassette is made by PCR using primer pairs containing recognition sequences of Xcm?I so that pMD-18Tman2XcmI can be easily converted into a T-vector by digestion of Xcm?I.Because man under control of lac promoter can be used as both stuff fragment and screen mark,mannanase can be expressed for degradation of mannan,then hydrolysis halos formed in screen plate indicate E.coli transformed by partial digestion of plasmid.In addition, the third unique Xcm?I restriction site locates in man,so background caused by remaind man is decreased.The plasmid can be stored for preparation of T vector, and then PCR cloning can be performed at any time.All these enhance the stability of PCR cloning and overcome the drawback of traditional T vector:instability,high cost ,low efficiency ,many false-positive colonies.The cloning efficiency of PCR products of ten genes is up to 90?%～100?%.

关 键 词：T载体 甘露聚糖酶基因 DNA克隆 PCR 

分 类 号：Q756[生物学—分子生物学]