融合酶表达载体的构建及出现问题的初探  

Constructing Expression Vector of New Fusion Enzyme and the Preliminary Investigation of Problems Appeared in the Process

作　　者：崔宝宁[1] 王芳[1] 王艳萍[1] 张治洲[1] 

机构地区：[1]泰达BIO-X系统生物技术研究中心天津科技大学,天津300457

出　　处：《生物技术》2010年第2期8-12,共5页Biotechnology

基　　金：教育部新世纪优秀人才计划项目("基因网络操作技术");天津科技大学引进人才科研启动基金(20080426)资助

摘　　要：目的:将限制性内切酶FokⅠ催化区域基因(631bp)和PI-SceⅠ识别区域的基因(546bp)连接到一起,克隆入载体质粒pET28a+中,为表达新的限制性内切酶融合酶做准备。方法:分别以啤酒酵母和海床黄杆菌作模板,PCR扩增PI-SceⅠ和FokⅠ基因片段,再将它们克隆入载体质粒pET28a+,然后对整合质粒进行双酶切检测。结果:整合过程中,无论是PI-SceⅠ还是FokⅠ基因片段,都能单独成功插入载体,但当插入第二段基因片段时,酶切结果显示大约600bp的基因片段缺失了。结论:缺失可能因为两段连在一起的新基因在转化过程中对宿主细胞有毒性,宿主细胞对其进行了剪切;也可能这两段基因会形成某种高级结构而导致其不能很好的连接,产生缺失现象。Objective：FokⅠ and PI-SceⅠ are two kinds of restriction endonuclease.The gene fragments of the splicing domain of PI-SceI（546bp）and endonucleolytic domain of FokⅠ（631）were together cloned into the expression vector pET28a＋,to prepare recombined vector for further expression of new fusion enzyme.Method：These two gene fragments were cloned by PCR and together cloned into vector pET28a＋,then examined by double digestion.Result：Each gene fragment could be successfully cloned into vector,while 600bp gene deletion was found by double digestion following the second fragment＇s cloning.Conclusion：The deletion might be caused by a cut owing to the host cell in the conversion process due to their toxicity to the cell while the two gene fragments were connected together.Another case might be that these two fragments could form into complex structure,which led to the phenomenon of deletion.

关 键 词：限制性内切酶 PI-SceⅠ FokⅠ pET28a+ 克隆 双酶切 

分 类 号：Q819[生物学—生物工程]