PMG-36e-CD::upp重组质粒的构建及鉴定

Construction and Identification of PMG-36e-CD::upp Recombinant Plasmid

目的：探讨利用分子生物学基因重组技术构建PMG-36 e-CD：：upp自杀基因重组质粒的方法。方法：用小量制备质粒DNA的方法分别提取表达型质粒载体PMG-36 e和自杀基因pORF-CD：：upp,以质粒pORF-CD：：upp为模板PCR扩增CD：：upp基因,表达型质粒载体PMG-36 e与自杀基因CD：：upp分别用限制性内切酶SacⅠ与SalⅠ进行双酶切,T4DNA连接酶16℃恒温水浴过夜连接,转化入感受态JM109,筛选单克隆提取重组质粒PMG-36 e-CD：：UPP进行1%琼脂糖凝胶电泳鉴定,双酶切鉴定,PCR鉴定,及测序鉴定。结果：重组质粒PMG-36 e-CD：：upp经1%琼脂糖凝胶鉴定,分子量为5.5Kb,与其标准分子量大小一致;重组质粒PMG-36 e-CD：：upp经双酶切鉴定及PCR初步鉴定构建成功;送上海生物工程技术服务有限公司测序认证,同源性达100%。结论：重组质粒PMG-36 e-CD：：upp可成功构建,可用于后续实验的表达研究。

Objective： To approach the application of suicide gene on the clinical tumor therapy by constructing recombinant plasmid PMG36e-CD ： ： upp using molecular biology gene engineering methods. Then transform the plasmid to Bacillus bifidus as preparation for the latter experiment. Methods： Extract the plasmid PMG-36e and suicide gene pORF-CD： ：upp by mini-preparation of plasmid DNA method and use pORF-CD ： ： upp as stencil PCR to amplify the CD ： ： upp gene. When designing the PCR primers of CD ： ： upp, PMG36e and suicide gene CD： ：upp were digested by Sac I and Sal I endonuclease. Put T4DNA ligase in the 16℃ thermostatic waterbath overnight. Select the monoclone serial subeuhivation and extract the recombinant plasmid, conform it with agarose gel electrophoresis test, enzyme digestion test, PCR test, sequence test. Results： After underlook 1% agarose gel electrophoresis test, the molecular weight of the recombinant plasmid PMG-36e-CD ： ： upp was 5.5Kb which equaled to molecular weight norm. Enzyme diges- tion test and PCR test showed PMG-36e-CD： ：upp successfully constructed. Samples of recombinant plasmid PMG-36e-CD： ：upp were sent to Shanghai Biotechnology Service Corporation for DNA sequence analyzing which showed 100% homology. Conclusion： Recombinant suicide gene-PMG36e-CD： ：upp can be constructed without shifting, changing, opening reading frames or gene mutation. The products are fit for later expression study.