摘要
目的利用pGEM-TEasyvector构建pET15b-SOD1重组质粒。方法以pBluescriptIISK-SOD1质粒为模板,应用pfuDNA聚合酶,聚合酶链反应法(PCR)扩增SOD1cDNA。将扩增的SOD1cDNA与三磷酸脱氧腺苷(dATP)反应,然后通过TaqDNA聚合酶具有的非模板依赖性末端转移酶活性在SOD1cDNA的3’末端加“A”并纯化,然后将纯化后的SOD1cDNA与pGEM-TEasyvector直接连接,连接产物转化大肠杆菌DH5α,蓝白筛选,挑选白色克隆扩增、鉴定,重组的质粒命名为pGEM-TEasy-SOD1。pGEM-TEasy-SOD1和pET15b分别用XhoⅠ、BamHⅠ双酶切,采用低熔点琼脂糖凝胶电泳回收所需片断,前者回收470bp的SOD1cDNA片段,后者回收线性化的pET15b片段,将两者连接后转化大肠杆菌DH5α,抽提后的重组体质粒命名为pET15b-SOD1,然后对pET15b-SOD1进行酶切和核苷酸序列测序鉴定。结果重组质粒pET15b-SOD1经测序分析证实克隆入pET15b-SOD1的人SOD1cDNA与GeneBank“AB087266”登录的人SOD1cDNA序列一致,从而成功构建了pET15b-SOD1重组质粒。结论pET15b-SOD1重组质粒的构建为细胞穿透肽介导的SOD1转导提供了一对照载体。
Objective To construct the recombinant pET15b-SOD1 plasmid by pGEM-T Easy vector. Methods Utilizing pfu DNA polymerase and pBluescript II SK-SOD1 plasmid as template, SOD1 cDNA was amplified by polymerase chain reaction(PCR).SOD1 cDNA was added'A'by using the template-independent terminal transferase activity of Taq DNA polymerase. The purified products of SOD1 cDNA whose 3’end was added A were ligated with pGEM-T Easy vector ,and were transformed into DH5α. The white clones were selected to be amplified and identified , the recombinant plasmid was named after pGEM-T Easy-SOD1. pGEM-T Easy-SOD1 and pET15b were further double-enzyme digested by Xho I and BamH I, respectively, and the purified 470bp SOD1 cDNA fragment of the former and the purified linear fragment of the latter were ligated and transfered into DH5α. The clones were randomly selected and the recombinant plasmids were purified and named after pET15b-SOD1, which were identified by double-enzyme digested and sequenced. Results Recombinant pET15b-SOD1 plasmid was proved that the SOD1 cDNA sequence of pET15b-SOD1 by sequencing was coincided with the human SOD1 cDNA sequence of GeneBank 'AB087266'. The recombinant pET15b-SOD1 plasmid were constructed successfully. Conclusion Construction of recombinant pET15b-SOD1 plasmid provides the control vector for transduction of Cu,Zn-superoxide dismutase mediated by cell-penetrating pepetides(CPPs).
出处
《郧阳医学院学报》
2005年第2期65-68,F002,共5页
Journal of Yunyang Medical College
关键词
超氧化物歧化酶
TA克隆载体
基因重组
原核表达载体
Superoxide dismutase
TA cloning vector
Gene recombination
prokaryotic expression vector
