SOD基因克隆及真核表达载体构建

To colone superoxide dismitase SOD gene of rat and construct recombinant of pLNCX-SOD expressive vector

关键词:  超氧化物歧化酶  逆转录病毒  基因

Key words:  superoxide dismutase(SOD)  retrovirus  gene

摘要:

克隆超氧化物歧化酶（SOD）基因，构建逆转录病毒（pLNCX）-SOD真核表达载体转染复合体，为基因转染及基因治疗进行准备。［方法］RT-PCR法扩增SOD基因，pLNCX载体进行限制性酶切、去磷酸化，电泳后回收切开的pLNCX及SOD片段，经连接反应后构建pLNCX-SOD真核表达载体。［结果］大鼠心肌细胞提取的总RNA电泳结果，28 s和18 s两种核糖体RNA的位置和亮度说明提取的RNA是完整的。SOD cDNA测序结果表明，克隆的SOD cDNA序列与大鼠SOD序列一致。质粒的所有克隆均具有同样大小，与计算结果一致，为7.0 kb。限制性酶切电泳结果显示，pLNCX-SOD经HindⅢ酶切后，得到了约500 bp的SOD片段，说明质粒的构建是正确的。［结论］克隆了大鼠SOD基因，构建了pLNCX-Cu/Zn SOD真核表达载体复合体。

Abstract:

To colone superoxide dismutase(SOD) gene, construct combinant of retrovirus SOD gene, make a basis of gene transfection and gene therapy. ［Methods］ Total RNA was extracted from the myocardium of SD rat and SOD gene was amplified by RT-PCR. The full leugth is 459 bp. The PCR product was linked with PT7 Blue T-Vector and E.Coli DH5α was transformed by PT7-SOD plasmid. The positive clone was screened and identified by restriction endonuclease digestion. The sequence of cloned SOD is consistent with the reported sequence of the genbank. SOD gene was digested from PT7-SOD at the site of HindⅢ. The digested product was blunted and inserted into the retrovirus vector of pLNCX at the site of HindⅢ. ［Results］ SOD gene was cloned and recombinant of pLNCX-SOD gene was constructed which make a basis for gene transduction and over expression of SOD and gene therapy of SOD. ［Conclusion］ We successfully