PTD4-Cu,Zn-SOD原核重组表达载体的构建  

Construction of prokaryotic recombinant expression vector of PTD4-Cu, Zn-SOD

作　　者：党莎杰 薛荣亮[2] 孟丽华[2] 杨毅猛 张小玲[2] 雷晓明[2] 韩丽春 

机构地区：[1]陕西省肿瘤医院麻醉科,西安市710061 [2]西安交通大学第二附属医院麻醉科

出　　处：《中华麻醉学杂志》2015年第4期486-489,共4页Chinese Journal of Anesthesiology

基　　金：国家自然科学基金（81071070）

摘　　要：目的 构建蛋白质转导结构域4-铜锌超氧化物歧化酶（PTD4-Cu,Zn-SOD）原核重组表达载体.方法 利用基因重组技术,设计Cu,Zn-SOD特异性引物和PTD4寡核苷酸序列,Cu,Zn-SOD基因进行PCR反应,产物进行鉴定、回收和纯化,以pET16b为载体,经Xho Ⅰ和BamH Ⅰ双酶切反应、连接反应和质粒转化,构建pET16b-Cu,Zn-SOD原核重组表达载体,再分别将PTD4基因和pET16b-Cu,Zn-SOD载体经Nde Ⅰ和Xho Ⅰ双酶切反应、连接反应和质粒转化,构建pET16b-PTD4-Cu,Zn-SOD原核重组表达载体,并进行限制性内切酶谱分析和基因测序.结果 成功构建了长度为6 207 bp的pET16b-PTD4-Cu,Zn-SOD原核重组表达载体,用Nde Ⅰ和BamH Ⅰ双酶切,得到约5.7 kb的载体片段和约510 bp的PTD4-Cu,Zn-SOD基因片段,与预期结果一致.pET16b-PTD4-Cu,Zn-SOD原核重组表达载体基因测序结果与预期的基因序列相比,碱基序列均正确.结论 成功构建了PTD4-Cu,Zn-SOD原核重组表达载体.Objective To construct the prokaryotic recombinant expression vector of PTD4-Cu,Zn-SOD.Methods By using the techniques of gene recombination,the primers of Cu,Zn-SOD and the oligonucleotide sequences of PTD4 were designed,PCR amplification was performed for Cu,Zn-SOD genes,the PCR products were identified,reclaimed and purified,and pET16b served as carrier.The prokaryotic recombinant expression vector of pET16b-Cu,Zn-SOD was constructed using double digestion with Xho Ⅰ and BamH Ⅰ,ligated reaction and plasmid transformation.Then PTD4 gene and pET16b-Cu,Zn-SOD carrier were double digested with Nde Ⅰ and Xho Ⅰ and ligated,and the plasmid was transformed,and the prokaryotic recombinant expression vector of pET16b-PTD4-Cu,Zn-SOD was constructed.The reconstructed vector was analyzed by restriction mapping and was verified by gene sequencing.Results The prokaryotic recombinant expression vector of pET16b-PTD4-Cu,Zn-SOD with a length of 6 207 bp was constructed successfully.The carrier fragment about 5.7 kp and PTD4-Cu,Zn-SOD gene fragment about 510 bp were obtained by double digestion with Nde Ⅰ and BamH Ⅰ,which was consistent with the expected results.The results of gene sequencing showed that the base sequences of pET16b-PTD4-Cu,Zn-SOD were correct when compared with the expected gene sequences.Conclusion The prokaryotic recombinant expression vector of pET16b-PTD4-Cu,Zn-SOD is constructed successfully.

关 键 词：tat基因产物 人免疫缺陷病毒 蛋白质结构 三级 超氧化物歧化酶 

分 类 号：R3416[医药卫生—基础医学]