CAJ | 학술논문

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.
목적 구건단백질전도결구역4-동자초양화물기화매(PTD4-Cu,Zn-SOD)원핵중조표체재체.방법 이용기인중조기술,설계Cu,Zn-SOD특이성인물화PTD4과핵감산서렬,Cu,Zn-SOD기인진행PCR반응,산물진행감정、회수화순화,이pET16b위재체,경Xho Ⅰ화BamH Ⅰ쌍매절반응、련접반응화질립전화,구건pET16b-Cu,Zn-SOD원핵중조표체재체,재분별장PTD4기인화pET16b-Cu,Zn-SOD재체경Nde Ⅰ화Xho Ⅰ쌍매절반응、련접반응화질립전화,구건pET16b-PTD4-Cu,Zn-SOD원핵중조표체재체,병진행한제성내절매보분석화기인측서.결과 성공구건료장도위6 207 bp적pET16b-PTD4-Cu,Zn-SOD원핵중조표체재체,용Nde Ⅰ화BamH Ⅰ쌍매절,득도약5.7 kb적재체편단화약510 bp적PTD4-Cu,Zn-SOD기인편단,여예기결과일치.pET16b-PTD4-Cu,Zn-SOD원핵중조표체재체기인측서결과여예기적기인서렬상비,감기서렬균정학.결론 성공구건료PTD4-Cu,Zn-SOD원핵중조표체재체.
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.