Construction of prokaryotic recombinant expression vector of PTD4-Cu, Zn-SOD / 中华麻醉学杂志

ABSTRACT

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.