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

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 XhoⅠand BamHⅠ, ligated reaction and plasmid transformation.Then PTD4 gene and pET16b-Cu, Zn-SOD carrier were double digested with NdeⅠ and XhoⅠand 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 NdeⅠand BamHⅠ, 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. Key words: tat Gene products, human immunodeficiency virus; Protein structure, tertiary; Superoxide dismutase