Cloning of human alpha1-antitrypsin (α_1-AT) and its expression in COS-7 cells.

Objective To clone the encoding gene of human alpha1-antitrypsin (α 1-AT), to construct recombinant human alpha1-antitrypsin (α 1-AT), and to detect expression of the gene in COS-7 cells. Methods Using the isolated total RNA from human embryo kidney cell line HEK293 as a template, the cDNA encoding α 1-AT was amplified by reverse transcription-polymerase chain reaction(PCR) method with upstream primer containing Kozak sequence and KpnⅠsite,and down primer containing XhoⅠsite. The PCR product was digested with KpnⅠand XhoⅠ,and cloned into eukaryotic expression vector pcDNA3.1(+) and then sequenced. Finally, the constructed recombinant plasmid pcDNA3.1(+)-α 1-AT was transformed into COS-7 cell line and α 1-AT expression in cell line COS-7 was determined by Western blotting. Results The sequence of α 1-AT cDNA obtained in this study was the same as that of reported previously. The recombinant plasmid pcDNA3.1(+)-α 1-AT was constructed and transformed into COS-7 cell line successfully. Secretive recombinant human α 1-AT was detectable in the supernatant of transfected COS-7 cells by Western blotting. Conclusion α 1-AT cDNA is obtained and its expression in cell line COS-7 is determined in this study successfully. These results might be helpful for further studies in function of α 1-AT.