关键词: 抗酶抑制因子, 原核表达, 抗体

Abstract: We aim to carry out prokaryotic expression and purification of human antizyme inhibition factor-1（AZIN1）,and to prepare as well as identify polyclonal antibody against AZIN1. Prokaryotic expression vector pET-28a/AZIN1 was transformed into Escherichia coli BL21（DE3）,in which the recombinant AZIN1 was expressed by IPTG induction and purified by affinity chromatography with Ni-NTA resin under denaturing condition. The purified recombinant AZIN1 was used as the antigen to prepare the antibody in the BALB/c mice. The titer and specificity of anti-sera were detected by ELISA,Western blot,cell immunofluorescence,and immunochemistry. As results,restriction analysis and DNA sequencing proved that the plasmid pET-28a/AZIN1 was constructed successfully. In E. coli,recombinant AZIN1 protein was expressed by IPTG induction and mainly existed in a form of inclusion body. The AZIN1 protein expressed in the E. coli was effectively purified using affinity chromatography. When used as the antigen to immune mice,this protein induced the production of specific antibody against AZIN1 with serum titer of 1:640 000. The prepared antiserum specifically recognized and bound to the AZIN1 protein expressed in human or mouse tumor cells. And this antiserum was efficiently used in the analysis for AZIN1 by Western blot,cell immunofluorescence,and cell immunochemistry. In conclusion,the prokaryotic expression and purification of human AZIN1 protein and preparation of polyclonal antibody against AZIN1 were successfully performed in this study. These results provide a basis for further research on the roles of AZIN1 in regulating cell proliferation and in disease prevention and treatment.

Key words: antizyme inhibition factor-1, prokaryotic expression, antibody