Construction of prokaryotic expression vector for human carboxypeptidase H and its application

Objective To get human recombinant carboxypeptidase H(CPH)protein,and to explore the application of the recombinant protein.Methods The human CPH gene fragment was amplified from plasmid pcDNAⅡ by PCR and cloned into prokaryotic expression vector pQE30.The recombinant expression plasmid was transfected into E.coli M15 and induced with IPTG.CPH protein was purified by Ni-NTA column and the antigenicity of the purified protein was confirmed by Western-blot.The purified recombinant protein was used in ELISA to evaluate the method for the detection of CPH-Ab in human sera.Results Prokaryotic expression vector for CPH was successfully constructed.The purified protein could be recognized by the CPH-Ab in sera of type 1 diabetic patients.When compared with radioligand assay,the more increased sensitivity and specificity of the ELISA test were 93.3% and 97.5%,respectively.Conclusions The purified CPH protein is one of suitable candidates of immunodiagnostic antigen.