Purification,antiserum preparation and antigenic characters of ClpC2 in Myco-bacterium tuberculosis / 中国免疫学杂志

ABSTRACT

Objective:To investigate the antigenicity of ClpC 2 and the feasibility of polyclonal antibodies of ClpC 2 as detected antibody.Methods:rClpC2 was induced with IPTG.The rClpC2 was identified by SDS-PAGE and Western blot ,and purified by affinity chromatography ,with which rabbit were immunized and the specificity of rabbit antiserum was detected by Western blot , the titer of rabbit antiserum against ClpC2 was detected by double immunodiffusion and indirect enzyme-linked immunosorbent assay (ELISA).The antigenicity of the rClpC2 was detected by ELISA.The polyclonal antibodies of ClpC 2 were prepared to detect the ClpC 2 in clinical serum of TB patients by ELISA.Results:SDS-PAGE showed specific protein band with a relative molecular mass of 46 kD.The rClpC2 could bind with the antibody in the blood serum of the mouse immuned by MTB.By Bandscan analysis rClpC 2 accounted for about 58.7%of the total bacteria protein ,the purity of rClpC2 was 88.5% after purification.The ClpC2 of BCG could bind with the rabbit antiserum.The titer of antiserum were 1∶32 and 1∶320 000 by double immunodiffusion and ELISA detected respectively.ELISA results showed that clinical serum positive rate of rClpC 2 antigen was 46%in TB patients,the sensitivity of this protein was 46%,and the spe-cificity of this protein was 90%.ELISA results showed that the sensitivity of rabbit antiserum against ClpC 2 was 40%, and the specificity was 90%.Conclusion: Successfully expressed and purified rClpC 2 and high titer polyclonal antibody were successfully prepared,and these results will provide basements for further study on the biological functions of ClpC 2 and its candidate potentiality as serological diagnosis and drug-target and biological functions of antiserum against ClpC 2.