Study On The Application Of CPAF Recombinant Protein From Chlamydophila Psittaci In The Serology Diagnosis

Objective:The sequence of CPAF were screened from Chlamydophila psittaci and the dominant antigen were analyzed and screened, and the recombinant protein was expressed and purified. The serological diagnosis method was established and applied to the early serology diagnosis of C. psittaci infection. The aim is to screen the antigen which can be applied in the C. psittaci antibody examination and lay bases for the fast diagnosis of C. psittaci infection.Methods:The major immunodominant region gene of 6BC CPAF protein were chosen, and the pGEX6p-2/CPAFm recombinant plasmid was constructed, the protein expression was induced by IPTG and analyzed by SDS-PAGE and Western blot analysis. The recombinant protein was purified by GST agarose gelatin FF. Indirect ELISA method of serological diagnosis was established with the reorganization protein as coating antigen. The immunity reactivity of recombinant protein was analyzed by Western blot and indirect ELISA method. The purified recombinant protein was then used to immunize New Zealand Whites rabbits to generate polyclonal antibodies and the immunogenicity of recombinant protein was analyzed. The sera were titried by ELISA and Western blot analysis.180 sera samples from ducks with respiratory tract infection symptoms were detected with a commercial ELISA-kit and the established indirect ELISA method based on C. psittaci CPAFm, to assess the value of the recombinant protein in serodiagnosis.Results:Restriction enzyme digestion analysis and sequencing showed that the inserted target gene were CPAF (586～1351bp,765bp); A fusion protein with molecular weight about 54kDa was attained after expression and purification. Western blot proved that the recombinant protein can specifically react with C. psittaci IgG positive sera.Indirect ELISA was successfully developed to detect the antibody to the C. psittaci in the duck sera. Results show that the recombinant protein has no cross reaction with Chlamydophila pneumoniae and Chlamydia trachomatis.The sensitivities and specificities of indirect ELISA are higher than that of Birds Chlamydia Psittaci IgG ELISA Kit. Diagnosticaccordancerate of the former is 100 %,the latter is 77.5～95%.Specific humoral response were elicited by recombinant protein in zealand rabbit and the specific antibody titer was above 1:8000 after immunization for 4 times.Conclusion:1. The pGEX6p-2/CPAFm restructuring plasmid was constructed, and CPAF recombinant protein with molecular weight about 45 KDa was successfully expressed in E.coli BL21.2. The purified CPAF recombinant protein, has good immunity activity, can specifically react with C. psittaci positive sera. And the recombinant protein, has good immunogenicity, can stimulate New Zealand rabbits produce high titer specific antibody.3. The indirect ELISA method was established with reorganization protein for envelope antigen and it's sensitivities and specificities is higher than Birds Chlamydia Psittaci IgG ELISA Kit. Thus the result can lay foundation for the development of quick diagnostic kit to detect C. psittaci infection.