Expression, Purification Of CPAF Immunodominant Domain Recombinant Protein Of Chlamydia Trachomatis And Its Clinical Diagnosis

Objective:To clone and express the Chlamydial Protease-like Activity Factor(CPAF)immunedominant domain gene of C.trachomatis, and assess the immunocompetence of the expressed product. Indirect ELISA and indirect immunofluorescent assay were constructed to evaluate its significance in the diagnosis of C.trachomatis.Methods:The immunedominant domain epitope of C.trachomatis CPAF was chosen by computer analysis, then was amplified from C.trachomatis serovar D complete genome by polymerase chain reactions, subcloned into the expression vector pGEX-6p-2 to generate recombinant plasmid pGEX-6p-2/CPAF, then expressed in E.coli BL21, and analyzed by SDS-PAGE and Western-blot. The recombinant protein was purified with GST agarose gel FF after renaturation, and the concentrations of the purified protein were determined by A280 ultraviolet spectrophotometry. The expressed and purified protein were identified by SDS-PAGE and Western-blot. NewZealand rabbits were tested for its immunogenicity. The immunoreactivity was evaluated by Western-blot and indirect ELISA. Indirect ELISA was developed to detect control sera and the antibodies to C.trachomatis in human sera. The polyclonal antiserum from immunized rabbits by recombinant protein was purifiedand and used to detect C.trachomatis antigen in the secretion of human gential tract by indirect ELISA and IIF. Results:Recombinant plasmid pGEX-6p-2/CPAF was successfully constructed. SDS-PAGE proved that the recombinant protein with molecular weight about 43×103 was expressed effectivly. Western-blot indicated that the recombinant protein could specifically react with GST mouse mAb and C.trachomatis IgG positive sera. Specific humoral response was elicited after introducing the recombinant protein to NewZealand rabbits and the specific IgG antibody titer was above 1:16 000. The result of detecting 60 control sera showed the sensitivities and the specificities of the Indirect ELISA were both 100%(30/30). The IgM and IgG concordance between the developed indirect ELISA test and the ELISA test by Jingmei corporation were 96.8% and 98.4% respectively. The antiserum could react with the C.trachomatis antigen in clinical secretion of human gential tract, the concordance of the antiserum detection C.trachomatis antigen by indirect ELISA and IIF to PCR method were 91.9% and 90.0%, respectively.Conclusions:1. Prokaryotic expression vector pGEX-6p-2/CPAF was constructed successfully and Ct CPAF with molecular weight near 43×103 was well expressed in E.coli BL21(DE3).2. Ct CPAF recombinant protein showed excellent immunongenicity and can induce the humoral responses in NewZealand rabbits efficiently.3. The expressed recombinant protein showed excellent immunoreactivity, can specifically react with C.trachomatis positive sera, and could be applied in C.trachomatis serodiagnosis.4. The antiserum could specifically detect the Ct antigen in clinical secretion of human gential tract by indirect ELISA and IIF, and coule be applied in detecting C.trachomatis antigen.