| Objective:The recombinant expression vector containting the immunodominant region (181～400aa, CPAFm) gene of the Chlamydial protease-like activity factor(CPAF)from Chlamydophila pneumoniae was constructed and expressed in E.coli. After purified, the immunocompetence of the expressed protein was analyzed. Indirect ELISAs were developed to detect antibodies and antigen respectively, and the value of the recombinant protein in clinical diagnosis was evaluated.Methods:The immunodominant region epitope gene of CPAF from C. pneumoniae CWL029 strain was chosen according to bioinformatics analyses and references, then was amplified by PCR. The PCR product was subcloned into pGEX6p-2 expression vector, and transformed into E.coli BL21 after being identified by PCR, enzymes cleavage analysis and sequencing. After the E.coli BL21 containing the recombinant plasmid was induced by IPTG, the recombinant protein was expressed, and then analyzed by SDS-PAGE and Western blot. The recombinant protein was purified with (glutathione S-transferase, GST) agarose gel FF after renaturation and its concentrations were determined by A280 ultraviolet spectrophotometry. Indirect ELISA was developed by coating microwell plates with the purified protein, then its reliability test and stability test were carried out after optimization, and its cross reaction with Chlamydia trachomatis was analyzed by detecting C. trachomatis positive sera. The immunogenicity of the recombinant protein was tested by immuning NewZealand rabbits and detecting the titers of specific antibodies in rabbits'sera with the indirect ELISA and the imported ELISA kit based on the whole C. pneumoniae bacterium. The immunoreactivity of the recombinant protein was tested by the indirect ELISA to detect C. pneumoniae reference sera and the Western blot using human anti-C. pneumoniae positive sera as the primary antibody. 300 sera samples of patients with respiratory tract infection were detected with gold standard method microimmunofluorescence (MIF) as reference method to assess the value of the recombinant protein in serodiagnosis. Anti-GST-CPAFm polyclonal antibody was used as primary antibody after purification, then another indirect ELISA was developed for detecting C. pneumoniae antigen. 120 sputum and throat swabs were detected with imported PCR reagent as reference test to investigate the value of the recombinant protein for detection C. pneumoniae antigen in early infection.Results:660 bp fragment of CPAF gene was selected and obtained by PCR amplification. The restriction enzymes cleavage analysis and nucleotide sequencing showed the target gene was successfully inserted into pGEX6p-2 prokaryotic expression vector. The similarity had 100% between the inserted gene with CPAF gene reported in Genbank by BLAST analysis. The SDS-PAGE demonstrated that the GST-CPAFm recombinant protein with relative molecular weight about 51.3×103 was expressed after the E.coli BL21 containing the recombinant plasmid was induced by IPTG, and mainly existed in the pattern of inclusion body. Its purity reached up to 95% after purification with GST agarose gel FF. The Western blot proved that it could specifically react with C. pneumoniae positive sera. The indirect ELISA used the purified recombinant protein as coating antigen was developed and optimized. The average inter-batch coefficient of variation(CV) and average intro-batch CV of its reliability test were 6.52% and 8.08% respectively. The average inter-batch CV of its stability test was 6.93%. The indirect ELISA showed that no cross reaction with C. trachomatis after detecting anti-C. trachomatis positive sera. After the NewZealand rabbits were immuned with the purified recombinant protein, the titers of the specific IgM and IgG antibodies were above 1﹕8 000 and 1﹕16 000 respectively.The sensitivities and specificities of the indirect ELISA were both 100%(40/40) for C. pneumoniae IgG and IgM reference sera. The concordance rate of IgG between the indirect ELISA test and the MIF test to 300 patients sera was 99.0% and that of IgM was 98.3%. The purified anti-GST-CPAFm polyclonal antibody was used to develope another indirect ELISA for C. pneumoniae antigen. The concordance rate of antigen detection between the indirect ELISA reagent and an imported PCR reagent to 120 sputum and throat swabs was 88.3%.Conclusion(1) Cpn CPAF immunodominant region epitope (181～400aa, CPAFm) gene was amplified by PCR, and pGEX6p-2/CPAFm prokaryotic expression vector was successfully constructed, and then transformed into E.coli. A recombinant protein with relative molecular weight near 51.3×103 was expressed.(2) Recombinant protein showed excellent immunogenicity, and could induce NewZealand rabbits to produce specific IgG and IgM antibodies with high titers.(3) Recombinant protein showed excellent immunoreactivity, and could specifically react with C. pneumoniae positive sera, and could be applied to C. pneumoniae serodiagnosis.(4) Recombinant protein showed excellent application value in serodiagnosis and antigen detection for C. pneumoniae infection.
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