Isolation, Cultivation, Identification And Preliminary Observation In Mice Inoculated Of C.pneumoniae From Clinical Specimens

Objective:This study attempts to isolate, culture and identify Chlamydophila pneumoniae(Cpn) from clinical specimens , observe the general condition and pulmonary lesions in mice after inoculation of Cpn,to provide high-quality bacteria for study of Cpn.Methods: Collected swab and lavage fluid specimens from patients with respiratory tract infections,select conserved sequence 16srRNA Cpn-specific primers, screening positive for Cpn infection samples using polymerase chain reactions (PCR) method ,Isolated and cultured Cpn from clinical specimens using cell culture,then identified them with Giemsa staining, indirect immunofluorescence, 16SrDNA sequence homology and phylogenetic analysis,established animal models to carry out a preliminary study of their virulence.Results: 15 cases of positive samples were amplified from 450 specimens,the positive samples were inoculate on the Hep-2 cells, two strains isolated from clinical specimens were identified as Cpn, 16SrRNA sequence homology and phylogenetic analysis showed a high homology with Cpn standard strains ,the lungs of mices were injected clinical isolates occurred significant pathological changes.Conclusion: Two Cpn clinical strains were isolated and cultured successfully,they will lay a foundation for further study of Cpn. Objective:This study attempts to explore the immune activity of Pmp6 and Pmp21 from Cpn, to investigate the futher significance in the clinical serology diagnosis.Methods:The immunodominant region epitope gene of Pmp6 and Pmp21 of Cpn AR-39 strain were chosen according to the bioinformatics analysis and references, then were amplified from Cpn AR-39 by polymerase chain reactions (PCR) respectively. The PCR products were directly cloned into pGEX6p-2 expression vector, then transformed into E.coli Blue21 after being identified by PCR and sequencing respectively. E.coli Blue21 with the recombinant plasmid were expressed, and analyzed by SDS-PAGE and Western blot. The recombinant proteins were purified with GST?Bind? Resin and their concentrations were determined by BCA assay. Indirect ELISA was developed by coating microwell plates with the purified protein, then its reliability test and stability test were carried out after optimization, the immunogenicity of the recombinant protein was tested by immuning BALB / c mice and the titers of specific antibodies in mices'sera were detected with the indirect ELISA. The immunocompetence of the recombinant protein was analyzed by Western blot and indirected ELISA respectively. 120 sera samples of patients with respiratory tract infection were detected with indirected ELISA and Bio-Quant C.pneumoniae IgG ELISA Kits to assess the value of the recombinant protein on clinic diagnosis.Results:The 448bp~1203bp base sequence in Pmp6 genes (fragment length of 752bp, encoding 252 amino acids) and 154bp~885bp base sequence in Pmp21 genes (732bp fragment length , encoding 244 amino acids) were selected as the interested epitope after analysized by software, then the fragments were amplified by PCR amplification, the nucleotide sequencing showed the target genes were successfully inserted into pGEX6p-2 prokaryotic expression vectors , and the sequences were correct. The recombinant proteins with molecular weight about 50kDa(Pmp6)and 52kDa(Pmp21)were obtained after expression and purification, and its purity reached up to 95% after purification with GST?Bind? Resin. Western blot proved that these two recombinant proteins could specifically react with C. pneumoniae pAb respectively. After the BALB/c mice were immuned with the purified recombinant protein, the pAb titers of the specific IgG antibodies were detected by indirect ELISA, and the titers of Pmp6 and Pmp21 were higher than 1:6 400, 1:128 00 respectively. And the indirect ELISA was successfully developed to detect the IgG antibody of Cpn in human sera, the coincidence rate of IgG between the indirect ELISA test and Bio-Quant C.pneumoniae IgG ELISA Kits to 120 patients sera were 91.7% (Pmp6) and 97.5% (Pmp21).Conclusion:1) The recombinant protein of GST-Pmp6 and GST-Pmp21 showed excellent immunongenicity and could efficiently induce the humoral responses in BALB/c mice.2) GST- Pmp6 and GST- Pmp21 showed specificity and sensitivity, which could be applied to the serological diagnostic antigen candidate of C. pneumoniae.