Preliminary Localization Of The Predicted Effector Proteins Cpn0423 And Cpn0710 Secreted Via T3SS Of Chlamydophila Pneumoniae

Objective:To localize the predicted effector proteins Cpn0423 and Cpn0710 secreted via T3SS in Hep-2 cells infected with Cpn, and evaluate their immunogenicity. To detect the distribution of proteins Cpn0423 and Cpn0710 in Hep-2 cells infected with Cpn. To deserve the morphologically of Cpn inclusion bodies at different time.Methods:The recombinant plasmid pGEX6p-2/Cpn0423 and pGEX6p-2/Cpn0710 were extracted, andtransformed to E.coli BL21 to express the fusion proteins Cpn0423 and Cpn0710, SDS-PAGE and Western-Blot were used to analyze and identify the expressional products after being purified by using Merck GST tag assay kit. The concentrations of the fusion proteins Cpn0423 and Cpn0710 were determined by BCA assay.The purified fusion proteins were used to immunize BALB/c mice to produce polyclonal antibodies.Antibodies were used to preliminarily localize the proteins Cpn0423 and Cpn0710 in Hep-2 cells infected with Cpn with indirect immunofluorescence assay(IFA). ELISA was used to detect the polyclonal antibodies titers, and to determine the immunogenicity of Cpn0423 and Cpn0710 proteins.The distribution of proteins Cpn0423 and Cpn0710 were observed in Hep-2 cells infected with Cpn at 6h, 12h, 24h, 36h, 48h, 72h, 96h, 120h. IFA was used to analyze the morphology of Cpn inclusion bodies.Results:The fusion proteins Cpn0423 and Cpn0710 were highly expressed after induced with IPTG. and the fusion proteins Cpn0423 and Cpn0710 were expressed in dissolvable form after ultrasonication. Cpn0423 and Cpn0710 were purified with Merck GST tag assay kit,and analysed by SDS-PAGE, and the purity of proteins was above 95%.The purified fusion proteins were used to immunize BALB/c mice, the ELISA result showed that proved the polyclonal antibodies were 1:16000 and 1:12000 respectively. The Hep-2 cells were infected successfully, we discovered that the singals of Cpn0423 and Cpn0710 were detected signals in the cytosol of the Hep-2 cells infected with Cpn. Morphologically analyze the change of Cpn inclusion bodies with the time.Conclusion:(1) The fusion proteins Cpn0423 and Cpn0710 were successfully expressed and purified.(2) Cpn0423 and Cpn0710 have good immunogenicity.(3) Cpn0423 and Cpn0710 may be the secreted proteins.