Screening,identification And Immunocompetence Of Effector Proteins Secreted Via Type Ⅲ Secretion System Of Chlamydophila Pneumoniae

Objective: This study attempts to predict and screen effector proteins secreted via type III secretion system of Chlamydophila pneumoniae , and study on characterization and intracellular localization of these putative Chlamydia pneumoniae effector proteins.Methods: Genes of effector proteins secreted via type III secretion system of Chlamydophila pneumoniae were predicted according to the bioinformatics analysis and references. RT-PCR was used to examine the transcription of these genes in Cpn infected cells at 72h pi. Eleven Pairs of Primers were synthesized according to these gene sequenees of Chlamydia pneumoniae strain AR39. PCR was used to amplify these genes,And then these genes were cloned into pGEX -6P-2 vector to induce the expression of corresponding recombinant proteins with IPTG in E.coli BL21,and then purified using Merck GST*tag assay kit. their concentrations were determined by BCA assay, Indirect ELISA were developed by coating microwell plates with the purified proteins, their reliability test and stability test were carried out after optimization.The purified fusion proteins were used to immunize BALB/c mice to produce polyclonalantibodies.Antibodies were used to loealize these proteins in Cpn infected cells at 72h pi.Results: Cpn0704,Cpn0710,Cpn1022,Cpn0810,Cpn0962,Cpn0960,Cpn0422,Cpn0425,Cpn0323,Cpn0322,Cpn0432,Cpn0431,Cpn0434,Cpn0661 were predicted as genes of effector proteins secreted via Cpn T3SS according to the bioinformatics analysis and references, Cpn0704,Cpn0710,Cpn1022,Cpn0810,Cpn0962,Cpn0960,Cpn0422,Cpn0425,Cpn0323,Cpn0322,Cpn0431 transcripted by RT-PCR analysis in Cpn infected cells at 72h pi,Then these genes fragments were amplified by PCR; Restriction enzyme digestion and sequencing showed that the inserted target genes were the purpose fragments, compared with genes reported by GenBank, it had 100% similarity.These fusion proteins was expressed after induced with IPTG,Cpn1022,Cpn0960,Cpn0810,Cpn0704,Cpn0425 were purified. Western blot proved that the recombinant proteins could specifically react with anti-GST polyclonalantibody. Indirect ELISA was successfully developed to detect the antibody to Cpn in human sera, all the positive sera were reacted with the Cpn0810;17 of the positive sera reacted with the Cpn0425;14 of the positive sera reacted with the Cpn0960 and Cpn07704;Only 11 of the positive sera reacted with the Cpn1022. While all the proteins were not reacted with uninfected human sera. Specific humoral response were elicited by recombinant proteins in BALB/c mice and the specific antibody titer was 1∶1 6000, 1∶32 000 and 1∶64 000 respectively. five kinds of recombinant proteins stimulated the BALB/c mice to show fever red skin in leg, disappearanced after 6~10d and delayed-type hypersensitivity were positive.Conclusion:1. Cpn0704,Cpn0710,Cpn1022,Cpn0810,Cpn0962,Cpn0960,Cpn0422,Cpn0425,Cpn0323,Cpn0322,Cpn0431 transcripted in Cpn infected cells at 72h pi;2. Prokaryotic expression vector pGEX-6P-2 / Cpn0704,Cpn0710,Cpn1022,Cpn0810,Cpn0962,Cpn0960,Cpn0422,Cpn0425,Cpn0323,Cpn0322,Cpn0431 were successfully constructed, and the corresponding recombinant proteins were expressed in E.coli BL21;3. Cpn1022,Cpn0960,Cpn0810,Cpn0704,Cpn0425 recombinant proteins showed excellent immunongenicity and could induce the humoral responses efficiently;4. Cpn1022,Cpn0960,Cpn0810,Cpn0704,Cpn0425 recombinant proteins showed better immunoreactivity, could specifically react with Cpn IgG positive sera;...