Study On Localization And Biological Characterization Of Plasmid Proteins And Predicted Inclusion Membrane Proteins Of Chlamydia Trachomatis

Chlamydia trachamatis(Ct)is the most prevalent sexually transmitted bacterial infection in the world and the infection caused by Ct is an important public-health concern.Asympotomatic and chronic infection may result in severe complications such as ectopic pregnancy,infertility, pelvic inflammatory,prostatitis etc.Studies on chlamydial pathogenesis and searching for an effective vaccine become an area of intensive investigation.Plasmid proteins are highly associated with chlamydial diseases, the plasmid-deficient strain could not cause disease in the oviduct, suggesting plasmid pay role in Chlamydia pathogenesis.On the other hand,proteins localized in the inclusion membrane can potentially play important roles in chlamydial interactions with host cells.Therefore,we carried out some experiments on chlamydial pathogenesis,identification new inclusion proteins and vaccine development in the current study.PartⅠStudy on Localization and Biological Characterization of Plasmid Proteins and the Immunological Effect of pORF5 Vaccine of Chlamydia TrachomatisObjectivesTo construct the prokaryotic expression vector pGEX-6p-pCTs with plasmid protein(pCT)genes of Ct and express the fusion proteins in E.coli.The purified proteins were used to immunize the BALB/c mice for producing polyclonal antibodies and monoclonal antibodies,the antibodies were used to localize the plasmid proteins in the Chlamydia-infected cells.To construct the eukaryotic expression vector pcDNA3.1-pORF5,and evaluate the immunological effect induced by pcDNA3.1-pORF5 combined with pcDNA3.1-IL-12 by intranasal immunization.Methods1.Eight pairs of primers were synthesized according to the plasmid gene sequences of Ct serovar D.PCR was used to amplify the plasmid genes.The genes were cloned into pGEX-6p vector.After induced with IPTG,the fusion proteins were purified using Glutathione Sepharose 4B beads.The purified fusion proteins were used to immunize mice for producing both polyclonal antibodies and monoclonal antibodies(mAbs). Both antibodies were used to localize the endogenous proteins during chlamydial infection.The purified pORF5 protein was used to stimulate RAW264.7 cells to secrete inflammatory cytokines.The distribution pattern,localization and immunogenicity ofpORF5 were also analyzed.2.pORF5 gene was subcloned to pcDNA3.1 eukaryotic expression vector,BALB/c mice were immunized with pcDNA3.1-pORF5, pcDNA3.1-pORF5 plus pcDNA3.1-IL12,pcDNA3.1-IL12,pcDNA3.1, PBS intranasally.The immune effects of pORF5 vaccine were evaluated by detecting antibodies,cytokines,proliferation response of spleen cells, clearance ability of MoPn challenge from genital tract and local histomorphology. Results1.pGEX-6p-pCTs were successfully constructed.The eight pORFs were expressed as GST fusion proteins in E.coli.The ELISA result showed that the 8 plasmid proteins had a good immunogenicity,the specific antibody titers all reached more than 1:6400.17 hybridoma strains were obtained which could stably secret mAbs specific for pCT proteins.2.8 plasmid fusion proteins were positively recognized antiserum,but there was a great variation in both the antibody binding frequency and titer among the plasmid proteins,The ORF5 fusion protein was reacted with 15 human antisera in an ELISA.F6 fragment that only lacks the N-terminal 66 amino acids was recognized by the human antibodies as strongly as the whole pORF5 protein was.3.pORF5 was detected a dominant signal in the cytosol of the Chlamydia-infected cells with a pattern similar to that of anti-CPAF, while the rest 7 proteins inside the chlamydial inclusions only.pORF5 also appeared in the purified RBs and EBs although in small quantity. Monomer,trimer,polymer of pORF5 could be detected in the chlamydial infected cells,pORF5 was expressed as early as 12 hours and prior expression of pORF5 as RFP fusion protein in HeLa cells did not alter the susceptibility of the transfected cells to the subsequent chlamydial infection(p＞0.05).4.pORF5 induced IL-6,IL-8,TNF-αproduction in the Raw264.7 macrophage cultures in a dose dependent manner.When the amount of pORF5 was increased to 10μg/mL,the level of TNF-α,IL-6,IL-8 were 1658.87±255.34pg/mL,7511.55±720.13pg/mL,4643.20±412.24pg/mL separately. 5.The antibody titers were increased as the inoculation times adding, several mice could be detected the specific antibody from mice immunized with pORF5 and pORF5 plus IL-12.Specific antibody increased markedly in two groups at six weeks.Antibody titers continued to rise until 9w after the first immunization,but there was almost unfluctuating in the control mice.The local antibody from genital tract, IFN-γand proliferation response of spleen cells were markly increased in pORF5 plus IL-12 group.6.The mice weights immunized with pORF5 plus IL-12 and pORF5 were slightly decreased and increase quickly compared with the control groups after challenge,the weights were recovered at day 15,12 and 21 in pORF5,pORF5 plus IL-12 and control groups separately.There was a significant reduction in the number of MoPn recovered from the mice vaccinated with pORF5 plus IL-12 compared to the number of MoPn recovered from PBS,pcDNA3.1 or pcDNA3.1-IL-12-treated mice. Moreover,100%of pORF5 plus IL-12 vaccinated mice had successfully resolved the infection by day 18,but the 3 control groups resolved the infection at day 30.7.Gross examination showed that hydrosalpinx was present in oviducts of the control mice,compared to pORF5 plus IL-12 group.The inflammatory response elicited by infection was characterized by a marked mucosal edema,focal erosion and necrosis,fibrosis.The mucosa and the surrounding tissue were infiltrated with lymphocytes in mice immunized with pcDNA3.1,pcDNA3.1-IL-12,PBS.The scores for pcDNA3.1,pcDNA3.1-IL-12,PBS,pORF5,pORF5 plus IL-12 were 3.0±0.4,2.6±0.4,2.4±0.3,1.5±0.3,0.8±0.2 separately. Conclusion1.The pGEX-6p-pCTs recombinants were successfully constructed and the fusion proteins were expressed in E.coli.17 hybridoma strains were obtained which can stably secret mAbs,the mAbs are specific to pORF proteins.2.Each of the 8 plamid fusion proteins has a good immunogenicity and immunoreactivity.3.The plasmid proteins are expressed during human infection, pORF5 is the most immunodominant antigen among the 8 plasmid proteins.It is highly conformation-dependent antigen.pORF5 exists in the Chlamydia-infected cells in the monoer,trimer,polymer manner.4.pORF5 is a secreted protein,which is the second secreted protein followed by CPAF in Ct,while the rest 7 proteins inside the chlamydial inclusions only.5.pORF5 could induce inflammatory cytokines TNF-α,IL-6,IL-8 in mouse macrophages in a dose dependent manner.6.Intranasal immunization with pORF5 plus IL-12 can induce systemic response and protection against MoPn-induced upper genital tract pathology. PartⅡStudy on Localization and Biological Characterization of Predicted Inclusion Membrane Proteins of Chlamydia TrachomatisObjectivesTo construct the prokaryotic expression vector pGEX-6p-InCs of 50 predicted inclusion(InC)membrane protein genes from Ct serovar D and to localize them in the Chlamydia-infected cells with the polyclonal antibodies raised with the fusion proteins.To study the biological characterization of the 50 predicted InC proteins which would benefit to further research on chlamydial pathogenesis.Methods1.Searching for 50 gene sequences encoded the predicted InC proteins from GenBank.Fifty pairs of primers were synthesized according to the InC protein gene sequences of Ct serovar D.PCR was used to amplify InC protein genes.The fragments were directly cloned into pGEX-6p vector to construct pGEX-6p-InC recombinants. Expression of the fusion proteins were induced with IPTG.The GST fusion proteins were purified using Glutathione Sepharose 4B Beads, Western blot was carried out to identify the purified products.2.The GST fusion proteins were used to immunize mice for producing polyclonal antibodies.The fusion protein-specific antibodies were then used to localize the endogenous proteins in Ct-infected cells via an indirect immunofluorescence assay(IFA).The immunogenicities of InC proteins were also carried out by ELISA.3.To further map the immunodominant regions,the 15 ORFs were also cloned into fragments.Expression of the fusion proteins was induced with IPTG.Western blot was used to map the immunogenicity. 4.The predicted InC protein genes were cloned into the pDsRed Monomer C1 mammalian expression vector.The recombinant plasmids were transfected into HeLa cells to express the RFP fusion proteins,then the transfected cultures were subsequently infected with Ct serovar D for analyzing the effect of prior expression of RFP fusion proteins on chlamydial infection after immunofluorescence staining.Results1.The prokaryotic expression recombinants,pGEX-6p-InCs were successfully constructed and all corresponding fusion proteins of 50 InC genes were expressed in E.coli XL1Blue.Most of the predicted InC proteins induced the high titer antibodies in mice.2.The InC protein genes were cloned into the pDsRed Monomer C1 vector successfully,all recombinants expressed RFP fusion proteins and located at cytoplasm.Cytosolic expression of InCs as RFP fusion proteins in HeLa cells did not alter the susceptibility of the transfected cells to the subsequent chlarnydial infection except CT119.The cells expressing RFP-CT119 fusion protein displayed an infection rate of 29.3±9.5% while the adjacent untransfected cells in the same coverslip displayed 64.1±15.4%in the Ct-infected culture.3.15 InC proteins including CT089,CT115,CT116,CT118,CT119, CT147,CT223,CT225,CT226,CT228,CT229,CT484,CT529,CT618,CT813 were the most immunodominant antigens among the predicted 50 InC proteins.To further compared the relative immunodominance between the N- and C-terminal portions of the immunodominant antigens, we found that the C-terminal portions seemed to be consistently more immunodominant than the N-terminal in most proteins with the exception of CT223,CT529 and CT618,on the contrary,these three proteins displayed a more immunodominant N-terminal region.4.The distribution pattern of CT225,CT228,CT358,CT440 were similar to CT119,while the distribution pattern of other 7 proteins including CT058,CT192,CT195,CT383,CT484,CT565,CT850 were similar to MOMP and HSP60.Conclusion1.CT225,CT228,CT358,CT440 were identified to localize in the inclusion membrane,CT058,CT192,CT195,CT383,CT484,CT565,CT850 were detected inside the inclusions for the first time.2.Most of InC proteins have good immunogenicity,suggesting they could be good candidates for developing vaccine.3.The immunogenicity of the InC proteins is mostly localized in the C terminal.4.Cytosolic expression of InCs as RFP fusion proteins in HeLa cells doesn't alter the susceptibility of the transfected cells to the subsequent chlamydial infection except CT119.