The Study Of Location, Immunogenicity And Expression Of Predicted T3SS Effector Proteins In Chlamydophila Psittaci

ObjectiveTo predict effector proteins of type III secretion system in Chlamydophila psittaci,and investigate the intracellular localization and immunogenicity and its expressioncharacters for further studying its structure and function.MethodsIn combination of computeral methods such as Effective T3and BPBAac tool, andthe relevent literatures, T3SS effector proteins were predicted in C.psittaci. DNA wasextracted as the temple, and specific primers were synthesized, then PCR was used toamplify these genes. After digestion, they were ligated into pGEX-6P-1vector, andcloned into to the vector. Sequencing was used to identified the positive recombinantplasmid. Recombinant proteins were induced with IPTG in E.coli BL21, thenanalyzed by SDS-PAGE and Western blot. After optimization, the recombinantproteins were induced in large scale and purified using GST*tag assay kit. After theconcentrations were determined by BCA assay, the recombinated proteins wereimmunized BALB/c mice to evaluate the immunogenicity of these proteins. Then theantibody titer in mice serum was measured by ELISA. Using these polycloneantibodies, the location of the predicted proteins was analyzed with indirectimmunofluorescence in C.psittaci infected cells. The expression characters of theseproteins were detected by Western blot after2,8,24,48h post infection.ResultsCPSIT₀930CPSIT₀767CPSIT₀749CPSIT₀580CPSIT₀420were predict.CPSIT₀930CPSIT₀767CPSIT₀749CPSIT₀580CPSIT₀420were expressedin the soluble state. Specific humoral response were induced by recombinant proteinsin BALB/c mice and the specific antibody titer of CPSIT₀930CPSIT₀767CPSIT₀749CPSIT₀580CPSIT₀420reach to18000. IFA was successfully todetect the asubcellular localization,and the results were showed as following: CPSIT₀930CPSIT₀767CPSIT₀749CPSIT₀580CPSIT₀420were in theinclusion. CPSIT₀930CPSIT₀767CPSIT₀749CPSIT₀580CPSIT₀420wereexpress after2h after CPS infection.Conclusions1. CPSIT₀930, CPSIT₀767, CPSIT₀749, CPSIT₀580and CPSIT₀420haveexcellent immunongenicity and could induce specific humoral immune responses.2. CPSIT₀930, CPSIT₀767, CPSIT₀749, CPSIT₀580and CPSIT₀420werelocalized in inclusion body.3. CPSIT₀930, CPSIT₀767, CPSIT₀749, CPSIT₀580and CPSIT₀420may playa role in C.psittaci prophase infection.