Study On Expression And Function Of CT703 Protein In Chlamydia Trachomatis-infected Cells

Objective:1. To investigate the expression pattern of CT703 protein in Chlamydia trachomatis-infected cells by Western Blot and immunofluorescence assay and investigate the expression change of CT703 protein under Chlamydia trachomatis persistent infection.2. To investigate whether CT703 protein can activate Raf/MEK/ERK signaling pathway and investigate the antiapoptotic avtivity of CT703 protein.Methods:1. The full length of CT703 gene of Chlamydia trachomatis was amplified by RT-PCR, and was cloned into the prokaryotic expression vector pGEX-6p-1.2. The fusion protein GST-CT703 was expressed in the E. coli BL21 strain induced with IPTG, and then was separated and purified with SDS-PAGE gel, purified fusion protein was used to raise antigen-specific antibodies.3. To detection the expression pattern of CT703 protein by Western Blot and immunofluorescence assaies under chlamydia trachomatis acute infection.4. To examine the expression change of CT703 mRNA by RT-PCR and that of protein by Western Blot assay under Chlamydia trachomatis persistent infection induced with IFN-y.5. To construct the recombinant eukaryotic expression plasmid of pcDNA4/CT703. To investigate whether CT703 protein can activate Raf/MEK/ERK signaling pathway and examine the apoptotic rate and caspase-3 activity induced by STS in HeLa cells transfected transiently with pcDNA4/CT703.Results:1. About 1.5kb DNA band amplified by RT-PCR was seen in the 1% agarose gel, which was consistent with the entire CT703 gene fragment. Digested with the restriction endonuclease EcoR?and No t?, about 1.5kp DNA fragment was cleaved off from the recombinant plasmid. Sequence analysis revealed that the fragment inserted into the pGEX-6p-1 vectors was 100% homologous to the published nucleotide sequence in GeneBank.2. The recombinant expression plasmid pGEX-6p-1/CT703 was overproductive in E. coli BL21 induced with IPTG. The product was an 81-kDa fusion protein composed of the 26-kDa pGEX-6p-1 vector expression protein and the 55-kDa CT703 protein, which was consistent with prediction Molecular Weight. The purified fusion protein showed one clear band on SDS-PAGE and was detected with anti-GST antibody by Western Blot. The mouse antiserum could bind to CT703 protein specially. The result showed the antibody titer examined by ELISA reached to 1:32000.3. The expression of CT703 protein in Chlamydia trachomatis-infected cells was examined by Western Blot assay; the result showed that CT703 protein was first detected at 24 h after infection, and the protein levels increased with progression of infection, but not in the parallel uninfected HeLa cells. In IF A, the CT703 protein was monitored as early as 12 h after infection. The expression of CT703 mRNA and protein was not time-dependent under persistent infection induced with IFN-?. Both the CT703 mRNA and protein production greatly decreased at the same time points under persistent infection, as compared with under acute infection.4. Localization of CT703 protein in Chlamydia trachomatis-infected cells by indirect immunofluorescence assay. The result showed that the localization of CT703 protein was different from CPAF and CT813 protein which were known plasmosin and Inc protein, respectively.5. Eukaryotic expression plasmid pcDNA4/CT703 was constructed successfully, which was demonstrated by PCR, restriction enzyme digestion and sequence analysis. The expression of CT703 protein in HeLa cells after the plasmid was transfected was demonstrated by Immunofluorescence assay and Western Blot.6. The eukaryotic expression plasmid pcDNA4/CT703 was transfected transiently into cells, and then cultured 36 h, the pRaf and pERK were detected by Western Blot assay, and the result demonstrated CT703 protein can not activate Raf and MEK. The apoptotic rate and caspase-3 activity induced by STS for 5h was examined in HeLa cells transfected transiently with pcDNA4/CT703 for 36 h. The HeLa cell induced with STS, Chlamydia trachomatis-infected HeLa cells induced with STS and alone HeLa cells were as the control groups. The apoptotic rates were (93.1±2.01)%?(91.3±1.67)%?(3.21±0.87)%?(2.08±0.76)% respectively. The apoptotic rate of STS-induced cells transfected transiently with pcDNA4/CT703 was significant difference when compared with HeLa cells group and Chlamydia trachomatis-infected cells group induced with STS (P<0.05), but no significant difference when compared with HeLa cells group induced with STS (P>0.05). The result of caspase-3 activity was consistent with that of apoptotic rate. Conclusions:1. The prokaryotic expression plasmid pGEX-6p-1/CT703 and eukaryotic expression plasmid pcDNA4/CT703 was constructed successfully.2. The expression of CT703 protein was time-dependent increase under Chlamydia trachomatis acute infection, but was not time-dependent under persistent infection. CT703 protein production greatly decreased at the same time points under Chlamydia trachomatis persistent infection, as compared with under Chlamydia trachomatis acute infection.3. CT703 protein can not activate Raf/MEK/ERK signaling pathway, and can not inhibit cell apoptosis induced with STS.