Objective To localize and characterize the plasmid protein pORF5 in the Chlamydia trachomatis(Ct) infected cells. Methods The open reading frame encoding for pORF5 protein from the Ct plasmid was amplified and cloned into the pGEX-6p vector. The recombinant plasmid pGEX-pORF5 was transformed into XL1-blue E. coli to express fusion protein with the glutathione-s-transferase (GST). After purified with Glutathione Sepharose 4B beads, the pORF5 fusion protein was used to immunize mice to make monoclonal and polyclonal antibody. The antibodies were used to localize the endogenous pORF5 protein and detect the expression pattern in Chlamydia-infected cells using an indirect immunofluorescence assay (IFA). At the same time, ELISA was used to determine whether pORF5 plasmid protein was expressed and immunogenic during Ct infection in humans. Results pORF5 was detected a dominant signal in the cytosol of the Chlamydia-infected cells with a pattern similar to that of anti-CPAF. pORF5 also appeared in the RBs and EBs in small quantity. Athough pattern was similarly, pORF5 did not overlap with CPAF. pORF5 protein was strongly recognized antiserum in an ELISA. Conclusion The pORF5 plasmid protein was identified as a secreted protein with good immunogenicity, pORF5 gene was to express the endogenous target protein during human infection.

To localize and characterize the hypothetical protein CT358 in the chlamydial infected cells.CT358 gene from the Chlamydia trachomatis(C.trachomatis) serovar D genome was amplified and cloned into the pGEX and pDSRedC1 vectors.The recombinant plasmid pGEX-CT358 was constructed and expressed as GST fusion proteins.The GST-CT358 fusion protein was used to immunize mice to raise the antibodies,which specifically recognized CT358 without cross-reacting with other unrelated proteins.The antibodies were then used to localize the endogenous CT358 protein and determine the expression pattern in Chlamydial infected cells using an indirect immunofluorescence assay(IFA).Meanwhile,pDSRedC1-CT358 recombinant plasmid was transfected to HeLa cells to evaluate the effect of CT358 expression on the subsequent chlamydial infection.The hypothetical protein CT358 was identified in the inclusion membrane of C.trachomatis-infected cells for the first time,and it was detected as early as 12 h after C.trachomatis infection and remained in the inclusion membrane throughout the rest of the infection cycle.Cytosolic expression of CT358 via a transgene failed to affect the subsequent chlamydial infection.These observations together have demonstrated that CT358 is a newly identified chlamydial inclusion membrane protein,giving the potentially importance for further understanding the mechanisms of chlamydial intracellular parasitism.

To localize and characterize the hypothetical protein CT358 in the chlamydial infected cells. CT358 gene from the Chlamydia trachomatis (C. trachomatis) serovar D genome was amplified and cloned into the pGEX and pDSRedCI vectors. The recombinant plasmid pGEX-CT358 was constructed and expressed as GST fusion proteins. The GST-CT358 fusion protein was used to immunize mice to raise the antibodies, which specifically recognized CT358 without eross-reacting with other unrelated proteins. The antibodies were then used to localize the endogenous CT358 protein and determine the expression pattern in Chlamydial infected cells using an indirect immunofluorescence assay (IFA). Meanwhile, pDSRedC 1-CT358 recombinant plasmid was transfected to HeLa cells to evaluate the effect of CT358 expression on the subsequent chlamydial infection. The hypothetical protein CT358 was identified in the inclusion membrane of C. trachomatis-infected cells for the first time,and it was detected as early as 12 h after C. trachomatis infection and remained in the inclusion membrane throughout the rest of the infection cycle. Cytosolic expression of CT358 via a transgene failed to affect the subsequent ehlamydial infection. These observations together have demonstrated that CT358 is a newly identified chlamydial inclusion membrane protein, giving the potentially importance for further understanding the mechanisms of chlamydial intracellular parasitism.

ObjectiveTo purify and characterize the monoclonal antibody (McAb) against Chlamydia trachomatis pORF5 plasmid protein.Methods The hybridoma cells stably secreting specific McAb against pORF5 were cultured in a large scale,and protein G purification by affinity chromatography was used to purify 2H4 McAb.ELISA was used to determine the antibody titer,and identify McAb isotype.Immunofluorescence assay (IFA) and Western blot were performed to detect McAb specificity.Results The purity of 2H4 antibody was 93％,the titer reached 1:1024,and 2H4 McAb was identified to belong to IgG2a isotype,2H4 McAb reacted strongly with the GST-pORF5 fusion protein and endogenous pORF5 protein expressed by Chlamydia trachomatis serovar A,D,L2,Chlamydia muridarum ( MoPn ),Chlamydia psittaci 6BC,but not other chlamydial plasmid proteins and Chlamydia pneumoniae(Cpn) AR39 strain.Conclusion2H4 McAb against pORF5 protein was successfully purified with a high titer and specificity which lay a foundation for further study on pORF5 protein structure and function.

Background:Occult invasive cervical cancer discovered after simple hysterectomy is not common, radical parame?trectomy (RP) is a preferred option for young women. However, the morbidity of RP was high. The aim of our study is to assess the incidence of parametrial involvement in patients who underwent radical parametrectomy for occult cervical cancer or radical hysterectomy for early?stage cervical cancer and to suggest an algorithm for the triage of patients with occult cervical cancer to avoid RP. Methods:A total of 13 patients with occult cervical cancer who had undergone RP with an upper vaginectomy and pelvic lymphadenectomy were included in this retrospective study. Data on the clinicopathologic characteristics of the cases were collected. The published literature was also reviewed, and low risk factors for parametrial involvement in early?stage cervical cancer were analyzed. Results:Of the 13 patients, 9 had a stage IB1 lesion, and 4 had a stage IA2 lesion. There were four patients with grade 1 disease, seven with grade 2 disease, and two with grade 3 disease. The median age of the entire patients was 41years. The most common indication for extrafascial hysterectomy was cervical intraepithelial neoplasia 3. Three patients had visible lesions measuring 10–30mm, in diameter and ten patients had cervical stromal invasions with depths ranging from 4 to 9mm; only one patient had more than 50% stromal invasion, and four patients had lymph?vascular space invasion (LVSI). Perioperative complications included intraoperative bowel injury, blood transfusion, vesico?vaginal ifstula, and ileus (1 case for each). Postoperative pathologic examination results did not show residual disease or parametrial involvement. One patient with positive lymph nodes received concurrent radiation therapy. Only one patient experienced recurrence. Conclusions:Perioperative complications following RP were common, whereas the incidence of parametrial involve?ment was very low among selected early?stage cervical cancer patients. Based on these results, we thought that patients with very low?risk parametrial involvement(tumor size≤2cm, no LVSI, less than 50% stromal invasion, nega?tive lymph nodes) may beneift from omitting RP. Further prospective data are warranted.