Development of ELISAs for the detection of urogenital chlamydia trachomatis infection targeting the pORF5 protein.

OBJECTIVE: To prepare antibodies against pORF5 plasmid protein of Chlamydia trachomatis and develop double-antibody sandwich enzyme-linked immunosorbent assays (DAS-ELISAs) for the detection of genital C. trachomatis infections. METHODS: The pORF5 protein was expressed in Escherichia coli and used to immunize BALB/c mice and New Zealand rabbits to produce monoclonal antibodies (mAbs) and polyclonal antibody (pAb) for DAS-ELISAs. Clinical samples from 186 urogenital infection patients (groups I) and 62 healthy donors (groups II) were detected in parallel by the DAS-ELISAs developed in this study and by IDEIA PCE commercial ELISA. RESULTS: Two hybridoma cell lines, named 2H4 and 4E6, stably secreting specific mAbs against pORF5 were obtained. The mAb 2H4 was recognized by 32 (17.20%, positive recognition rate) and 25 (13.44%), mAb 2H4 by 0 (0%) and 2 (3.22%) samples from groups I and II, respectively. The sensitivities of mAbs 2H4 and 4E6 were 92.11% and 77.78% and the specificities were 100% and 96.88%, respectively in relation to the IDEIA PCE commercial ELISA. The sensitivities of detection for the DAS-ELISAs were 10 ng/mL (based on 2H4) and 18 ng/mL (based on 4E6). CONCLUSION: Two DAS-ELISAs were developed in this study that provided a feasible and effective assay that could be considered alternative tools for the serodiagnosis of C. trachomatis infection.

[Cloning and cellular localization of pORF8 plasmid protein of Chlamydia trachomatis].

OBJECTIVE: To clone the plasmid protein pORF8 of Chlamydia trachomatis and localize its expression in Chlamydia-infected cells. METHODS: pORF8 gene was amplified and cloned into pGEX-6p vector, and the pORF8 fusion protein was expressed in E.coli XL1 Blue. After purification with glutathione-conjugated agarose beads, the pORF8 fusion protein was used to immunize BALB/c mice to generate polyclonal antibodies against pORF8 protein. The antibodies obtained were used to localize the plasmid protein pORF8 in Chlamydia-infected cells with immunofluorescence assay (IFA). RESULTS: The pORF8 gene 744 bp in length was successfully cloned and the GST fusion protein with a relative molecular mass of 54 000 was obtained. The cellular distribution pattern of the plasmid protein pORF8 was similar to that of the major outer membrane protein (MOMP), a known C. trachomatis inclusion body protein, but not to that of chlamydial protease-like activity factor (CPAF, a secreted protein). CONCLUSION: The plasmid protein pORF8 is localized on the bacterial organism as an inclusion body protein in C. trachomatis-infected cells. The cellular location of pORF8 protein can potentially provide important insights into the pathogenesis of C. trachomatis.

[Preparation and identification of monoclonal antibodies against Chlamydia trachomatis Tarp protein].

OBJECTIVE: To obtain monoclonal antibodies (mAbs) against Chlamydia trachomatis Tarp protein. METHODS: Chlamydia trachomatis serovar D recombinant Tarp fusion protein was cloned and expressed. Balb/c mice were immunized with recombinant Tarp fusion protein, and the spleen cells of the immunized mice were fused with SP2/0 mouse myeloma cells. The hybridoma cell lines secreting mAbs against Tarp protein were screened by an indirect immunofluorescence assay and subcloned by limiting dilution culture. The specificities of these mAbs to Tarp were determined by ELISA, and their isotype and chlamydial species specificity identified by an indirect immunofluorescence assay. RESULTS: Recombinant GST-Tarp fusion protein with a relative molecular mass of about 136 000 was successfully cloned and expressed. Seven hybridoma cell lines stably secreting specific mAbs against Tarp protein were obtained. All the 7 mAbs reacted strongly with Tarp protein but not with other chlamydial proteins. Two mAbs were identified to belong to IgG2a isotype and the other 5 to IgG1 isotype. All the 7 mAbs reacted strongly with chlamydia serovar A, D, and L2, but not with MoPn, 6BC, or AR39. CONCLUSION: The highly specific mAbs against Tarp protein have been obtained to facilitate further study of the structure and function of Chlamydia Tarp protein.

[Early production of interleukin-17 in airway upon Chlamydia trachomatis infection increases the local secretion of IL-6 and MIP-2].

OBJECTIVE: To evaluate the early interleukin-17 (IL-17) production in airway upon Chlamydia trachomatis infection and its relationship with the secretion of interleukin-6 (IL-6) and macrophage inflammatory protein 2 (MIP-2) in local site. METHODS: In vivo, a murine model of pneumonia induced by intranasal inoculation with Chlamydia trachomatis mouse pneumonitis (MoPn, now classified as a new species C. muridarum) was used for the study. Chlamydial growth in the lung was assessed by inoculating HeLa cell monolayer with lung homogenates followed by enzyme-linked immunosorbent assay (IFA). IL-17, IL-6 and MIP-2 were measured by enzyme-linked immunosorbent assay (ELISA). Mice without infection acted as the control group. In vitro, L929 cells were pretreated with recombinant murine IL-17 (rmIL-17) at a dose ranging from 20, 100 to 500 µg/L for 24 h then infected with MoPn for 24 h. The supernatants were harvested and tested for IL-6 and MIP-2 concentration using ELISA. The cells were assayed for the number of inclusion-forming unit (IFU) by IFA. L929 cells without pretreatment with rmIL-17 but infected with MoPn was the control group. RESULTS: The study showed that in vivo, Chlamydial growth in the lung was found on day 1 after infection, and reached its peak at day 8 (6.49±0.19, lg IFU/lung) with subsequent decline in quantity. IL-17 peaked at 48 h (83.0 ng/L±35.8 ng/L) while IL-6 peaked on day 3 [(3.98±0.04) µg/L], MIP-2 peaked on day 8 [(2.19±0.71) µg/L]. The study showed that in vitro, compared with control group [(55.10±16.54) ng/L for IL-6 production and (13.71±0.84) ng/L for MIP-2], L929 cells pretreated with rmIL-17 at the different concentrations of 20, 100 and 500 µg/L for 24 h then infected with MoPn for 24 h, could significantly increase IL-6 (P <0.01) and MIP-2 secretion (P <0.05). The productions of IL-6 in the supernatants were (531.65±24.40), (629.95±7.71), and (646.51±35.92) ng/L. Meanwhile, the productions of MIP-2 were (107.21±28.40), (181.95±25.51), and (221.90±17.32) ng/L, respectively. RmIL-17 alone had no effect on IL-6 and MIP-2 secretion, and no direct effect on growth of chlamydial inclusion body was demonstrated either. CONCLUSION: IL-17 was produced early in airway upon Chlamydia trachomatis, and rmIL-17could induce IL-6 and MIP-2 production in L929 cells after infection with MoPn. These suggest that an early IL-17 response may play an important role by inducing the secretion of IL-6 and MIP-2 in initiating host defense against infection with Chlamydia trachomatis in the airway.

[Cloning and expression of Chlamydia trachomatis OmcBc gene and antigenicity analysis of the protein].

OBJECTIVE: To investigate the antigenicity of recombinant Chlamydia trachomatis (Ct) OmcBc protein and search for the new target for early diagnosis of Chlamydia infection and Chlamydia vaccine development. METHODS: The C fragment of OmcB encoding the amino acids from T270 to T553 was amplified from Chlamydia serovar D genomic DNA. The pGEX-6p-Ct OmcBc expression plasmid was constructed and transformed into E.coli XL-1blue. The expression of recombinant Ct OmcBc protein was induced by IPTG. Serum samples were collected from 120 patients with urogenital Chlamydia infection. The antiserum samples were collected from 7 New Zealand white rabbits and 5 Balb/C mice immunized subcutaneously and intraperitoneally with Ct serovar D inactivated EB, respectively, and from 9 Balb/C mice intranasally infected with Ct serovar D live EB. The anti-Chlamydia specific antibody were titrated by an immunofluorescence assay (IFA). The reactivity of the recombinant OmcBc protein with all the above antisera was detected by ELISA. RESULTS: The pGEX-6p-Ct OmcBc expression plasmid was successfully constructed. DNA sequencing showed that the inserted OmcBc was about 852 bp, encoding a protein with 284 amino acids. The expression of the recombinant GST-OmcBc was induced by IPTG, producing a fusion protein with a molecular weight of about 57 kD. The titer of the specific antibodies to Chlamydia in all the antisera was high. ELISA results showed strong reactivities of the recombinant GST-OmcBc fusion protein with all the above antisera. CONCLUSIONS: OmcBc protein is an immunodominant protein of Chlamydia. The recombinant GST-OmcBc with strong antigenicity may provide a basis for further study of early diagnosis of chlamydia infection and development of Chlamydia vaccine.

Three-dimensional reconstruction of the uterine vascular supply through vascular casting and thin slice computed tomography scanning.

It was the objective of this study to construct a model of the uterine vascular supply through vascular casting and thin slice computed tomography scanning. This will provide a teaching aide for the understanding of uterine artery embolization (UAE) procedures, as well as normal uterine and ovarian arterial anatomy. Using 20% chlorinated poly vinyl chloride, we infused and cast a set of a normal uterus, vagina and bilateral adnexa through the uterine artery and ovarian artery. After thin slice CT scanning, we obtained the three-dimensional (3D) reconstruction by maximum intensity projection (MIP) and surface-shaded display (SSD), and then observed its figure and characteristics. A model of the uterine vascular supply can be successfully reconstructed by vascular casting and thin slice CT scanning. The 3D reconstruction offers a clear view of the course of the uterine artery and its blood supply distribution. It has two major branches: The intramuscular uterine branch and the cervicovaginal branch (1). Blood supply is generally unilateral, with communicating branches between the two sides and possible anastomoses between the arterial blood supply of the uterus and the ovaries. The major blood supply of the cervix comes from the cervicovaginal branch of the uterine artery, while the vaginal arterial supply derives directly from the internal iliac artery. The CT technique allows real-time 360 degrees rotation and changes in model for in-depth study of the vascular network and its adjacent tissues. It is possible to construct an in vitro uterine arterial network by vascular casting and CT scanning, which can provide unique insight into the female genitourinary system arterial network. Based on this, we can create reconstructions as well as models for different diseases such as leiomyomata, adenomyosis, and endometrial cancer. These models will provide morphological evidence to the interventional therapy and UAE teaching in Obstetrics and Gynecology.

[Localization of the hypothetical protein CT249 in the Chlamydia trachomatis inclusion membrane].

To characterize the hypothetical protein CT249 using antibodies raised with CT249 fusion protein. The open reading frame (ORF) coding for CT249 in the Chlamydia trachomatis serovar L2 genome was cloned into the pGEX-6p2 vector using the restriction enzymes BamH I and Not I. The recombinant plasmid pGEX-6p2-CT249 was transformed into XL1-blue bacteria and the gene CT249 was expressed as fusion proteins with the glutathione-s-transferase (GST) tagged to the N-terminus. The GST-CT249 fusion protein was used to immunize mice and the mouse anti-fusion protein antibody was used to localize the endogenous CT249 protein in Chlamydia-infected cells using an indirect immunofluorescence assay (IFA). The CT249 gene with 351bps in length was successfully cloned and expressed as GST fusion protein with molecular weight of 38.2kDa. The anti-fusion protein antibodies produced from mice detected the hypothetical protein CT249 in the inclusion membrane of Chlamydia trachomatis-infected cells. Using antibodies raised with GST-CT249 fusion protein, the hypothetical protein CT249 have been identified as a Chlamydia trachomatis inclusion membrane protein. Given the potentially important role of inclusion membrane proteins in chlamydial interactions with host cells, this finding has provided a useful tool for further understanding the mechanisms of chlamydial intracellular parasitism.