We compared genotyping by restriction fragment length polymorphism (RFLP) analysis of the amplified omp1 gene with serotyping by dot enzyme-linked immunosorbent assay (dot-ELISA) to determine the suitability of RFLP analysis for epidemiologic study. Fifteen prototypes of Chlamydia trachomatis and 30 clinical isolates were used in this study. To serotype with dot-ELISA, chlamydia antigen was spotted onto a series of replicate nitrocellulose membrane patches and reacted with 11 mAbs that distinguish the 15 known serovars of C. trachomatis. For RFLP analysis, the amplified chlamydia omp1 gene was digested with AluI to differentiate serovars A to K and L1 to L3. Serovars of C, H, I, J, and L3 were further typed by RFLP analysis after digestion with HinfI, and a combination of EcoRI and DdeI. PCR-based RFLP could identify serotype of 28 among 30 clinical isolates tested. The remaining two untypical isolates were probably due to double infections or mechanical transferring error. Serotyping of C. trachomatis isolates shows that serovars E, D, F, and H are the most prevalent types found in urogenital samples in Korea. In this study, we show that RFLP analysis of amplified omp1 gene may be useful in genotyping C. trachomatis isolates.
Animal ; Antibodies, Monoclonal/immunology* ; Bacterial Outer Membrane Proteins/genetics ; Chlamydia trachomatis/immunology ; Chlamydia trachomatis/genetics ; Chlamydia trachomatis/classification* ; Genotype ; Mice ; Mice, Inbred BALB C ; Polymerase Chain Reaction* ; Polymorphism, Restriction Fragment Length* ; Serotyping

OBJECTIVETo construct a recombinant eukaryotic expression plasmid pcDNA3.1-Ct MOMP168 including Ct MOMP multi-epitopes gene, and evaluate the Ct MOMP-specific humoral and cellular immune response induced by pcDNA3.1-Ct MOMP168 in BALB/c mice.

METHODSRecombinant plasmid pcDNA3.1-Ct MOMP168 including Ct MOMP multi-epitopes gene was constructed. Then, BALB/c mice were randomly assigned to receive (intramuscular injection) either pcDNA3.1-Ct MOMP168 or pcDNA3.1 or PBS (n = 12, 100 microg/time per mouse), and the same immunization schedule was repeated for the third time at 2 week intervals. The titers of anti-Ct MOMP antibody and its antibody subtypes in sera, the cytotoxicity of Ct MOMP-specific cytotoxic T lymphocyte (CTL) in spleen, and the level of cytokine (IFN-gamma, IL-4, IL-10)-producing CD3(+) T cells in spleen were detected by ELISA, LDH release assays and intracellular cytokine staining-fluorescence activated cell sorter (ICS-FACS), respectively.

RESULTSThe recombinant plasmid pcDNA3.1-Ct MOMP168 was able to induce Ct-specific antibody response (A(490) = 0.973 +/- 0.136; serum titer was 1:1000) as compared with pcDNA3.1 (A(490) = 0.180 +/- 0.025) and PBS (A(490) = 0.110 +/- 0.015), and the major antibody subtype was IgG2a with statistical significance (F = 106.884, P < 0.05). When the ratio of effector cells and target cells reached to 50:1, the activity of cytotoxic T-lymphocyte in pcDNA3.1-Ct MOMP168 immunized mice (41.71% +/- 8.34%) was significantly higher (F = 22.315, P < 0.05) than that in pcDNA3.1 immunized mice (18.40% +/- 3.45%) and PBS immunized mice (14.50% +/- 2.42%). The levels of CD3(+) IFN-gamma(+) T cells in pcDNA3.1-Ct MOMP168 immunized mice (1.15% +/- 0.16%) were significantly higher (F = 99.638, P < 0.05) than that in pcDNA3.1 immunized mice (0.12% +/- 0.08%) and PBS immunized mice (0.09% +/- 0.03%), while the significant difference in the levels of IL-4(+) CD3(+) T cells and IL-10(+) CD3(+) T cells was not observed (F = 0.886 and 1.112, P > 0.05) between pcDNA3.1-Ct MOMP168 immunized mice (0.13% +/- 0.08% and 0.14% +/- 0.08%) and pcDNA3.1 (0.07% +/- 0.05% and 0.13% +/- 0.06%) or PBS immunized mice (0.08% +/- 0.04% and 0.07% +/- 0.04%).

CONCLUSIONIn BALB/c mice, the recombinant plasmid pcDNA3.1-Ct MOMP168 might induce not only the generation of Ct-specific antibody, but also the high level of Ct MOMP-specific CD3(+) IFN-gamma(+) T cells.

Animals ; Bacterial Outer Membrane Proteins ; genetics ; immunology ; Bacterial Vaccines ; immunology ; Chlamydia trachomatis ; genetics ; immunology ; Immunization ; Male ; Mice ; Mice, Inbred BALB C ; Porins ; genetics ; immunology ; T-Lymphocytes, Cytotoxic ; immunology

To predict the B cell epitopes for major outer membrane protein (MOMP) of Chlamydia trachomatis (CT), the secondary structure of CT MOMP was predicted by the methods of GOR based on the sequence of amino acids of E serotype CT MOMP. By combining the comprehensive analysis of transmembrane domain, hydrophilicity profile, surface probability, antigenic index and average flexibility, the B cell predominant epitopes of CT MOMP were further predicted. The N-terminal No. 73-81, 217-225, 377-386, 261-270 and 161-175 were the predominant B cell epitopes. Prediction of the B cell epitopes for the CT MOMP by the multi-parameters is helpful for the identification of B cell epitopes.
Amino Acid Sequence ; Antigens, Bacterial ; immunology ; Chlamydia trachomatis ; classification ; immunology ; Epitopes, B-Lymphocyte ; immunology ; Molecular Sequence Data ; Porins ; immunology ; Protein Conformation ; Protein Structure, Secondary ; Serotyping

OBJECTIVESTo evaluate the clinical significance of the detection of IgG and IgM antibodies against Chlamydia trachomatis (CT) in semen of asymptomatic infertile patients.

METHODSOne hundred and sixteen asymptomatic infertile patients and eighteen fertile males were selected randomly. The routine parameter analysis of semen was fulfilled by computer aided semen analysis(CASA). Then the seminal plasma was separated and the IgG, IgM antibodies against CT in seminal plasma were determined with ELISA method.

RESULTSIgG and IgM antibodies against CT were present in 13.8% (16/116) and 3.4% (4/116) of the semen of infertile patients, while for the fertile males the percentages were 11.1% (2/18) and 0, respectively. There were no differences between the two groups(P > 0.05). In the infertile patients, 22 patients were azoospermia. And in the rest 94 infertile patients, the percentages of IgG and IgM antibodies in abnormal sperm density group were 21.4% (6/28) and 7.1% (2/28), which were higher than those in normal group, but there were no statistical differences(P > 0.05). Similarly, the IgG, IgM antibodies were not correlated with the sperm motility(P > 0.05). The positive percentage of CT in 116 patients was 25.9% (30/116).

CONCLUSIONSThe percentages of IgG and IgM antibodies against CT in semen of asymptomatic infertile patients are similar to that in fertile males, which do not correlate with the changes of semen parameters, and may not be used for indication of CT infection.

Adult ; Antibodies, Bacterial ; blood ; Chlamydia trachomatis ; immunology ; Humans ; Immunoglobulin G ; blood ; Immunoglobulin M ; blood ; Infertility, Male ; microbiology ; Male ; Sperm Count ; Sperm Motility

No abstract available.
Adolescent ; Adult ; Antigens, Bacterial/analysis ; Chlamydia Infections/*diagnosis/epidemiology/microbiology ; Chlamydia trachomatis/immunology ; DNA, Viral/analysis ; Female ; Humans ; Mass Screening/*methods ; Papillomavirus Infections/*diagnosis/epidemiology/virology ; Papillomavirus, Human/genetics ; Poland/epidemiology ; Prevalence ; *Sexual Behavior ; Urban Population

Heat shock proteins (HSP) have been identified as an important factor of a very complex and highly conserved cellular defense mechanism to preserve cell survival under adverse environmental conditions. HSP 60 are immunodominant antigens of microbe such as Chlamydia trachomatis and have a potentiality to become a target antigen due to antigenic similarity between chlamydial and human HSP. This study was conducted to investigate the effects of Vero cell coculture to anti-HSP 60 on the early mouse embryo development in vitro. The 2-cell mouse embryos (ICR) were cultured and mouse embryo development was observed every 24 hr for 3 days. 45% and 22.1% of the embryos cultured in Ham's F-10 plus anti HSP 60 with Vero cells developed to the 4- to 8- cell stage (day 1) and morular stage (day 2) as compared with 29.2% and 2.7% of those cultured without Vero cells respectively. But at day 3, the beneficial effect of Vero cells was not noted. These findings suggest that Vero cells have some roles to overcome the detrimental effect of anti-HSP 60 to some degree. These results suggest that Vero cells coculture will promote reproductive outcome in patient previously sensitized to microbial (e.g. Chlamydia trachomatis) HSP 60.
Vero Cells ; Pregnancy ; Mice, Inbred ICR ; Mice ; Male ; Infertility, Female/etiology/immunology/therapy ; Immunodominant Epitopes ; Female ; Embryonic Development/*immunology ; Coculture Techniques ; Chlamydia trachomatis/immunology/pathogenicity ; Chaperonin 60/*immunology ; Cercopithecus aethiops ; Antigens, Bacterial ; Antibodies, Monoclonal/*administration & dosage ; Animals

OBJECTIVETo obtain monoclonal antibodies (mAbs) against Chlamydia trachomatis Tarp protein.

METHODSChlamydia 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.

RESULTSRecombinant 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.

CONCLUSIONThe highly specific mAbs against Tarp protein have been obtained to facilitate further study of the structure and function of Chlamydia Tarp protein.

Animals ; Antibodies, Monoclonal ; biosynthesis ; Antibody Specificity ; Bacterial Proteins ; immunology ; Cell Line, Tumor ; Chlamydia trachomatis ; immunology ; HeLa Cells ; Humans ; Mice ; Mice, Inbred BALB C ; Nuclear Proteins ; immunology ; Recombinant Fusion Proteins ; immunology

OBJECTIVETo 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.

METHODSThe 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.

RESULTSThe 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.

CONCLUSIONSOmcBc 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.

Animals ; Antibodies, Bacterial ; blood ; Antigens, Bacterial ; immunology ; metabolism ; Bacterial Outer Membrane Proteins ; genetics ; immunology ; Chlamydia trachomatis ; genetics ; immunology ; metabolism ; Cloning, Molecular ; Genes, Bacterial ; Humans ; Immune Sera ; immunology ; Mice ; Mice, Inbred BALB C ; Plasmids ; Rabbits

OBJECTIVETo obtain Chlamydia trachomatis heat shock protein (cHSP) 10 gene from clinical secretion samples.

METHODScHSP10 gene was amplified from 20 cases of clinical secretion samples with positive gold-labelling by specific primers of cHSP10 and identified by sequence analysis.

RESULTScHSP10 full-length gene was amplified from 1 of 20 cases of clinical secretion samples with positive gold-labelling. cHSP10 gene encoding 102 amino acids contains 306 bp, which nuclotide at position 194 changes from T to A, leading to the change of corresponding amino acid.

CONCLUSIONScHSP10 gene may be cloned from clinical secretion samples with positive gold-labelling, which make it possible to further construct expression plasmid of recombinant cHSP10.

Antibodies, Bacterial ; blood ; Bacterial Proteins ; genetics ; Base Sequence ; Chaperonin 10 ; chemistry ; genetics ; immunology ; Chlamydia trachomatis ; chemistry ; immunology ; Cloning, Molecular ; Female ; Humans ; Infertility, Female ; etiology ; Male ; Molecular Sequence Data ; Polymerase Chain Reaction

OBJECTIVETo clone the plasmid protein pORF8 of Chlamydia trachomatis and localize its expression in Chlamydia-infected cells.

METHODSpORF8 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).

RESULTSThe 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).

CONCLUSIONThe 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.

Animals ; Antibodies ; immunology ; Bacterial Proteins ; biosynthesis ; genetics ; Chlamydia Infections ; metabolism ; Chlamydia trachomatis ; chemistry ; genetics ; metabolism ; Cloning, Molecular ; Escherichia coli ; genetics ; metabolism ; Genetic Vectors ; genetics ; HeLa Cells ; Humans ; Mice ; Mice, Inbred BALB C ; Plasmids ; biosynthesis ; genetics ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; immunology