Chlamydia trachomatis is one of the major causes of sexually transmitted diseases (STDs). It has also been shown to be associated with ocular, neurological and urogenital diseases (Hausler, 1998). Trachoma is endemic in the Middle East, North Africa, and India. An estimated 400-500 million people world wide are infected with the serovar associated with trachoma, and 7-9 million blinded as a result (Mandell, 2001). So recognition and exact identification of the infection are very important. Detection of this organism by multiplication in a wide range of animal derived cell lines or the chick embryo yolk sack takes a long time and thus it is impossible to implement them as routine diagnostic methods. So the molecular and serological tests using specific recombinant antigens are the best diagnostic procedures (Hausler, 1998; Mandell, 2001).
Chlamydia is a spherical or ovoid obligatory intracellular bacterium that undergoes a characteristic and well defined dimorphic life cycle within eukaryotic host cells. The infective form is the elementary body (EB), 200-300 nm in diameter, which survives outside the host. EB form develops within the host cell into the intracellular replicative form known as the reticulate body (RB), which is 600-1000 nm in diameter (Balous and Dwerden, 1998).
The dominant antigen at the surface of the infectious Chlamydia EB is the MOMP, encoded by the omp1 gene, which was identified simultaneously by tjree independent groups in 1981 (Caldwell et al., 1981). This antigen of approximately 40 KDa is the basis for the serological classification of C. trachomatis into 15 or more serotypes (Hoelzle et al., 2004). In approaches involving the detection of Chlamydia antigens in clinical specimens, such as direct immunofluorescence staining (DFA) with fluorescein-conjugated monoclonal antibodies and enzyme-linked immunosorbent assays (ELISA), antibodies have been prepared against either the Chlamydia MOMP or the cell wall Lipopolysaccharide (Mandell, 2001).
The ELISA test with a sensitivity of 80% and a specificity of 91% is the best approach for detection of Chlamydia as reported by Mygind et al. (2000). In this study a molecular genetic approach was used to generate and evaluate recombinant antigens for use in a species-specific C. trachomatis immunoassay. A fragment of the MOMP of C. trachomatis was produced as a species specific recognition antigen by ELISA.
MATERIALS AND METHODS
Sample and DNA extraction: The C. tachomatis cell culture was a gift from Dr. Badami, Tehran University of Medical Sciences, and was used in the following DNA extraction procedure. Briefly, the cell culture containing chlamydia trachomatis was incubated in lysis buffer (320 mM sucrose, 10 mM Tris base, 5 mM MgCl2, 1% SDS, 40 µg/ml of proteinase K) for 4h, boiled for 15 min. and centrifuged at 10000 ×g for 10 min. The supernatant was transferred to a new microtube and used for the PCR reaction.
Primer design: Specific primers were designed based on the gene sequence of the Chlamydia MOMP (1183 bps) containing start (ATG) and termination codons. We designed SacI and BamHI restriction sites at the 5´ end of forward and reverse primers respectively. CtmoF 5´-GAG CTC ATA TGA AAA AAC TCT TGA AAT CGG-3´ and CtmoR 5´-GGA TCC TTA GAA GCG GAA TTG TGC ATT TA-3´ .
PCR reaction: The PCR reaction was conducted as follows: 1 × PCR buffer, 0.1mM of dNTPs, 1.5 mM MgCl2, 0.1 mg of DNA, 20 pmol each of forward and reverse primers and 1 unit of Taq DNA polymerase (Cinnagen, Iran). The final volume of the reaction was set to 50 ml by adding distilled water. The reaction was transferred to thermocycler machine for PCR amplification. The PCR cycling program was: denaturation at 94ºC for 30 sec, annealing at 52ºC for 30 sec and extension at 72ºC for 45 sec, and repeated for 30 times. Reaction was settled at 94ºC and 72ºC for 5 min before and after PCR cycling, respectively (Pherson et al., 2000). The PCR product was electrophoresed on 1% agarose gel (Fermentas), stained by ethidium bromide and the DNA band was observed under a UV transilluminator. PCR product was confirmed by restriction analysis.
Gene cloning: PCR product (MOMP gene) was electrophoresed on 1% low melting point (LMP) agarose gel and the DNA band was sliced under long wave UV. DNA was recovered using the DNA purification kit (Fermentas Cat. No. k0513). Recovered PCR product and the Eco RV blunt digested pBluescript were 3´ tailed with dATP and dTTP respectively using terminal deoxy nucleotidyl transferase (Eun, 1996; Gaastra and Klemm, 1984). PCR product was ligated (Gaastra and Hansen 1984) into pBluescript via the T/A cloning method, transformed into competent cells of the Escherichia coli XLI-blue strain (Hanaham, 1983) and dispensed onto LB agar plates containing 100 mg/ml ampicillin. Colonies were screened on agar plates supplemented with X-gal or IPTG to discriminate between recombinant (white) and non-recombinant (blue) forms.
The recombinant plasmid was digested by SacI and BamHI restriction enzymes, which established on 5´ ends of forward and reverse primers respectively, generating a 1183 bp DNA band fragment encoding the MOMP gene. Digested plasmid was electrophoresed on 1% LMP agarose gel and the resulting DNA fragment (MOMP gene) was sliced and recovered by the DNA purification kit (Fermentas). The purified DNA fragment was sub cloned in to digested PGEMEX1 (Pasteur, Iran)expression vector using SacI and BamHI restriction enzymes (Fermentas), and transformed in E. coli XL1-blue competent cells. Positive colonies containing the plasmid were mass cultured in LB medium. Recombinant plasmid was extracted (Feliciello and Chinali, 1993) and confirmed by restriction analysis.
Gene expression: Expression was performed as described previously (Spiro et al., 1997), but with some modifications. Briefly, the E. coli strain JM109 was transformed with the pGemex-1-MOMP plasmid and selected on LB agar containing 50 mg/ml of ampicillin. The transformant was inoculated into 3 ml culture tube containing modified YT medium [1.2% (w/v) bacto trypton, 2.4% (w/v) yeast extract, 0.04% (v/v) glycerol, 1% (w/v) M9 salts] [M9 salts medium contains: 6.4% (w/v) Na2H2O4. 7H2O, 1.5% (w/v) KH2PO4, 0.025% (w/v) NaCl, 0.05% (w/v) NH4Cl] (Merk) and allowed to grow at 37ºC in a shaker at 160 rpm, over night. The following day, the cultured bacteria were inoculated into a 50 ml flasks containing YT medium and allowed at 37ºC in a shaker, at 200 rpm.
Cultures in the logarithmic phase (at OD600 of 0.6) were induced for 6 hour with 1 mM IPTG. After induction cells were lysed in 5x sample buffer [100 mM Tris HCl pH 8, 20% (w/v) glycerol, 4% (w/v) SDS, 2% (w/v) beta-mercaptoethanol, 0.2% (v/v) bromo phenol blue] (sigma) and analyzed on 12% (v/v) SDS-PAGE (Smith, 1984a). The resulting gel was stained with coomassie brilliant blue R-250 (Smith, 1984b). The uninduced control culture was analyzed in parallel.
Protein purification: Purification was performed as described previously (Spiro et al., 1997), but with some modifications. Briefly, transformant from LB agar plates were used for preparing the pre inoculation in modified YT medium containing 50 mg/ml ampicillin. The cells were grown at 37ºC to OD600 of 0.6-0.8, followed by IPTG (1mM) induction for 6 h at 37ºC. After centrifugation at 6500 rpm for 10 min. the cell pellet was suspended in 50 ml of equilibration buffer (50 mM Tris, 0.5 M NaCl) containing protease inhibitor cocktail (Sigma). The cell suspension was sonicated (2 × 30 S) on ice. The cells were harvested by centrifugation at 4000 ×g for 15 min, then resuspended in 5 ml of ice-cold buffer containing 6M urea and incubated on ice for one hour. The insoluble material was removed by centrifugation at 12000 ×g for 20 min. The supernatant was filtered through a 0.45 mm membrane before binding to the resin. The recombinant protein was purified on its N-terminal T7 Tag by affinity chromatography (Jack, 1998). The column was equilibrated with 15 ml of Bind Wash buffer (42.9 mM Na2HPO4, 14.7 mM KH2PO4, 27 mM KCl, 1.37 mM NaCl, 1% tween 20 and 0.02% (v/v) Na3N). The filtered supernatant was dialyzed in order to remove urea and then applied to the column and allowed to bind during which the flow rate was 15 drops/min. The bound protein was eluted by Elution buffer (1M citric acid, pH 2). The eluted protein fraction was neutralized using 1.5% neutralization buffer (2M Tris base, pH 10.4). The sample was dialysed overnight in 500 ml of bind/wash buffer that was replaced four times over a period of 24h. Dot blot analysis was carried out using T7-Tag monoclonal Ab or patients’ serum and Horse Radish Peroxidase (HRP) conjugated goat anti-mouse IgG to estimate the expressed protein in E. coli cells collected 6 h post IPTG induction (Novagen Company Kit No. 69025-3).
Western blotting: Purified recombinant protein was electrophoresed on SDS-PAGE, transferred electrophoretically to nitrocellulose sheet and detected by colorimetric method. Briefly, nitrocellulose sheet was immersed in 3% bovine serum albumin in Tris-buffered saline, 0.1% Tween 20 at room temperature for 1 h to block excess protein-binding sites. The nitrocellulose sheet was reacted with a human serum infected Chlamydia at room temperature and washed in TBS 0.1% Tween 20. Immune reactions were identified with goat anti-human immunoglobulin conjugated to horseradish peroxidase. Color development was observed by addition of 4-chloro-1-naphthol as substrate (Campbell et al., 2001).
Dot blotting: Induced cells were lysed and dot blotted on nitrocellulose membrane (Shewry, 1998). Nitrocellulose membrane blot was exposed to the primary antibody (Chlamydia infected human serum) followed by the secondary antibody (human anti IgG peroxidase conjugated) and detected as described in previous section.
Applying of recombinant protein for ELISA: Polystyrene microtiter plates were coated with purified recombinant protein as described (Bora et al., 2002). To exchange the chromatography elution buffer and to remove any urea, the purified recombinant protein was dialysed for 2h at 4ºC with carbonate/bicarbonate sodium buffer (0.1 M, pH 9.5). The absorbance dialysed protein (OD=1.6) was diluted up to 20-fold to reach to OD= 0.07 (as determined by antigen serial dilution testing to obtain the optimum amount of antigen to be coated onto the Nunc Maxcisorb flat 96-well plates). After coating on the plates and incubating overnight at 4ºC. ELISA procedure was carried out with OPD ( Dako) +H2O2 as substrate. This project had 11 negative and 6 positive volunteers (confirmed by the Viro-Immun Anti- C. trachomatis-IgG Kit) as control which were diluted up to 1:40 and 100 ml added to each well . The OD of the reaction was measured at 492 nm (620 nm reference filter) with a Tekan ELISA reader. The mean value of the negative serum was 0.75 and all positive samples were higher than mean discriminating the positive serums from negative ones.
Chlamydia MOMP gene obtained from cultured C. trachomatis was amplified using specific primers. PCR product was tested by restriction analysis. Figure 1 illustrates the PCR product.
Recombinant plasmid (pGemex-1-momp) was transformed in JM109 E. Coli and was induced using 1 mM IPTG. Bacterial samples were collected before induction and at 3h intervals after induction and confirmed by SDS-PAGE, gel diffusion, dot blot and western blot analysis. Protein expression was optimized at 6h after induction. Figure 2 shows expressed protein on SDS-PAGE, Figures 3 and 4 show the results of dot blotting and gel diffusion confirmatory tests, respectively. Western blot analysis of purified protein is shown in Figure 5. In this study, the cloning, expression and purification conditions of the 39 KDa protein belonging to the C. trachomatis major outer membrane protein were accomplished. ELISA test: The purified protein was coated onto Nunc Maxcisorb flat 96-well plates and tested by ELISA. We compared 11 negative and 6 positive women sera who were confirmed by PCR and reconfirmed by Medoc kit (specific diagnostic kit based on a synthetic peptide of an immunodominant region of MOMP) at the 1/40 dilution, the results were significantly higher for positives. The PCR product of C. trachomatis major outer membrane protein gene was sequenced and deposited to Gene Bank under accession number: EF363779.
The organism, C. trachomatis, most clearly associated with non gonococcal uretritis (NGU) is an obligate intracellular parasite that causes as many as 50% of cases of NGU. Also trachoma, one of the leading causes of blindness in the world is caused by C. trachomatis. Urogenital tract infections are very common among sexually active people, partly due to C. trachomatis causing 30-50% of NGU and lymphogranoloma venerum (LGV). C. trachomatis is also responsible for non-genital diseases such as trachoma and keratoconjunctivitis which are major problems in societies with low hygiene.
The specific way of bacterial detection is sample inoculation into yolk sack of 7-8 day old chicken embryo (Hausler, 1998 and Mandell, 2001). Chlamydia can also be grown in a wide range of animal derived cell lines (McCoy, Hella 229, BAMK and SHK2), but this is not applicable since it requires experience, talent and is very time consuming (Campbell et al., 2001a; Bas et al., 2001; Gdoura et al., 2001; Bas et al., 2001b). Immunologic tests are now of more use, taking advantage of recombinant technology to produce specific bacterial proteins.
Bas et al. (2001a) presented the MOMP protein as a sensitive and specific antigen to detect the antibacterial antibodies. Gdoura et al. (2001) used the recombinant proteins in as ELISA to detect Chlamydia in seminal fluid. They compared the detection results of chlamydial infection by ELISA with the results obtained by PCR and cell culture. In this study the MOMP gene of C. trachomatis was cloned and expressed. Because the serological methods are preferred for detection of chlamydial infection. So we designed a diagnostic kit rely on species specific antigen. This study showed that the recombinant Chlamydia MOMP protein is suitable for differentiating completely between positive and negative sera by ELISA method.
The biologically active recombinant major outer membrane protein of Chlamydia trachomatis expressed in E. coli is useful in detection of species specific antigen in immunoassays.
The authors would like to express their gratitude to the Iranian Medical Biotechnology Network for providing financial support (grant No. 1192) to carry out this research.