Production Of Monoclonal Antibody Against Recombinant Protein CPAFm From Chlamydophila Pneumoniae

Objective: To prepare monoclonal antibodies against protein encoding immuno-dominant epitope (181～400aa, CPAFm) of Chlamydial protease-like activity factor(CPAF)from Chlamydophila pneumoniae(Cpn) and establish indirect ELISA to detect samples from patients who suffering respiratory passage disease and evaluate the value of the mAb in Cpn infection diagnosis.Methods: The immunodominant region epitope gene of CPAF from C.pneumoniae CWL029 was chosen and amplified by PCR. The PCR product was subcloned into pGEX6p-2 expression vector, and transformed into E.coli BL21. After being identified by PCR, enzymes cleavage analysis and sequencing the recombinant protein was induced to expressed by IPTG, and then analyzed by SDS-PAGE and Western blot. The recombinant protein was purified with glutathione S-transferase (GST) affinity chromatography, then analyzed by SDS-PAGE and Western blot. The concentration of the recombinant protein was detected with Nucleic Acid and proteinum analysator. After BALB/c mice immunized with the purified recombinant CPAFm,spleen cells of the immunized mice with higher antibody titer was fused with SP2/0 cells, and hybridoma cells secreting specific antibody were screened. Ascites were induced to produce mAbs and purified with ammonium sulfate precipitation method. The titer and isotype of the antibodies were identified with indirect ELISA. Then indirect ELISA was established with the mAb and used to detect 500 throat swabs from patients who suffering repiratory passage diseases.Results: Recombinant plasmid of pGEX6p-2/CPAFm was constructed and a recombinant protein with an estimated Mr of 51kDa was expressed in E.coli BL21. The mice serum Ab titer was above 1:16000 after immunized with the purified CPAFm. We got four clones that can stably secret mAbs named 8E3, 9G4, 13D1 and 13D10. Indirect ELISA and Western-blot analysis indicated that all the four mAbs can react with recombinant CPAFm. The four positive clones secreting mAb were injected in the abdominal cavitise of BALB/c mice and the ascites were collected and titrated after purified by salt fractionation. The isotype of mAb secreted by 8E3 and 13D1 was IgG1, the isotype of mAb secreted by 13D10 and 9G4 strains was IgG2b. Of the 550 clinic samples, no one was positive with indirected ELISA using CPAFm mAbs as first antibody, but seven were positive with pstⅠgene amplification.Conclusions:1. Four hybridoma cells secreting specific antibody were screened and all belonged to IgG isotypes, and all the mAbs could react with the recombinant CPAFm and native CPAF.2. The titer of the ascetic mAbs is low, and may be the possible reason of low sensitivity of the indirect ELISA based on the mAbs in clinical diagnosis. Objective: To study the propagation pattern of Chlamydia trachomatis serotype L2 in vitro, further explore the continuous culture method, and optimize the growth conditions of serotype L2 in vitro, and lay basis for L2 isolation from clinical specimens and further studies of the related experiments about L2.Methods: Human epidermoid carcinoma-2(HEp-2), Henrietta lacks strain of cancer cells(HeLa229), Human hepatoma cell line(HepG-2), Gastric cancer cells (SGC-7901) and African green monkey kidney aneuploid cells (Vero cells) were infected with C. trachomatis L2, then stained with fluorescent antibody, and inclusion bodies were observed at different times after infection under a fluorescence microscope to compare the sensitivity of the five cells to L2 infection, At the same time the influence of DEAE-dextran and cycloheximide were detected by comparing the inclusion shapes and the quantity of nucleic acid by quantitative Real-Time PCR at 12h, 24h, 36h and 48h after L2 infection.Results: After 24h post infection, HEp-2 cells, Vero cells, HepG-2 cells, HeLa cells and gastric cancer cells were swelled at different degrees, and inclusion bodies could be found in cytoplasm and occupied the entire cytoplasm at about 40-48h after infection. Fluorescent antibody staining and Real-Time PCR results showed that HeLa cells were in the highest infection rate among the 5 cell lines and in which L2 growed fastest, while HepG-2 cells were in the lowest infection rate. DEAE-dextran had no obvious influence on the infection rate and growth. In cycloheximide-treated group, L2 growed faster and the quantity of Chlamydia nucleic acid were higher than the control group. Conclusions:1) HEp-2 cells, Vero cells, HepG-2 cells, HeLa cells and SGC-7901 cells were successfully infected with Chlamydia trachomatis L2, and showed different sensitivity to L2, and the life cycle was different in different cell line;2) Cycloheximide had significant effects on the shapes and densities of the inclusion bodies;3) DEAE dextran showed no significan influences on the infection rate of L2.