[Studies on the establishment of a method to detect HPV 6b E7-specific antibodies of serum and cervical secretion from patients with condyloma acuminatum and its epidemiological significance].

OBJECTIVE: To establish a method for detection of the human papi11omavirus (HPV) 6b E7-specific antibodies in serum and cervical secretion from patients with condyloma acuminatum (CA). METHODS: A full-length HPV 6b E7 gene was amplified by PCR from the CA tissue to construct the recombinant plasmid pET32a(+)/HPV 6b E7. The expression of prokaryotic protein was analyzed by SDS-PAGE and Western blot, then purified with Ni-NTA Agarose affinity column and used as an diagnostic antigen for establishing indirect ELISA method, to detect specific serum IgG and specific cervical secretion sIgA from 56 CA patients, 81 healthy control. Sera from 43 cervical cancer was served as control. HPV 6b DNA from 56 CA patients was identified by PCR. RESULTS: Data showed that the nucleotide homology of cloned sequence was 99.5%, compared to the standard sequences of HPV 6b E7 gene (GenBank accession number: NC001355). A high level expression of E7 fusion protein was obtained in prokaryotic expression system (40 µg/ml). Based on HPV 6b E7 fusion protein being used as coating antigen, results from ELISA showed that the absorbance rates (A) of serum IgG from CA, cervix cancer and healthy control groups were 1.82 ± 0.48, 1.36 ± 0.39 and 1.39 ± 0.27, respectively. The level of IgG antibody in the serum of CA group was significantly higher than that in cervix cancer group and healthy control (P < 0.05). The A values of cervical secretion sIgA in CA and healthy control groups were 0.63 ± 0.26 and 0.53 ± 0.06, respectively, while the level of sIgA antibody in the cervical secretion of CA group was also significantly higher than that in healthy controls (P < 0.05). The positive rate of HPV 6b E7 DNA in CA tissue was 78.6% (44/56) by PCR method. When compared the results measured by PCR, the HPV 6b E7-specific IgG and sIgA antibodies by ELISA used to detect the patients infected with HPV 6b infection, showed that the sensitivity rates were 68.2% (30/44) and 54.6% (24/44) respectively, and the specificity were all 100% (12/12). CONCLUSION: Based on the serum and cervical secretion specific HPV 6b E7 antibodies from patients with CA to diagnose HPV 6b infection, results showed medium sensitivity and high specificity, and could further be used to investigate the epidemiological characteristics of HPV 6b infection.

[Immunogenicity of multi-epitopes gene of major outer membrane protein of Chlamydia trachomatis].

OBJECTIVE: To 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. METHODS: Recombinant 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. RESULTS: The 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%). CONCLUSION: In 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.

[Preparation and identification of immune serum against recombinant fusion protein of rhoptry 2 and major surface protein 1 from Toxoplasma gondii].

OBJECTIVE: To prepare and identify immune serum against the recombinant fusion protein of rhoptry 2 (ROP2) and major surface protein 1(P30) from Toxoplasma gondii. METHODS: The constructed recombinant plasmid of pET28b/ROP2-P30 was transformed to a bacterium BL21-Codon Plus (DE3)-RIL strain and was expressed under IPTG induction. Cells were lysed by multiple rounds of sonication to obtain supernatant and inclusion body respectively. The inclusion body was washed with 2 mol/L and 4 mol/L urea to remove the nonspecific protein. The washed products dissolved in 8 mol/L urea were received by SDS-PAGE. Two rabbits were immunized with the fusion protein rROP2-P30 and sera from the rabbits were collected. Immune diffusion test, indirect ELISA and Western-blot were used to detect antibody titer and specificity of the immune serum against rROP2-P30. RESULTS: Immune diffusion test demonstrated that specific immune serum were obtained. Indirect ELISA confirmed that the antibody titer in the serum reached 1:12 800 and the rROP2-P30 was recognized by specific IgG in this serum by Western-blot analysis. CONCLUSION: Specific immune serum against the recombinant fusion protein rROP2-P30 has been prepared.