沙眼衣原体主要外膜蛋白多表位基因的免疫原性研究

目的 构建包含沙眼衣原体(Chlamydia trachomatis,Ct)主要外膜蛋白(major outermembrsne protein,MOMP)多表位基因的真核表达质粒PcDNA3.1-Ct MOMP168,检测其诱导BALB/c小鼠产生Ct MOMP特异性体液和细胞免疫反应的水平.方法 构建包含Ct MOMP多表位基因的重组质粒PcDNA3.1-Ct MOMP168并以肌肉注射方式免疫BALB/c小鼠,同时设置pcDNA3.1组和PBS组对照(每组12只,每只100μg/次).采用酶联免疫吸附试验(ELISA)、乳酸脱氢酶释放法及细胞内细胞因子染色-流式细胞术(ICS-FACS)分别检测免疫小鼠血清中Ct MOMP特异性抗体的滴度及其抗体亚型、脾脏中细胞毒性T淋巴细胞(CTL)的杀伤活性及特异性分泌细胞因子,如γ-干扰素-(IFN-γ)、白细胞介素-4(IL-4)、白细胞介素-10(IL-10)等的CD3+T细胞的水平.结果 同pcDNA3.1(A490=0.180±0.025)和PBS免疫(A490=0.110±0.015)相比,PcDNA3.1-Ct MOMP168免疫可刺激小鼠产生高效价Ct特异性IgG抗体(A490=0.973±0.136;血清滴度1:1000),且抗体亚型以IgG2a为主,差异有统计学意义(F=106.884,P<0.05);pcDNA3.1-Ct MOMP168免疫组小鼠脾细胞中CTL杀伤活性(41.71%±8.34%)高于pcDNA3.1组(18.40%±3.45%)和PBS组(14.50%±2.42%),差异有统计学意义(F=22.315,P<0.05;效靶比为50:1);pcDNA3.1-Ct MOMP168免疫组小鼠的脾细胞中Ct MOMP特异性CD3+IFN-γ+T细胞的水平(1.15%±0.16%)高于pcDNA3.1组(0.12%±0.08%)和PBS组(0.09%±0.03%),差异有统计学意义(F=99.638,P<0.05),而PcDNA3.1-CtMOMP168组IL-4+CD3+T细胞(0.13%±0.08%)和IL-10+CD3+T细胞(0.14%±0.08%)的水平与pcDNA3.1(0.07%±0.05%,0.13%±0.06%)和PBS(0.08%±0.04%,0.07%±0.04%)组比较差异无统计学意义(F值分别为0.886、1.112,P值均>0.05).结论pcDNA3.1-Ct MOMP168重组质粒能诱导BALB/c小鼠产生Ct MOMP特异性的体液免疫和细胞免疫应答.

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 MOMPI68 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 μg/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-γ, 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 O-specific antibody response (A490=0.973±0.136; serum titer was 1: 1000) as compared with pcDNA3.1 (A490=0.180±0.025) and PBS (A490=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-γ+ 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-γ+ T cells.