中文摘要：

目的 探讨人乳头瘤病毒（HPV）6b结构蛋白L1和沙眼衣原体（Ct）主要外膜蛋白（MOMP）多表位嵌合DNA（HPV6b L1/Ct MOMP）诱导BALB/c小鼠产生特异性细胞免疫效应,及HPV6b L1对Ct MOMP多表位基因诱导细胞免疫的增强作用.方法 将Ct MOMP多表位基因连接在经真核密码子优化的HPV6b L1羧基端,构建嵌合重组质粒pcDNA3.1（＋）/HPV6b L1/Ct MOMP多表位,经酶切和测序证实后,转染COS-7细胞,间接免疫荧光鉴定其在真核细胞中的表达;将纯化后的嵌合重组质粒肌肉注射免疫BALB/c小鼠,并设置pcDNA3.1（＋）/Ct MOMP多表位质粒、pcD-NA3.1（＋）和PBS三组对照.采用0、2、4周免疫程序,DNA剂量为每只150μg/次.于末次免疫后2周,分别用乳酸脱氢酶（LDH）释放法和细胞内细胞因子染色-流式细胞术（ICS-FAGS）检测小鼠脾细胞对Ct MOMP多表位蛋白和HPV6b L1蛋白的特异性CTL活性,胞内细胞因子IFN-γ、IL-4和IL-10产生水平等细胞免疫应答指标.结果免疫后,在效靶比（E∶T）分别为40∶1、20∶1时,pcDNA3.1（＋）/HPV6b L1/Ct MOMP多表位嵌合重组质粒免疫组小鼠产生了针对Ct MOMP多表位蛋白特异性CTL杀伤活性（44.56%±4.02%,35.35%±2.89%）和HPV6b L1蛋白特异性CTL杀伤活性（27.08%±2.04%,21.68%±4.06%）,与各对照组比较差异有统计学意义（F=72.87,F=114.55,P＜0.05;F=30.04,F=10.47,P＜0.05）,且显著高于pcDNA3.1（＋）/Ct MOMP多表位质粒组（35.50%±2.68%,30.24%±1.75%;12.27%±3.36%,9.32%±3.07%;P＜0.05）;而pcDNA3.1（＋）/Ct MOMP多表位质粒组小鼠仅显示了Ct MOMP多表位特异性的CTL杀伤活性,与对照组比较差异也有统计学意义（F=58.85,F=120.21;P＜0.05）.胞内细胞因子IFN-γ的产生水平,pcDNA3.1（＋）/HPV6b L1/Ct MOMP多表位嵌合质粒组（4.34%±0.06%）高于pcDNA3.1（＋）/Ct MOMP多表位质粒组（3.14%±0.18%）,与对照组比较差异有统计学意义（F=473.83,P＜0.05）,而重组质粒组较空载体组和PBS对

英文摘要：

Objective To study on the specific cellular immune response produced in BALB/c mice immunized with human papillomavirus （HPV） type 6b capsid protein L1 and Chlamydia trachomatis （Ct） major outer membrane protein（MOMP） multi-epitope chimeric DNA （HPV6b L1/Ct MOMP multiepitope） , and the enhancement of the specific cellular immune response to Ct MOMP multi-epitope by HPV6b L1. Methods The Ct MOMP multi-epitope gene was connected to the C terminal of HPV6b L1,the gene of HPV6b L1 had been optimized according to the codon usage of eukaryotic system, and then the HPV6b L1/Ct MOMP multi-epitope chimeric gene was cloned to pcDNA3.1 （ ＋ ） vector. After identification by restriction enzyme digestion and DNA sequencing, the recombinant plasmid was transfected into COS-7 cells, Indirect Immunofluorescence （IIF） was used to confirm the expression of proteins. Then, BALB/c mice were randomly assigned to receive （intramuscular injection） either pcDNA3.1 （ ＋ ）/HPV6b L1/Ct MOMP or pcDNA3.1 （ ＋ ）/Ct MOMP or pcDNA3.1 （ ＋ ） or PBS （ n = 12, 150 μg/time）, and the same immunization schedule was repeated third times at 2 week intervals. The level of cytokine（ IFN-γ, IL-4, IL-10） -producing CD3＋ T cells in spleen, the cytotoxicity of Ct MOMP-specific and HPV6b L1-specific cytotoxic T lymphocyte （CTL） in spleen were detected by intracellular cytokine staining-fluorescence activated cell sorter （ICS-FACS） and LDH release assays, respectively. Results After immunization, when the efCTL （44.56%±4.02%, 35.35% ±2.89% ） and HPV6b L1 specific cytotoxicity of CTL （27.08% ±2.04%, 21.68% ±4.06% ） in pcDNA3.1 （ ＋ ）/HPV6b L1/Ct MOMP multi-epitope chimeric DNA immunized mice, were significantly higher than that in pcDNA3.1 （ ＋ ）/Ct MOMP multi-epitope DNA （35.50%±2.68%, 30.24% ±1.75%; 12.27% ±3.36%, 9.32% ±3.07%） and other control groups（F=72.87, F=114.55, P〈0.05; F=30.04, F=10.47, P〈0.05）, and Ct MOMP multi-epitope specific cytotoxicity of CTL