淋球菌NspA核酸疫苗的构建及其免疫活性的初步研究

发布时间：2019-04-12 06:39

【摘要】：目的：根据GenBank登录的淋球菌WHO—A株的NspA核酸序列设计引物，扩增出NspA基因，构建真核表达载体pcDNA3.1(+)／NspA，直接肌注免疫BALB／c小鼠，观察其所诱导产生的体液免疫和细胞免疫应答水平，为研制新型、高效的淋球菌核酸疫苗提供实验依据。方法：用PRIMER5.0软件设计引物，PCR扩增NspA全基因；将PCR产物纯化后与pUCm-T载体相连，经蓝白斑筛选、双酶切鉴定及测序鉴定后，亚克隆至pcDNA3.1(+)真核表达载体中；阳性克隆经双酶切及测序鉴定后转染RAW264.7和COS-7细胞，用RT-PCR检测NspA mRNA的水平，免疫细胞化学法检测蛋白表达。以pcDNA3.1(+)／NspA肌注免疫6w龄BALB／c小鼠，试管凝集法检测免疫小鼠血清中抗NspA抗体水平，ELISA双抗体夹心法检测脾淋巴细胞培养上清中IFN-γ水平，MTT比色法检测脾淋巴细胞增殖反应，PCR检测淋球菌NspA基因在小鼠股四头肌的存在。结果：成功构建了pcDNA3.1(+)／NspA真核表达载体，以脂质体转染RAW264.7和COS-7细胞后，用RT-PCR检测NspA mRNA的水平，免疫细胞化学法检测蛋白表达，表明重组质粒能在真核细胞有效转录和表达NspA。小鼠接种pcDNA3.1(+)／NspA核酸疫苗后，能产生抗NspA的特异性抗体，其滴度随着时间的增加和加强免疫而增高，6w后抗体最高滴度达1∶640。核酸疫苗免疫组小鼠脾淋巴细胞经PHA刺激后，，培养上清中IFN-γ含量明显升高(169.71±30.52pg／mL)，与空质粒组(23.79±11.85pg／mL)和PBS组(8.71±2.50pg／mL)之间有显著性差异(P＜0.01)。脾淋巴细胞增殖反应测定，未加PHA时，T淋巴细胞呈单个分布，无细胞增殖；加入PHA后，核酸疫苗组细胞增殖明显活跃，细胞成团增长，而对照组细胞增殖不
[Abstract]:Objective: according to the NspA sequence of Neisseria gonorrhoeae WHO-A strain logged in by GenBank, primers were designed to amplify NspA gene and construct eukaryotic expression vector pcDNA3.1 () / NspA, to immunize BALB/c mice by direct intramuscular injection. The humoral and cellular immune responses were observed in order to provide experimental basis for the development of a new and efficient nucleic acid vaccine of Neisseria gonorrhoeae. Methods: the whole NspA gene was amplified by PCR with PRIMER5.0 software, and the PCR product was purified and linked to pUCm-T vector. After blue and white spot screening, double enzyme digestion and sequencing identification, the product was subcloned into pcDNA3.1 () eukaryotic expression vector. The positive clones were transfected into RAW264.7 and COS-7 cells by restriction endonuclease digestion and sequencing. The expression of NspA mRNA and protein were detected by RT-PCR and immunocytochemistry respectively. Six-week-old BALB/c mice were immunized with pcDNA3.1 () / NspA intramuscular injection. The levels of anti-NspA antibody in serum and IFN- 纬 in supernatant of spleen lymphocyte culture were detected by test tube agglutination method and ELISA double antibody sandwich method, respectively. MTT colorimetry was used to detect the proliferation of splenic lymphocytes and PCR was used to detect the presence of Neisseria gonorrhoeae NspA gene in quadriceps femoris of mice. Results: the eukaryotic expression vector of pcDNA3.1 () / NspA was constructed successfully. After transfection with liposome into RAW264.7 and COS-7 cells, the level of NspA mRNA was detected by RT-PCR and the expression of protein was detected by immunocytochemistry. The results show that the recombinant plasmid can effectively transcribe and express NspA. in eukaryotic cells. Mice inoculated with pcDNA3.1 () / NspA nucleic acid vaccine produced specific antibodies against NspA, and the titers of the antibodies increased with the increase of time and enhanced immunization. The highest titers of anti-NspA antibodies reached 1? 640 at 6 weeks after inoculation. After stimulated by PHA, the content of IFN- 纬 in the supernatant of mice immunized with nucleic acid vaccine increased significantly (169.71 卤30.52 PG / mL),). There was a significant difference between the control group (23.79 卤11.85 PG / mL) and the PBS group (8.71 卤2.50pg/mL) (P < 0.01). The proliferation reaction of spleen lymphocytes showed that without PHA, T lymphocytes showed a single distribution and no cell proliferation. After adding PHA, the proliferation of T lymphocytes in nucleic acid vaccine group was obviously active and the cells in the control group did not proliferate.
【学位授予单位】：南华大学
【学位级别】：硕士
【学位授予年份】：2006
【分类号】：R392

【参考文献】