肺炎嗜衣原体MOMP核酸疫苗的构建及其免疫效果的初步研究
发布时间：2019-01-30 16:09
【摘要】： 目的:构建肺炎嗜衣原体(Chlamydophila pneumoniae, Cpn)重组单基因疫苗和双基因融合疫苗,建立动物感染模型,比较两种核酸疫苗在抗Cpn感染中的免疫效果。为研制高效、新型的Cpn核酸疫苗奠定实验基础。 方法:用Prime软件分析Cpn外膜蛋白MOMP和人IL-2基因序列,设计相应特异性引物,分别用PCR和RT-PCR扩增MOMP全基因及人IL-2基因,同时以编码6个氨基酸的18个寡核苷酸作为接头,通过重组PCR法将MOMP与IL-2基因融合,PCR产物经酶切纯化后克隆至pcDNA3.1(+)真核表达载体中;阳性克隆经双酶切及测序鉴定后,大量制备纯化后作为核酸疫苗,分别将重组质粒pcDNA3.1(+)-MOMP、pcDNA3.1(+)-IL-2、pcDNA3.1(+)-MOMP-IL-2及对照空质粒pcDNA3.1(+)分组注射于BALB/c小鼠后腿股四头肌,每次剂量为100μg,末次免疫后2w,免疫荧光组化法检测肺炎嗜衣原体MOMP基因在小鼠组织内的表达情况,无菌分离小鼠脾细胞,经特异性重组蛋白抗原刺激后, ELISA双抗体夹心法测定培养上清中IFN-γ水平,ELISA间接法测定BALB/c小鼠血清中抗MOMP和抗MOMP-IL-2特异性抗体水平;MTT法、淋巴细胞转化试验测定脾淋巴细胞特异性增殖反应。 结果: 1.成功构建了MOMP、IL-2以及MOMP-IL-2融合基因的原核表达载体和真核表达载体。 2.成功构建pcDNA3.1(+)-MOMP、pcDNA3.1(+)-MOMP-IL-2融合基因核酸疫苗,其均能在组织细胞内有效表达融合靶蛋白。 3.小鼠接种疫苗后,能产生特异性抗体,6w后抗体的最高滴度均达1:1280 4. MOMP单基因核酸疫苗组和MOMP-IL-2融合基因核酸疫苗组的鼠脾淋巴细胞经相应特异性抗原刺激后,培养上清中IFN-γ均显著升高(分别升至586±42.3pg/mL、695±43.7pg/mL)。 5. MOMP单基因核酸疫苗组和MOMP-IL-2融合基因核酸疫苗组小鼠脾淋巴细胞经相应特异性抗原刺激后,其刺激指数分别为(1.508±0.010、1.573±0.012)。 6.在小鼠肌组织中用PCR可检测到MOMP基因。 结论: 1. Cpn MOMP单基因核酸疫苗和MOMP-IL-2融合基因核酸疫苗均能刺激机体产生较强细胞免疫应答和体液免疫应答。 2.双基因核酸疫苗诱生特异性抗体的效率与单基因核酸疫苗有明显差异;且能诱导更强,更持久的细胞免疫应答。
[Abstract]:Aim: to construct recombinant single gene vaccine and double gene fusion vaccine of Chlamydia pneumoniae (Chlamydophila pneumoniae, Cpn) and to establish animal infection model and compare the immune effect of two nucleic acid vaccines against Cpn infection. For the development of high-efficiency, new Cpn nucleic acid vaccine to lay the experimental foundation. Methods: the sequences of Cpn outer membrane protein MOMP and human IL-2 gene were analyzed by Prime software. Specific primers were designed to amplify the whole MOMP gene and human IL-2 gene by PCR and RT-PCR, respectively. At the same time, 18 oligonucleotides encoding 6 amino acids were used as the junction, the MOMP gene was fused with IL-2 gene by recombinant PCR method, and the PCR product was purified by enzyme digestion and cloned into pcDNA3.1 () eukaryotic expression vector. The positive clones were digested by double enzyme and sequenced, and then purified as nucleic acid vaccine. The recombinant plasmids pcDNA3.1 ()-MOMP,pcDNA3.1 ()-IL-2, were prepared respectively. PcDNA3.1 ()-MOMP-IL-2 and control plasmid pcDNA3.1 () were injected into the quadriceps femoris of the hind leg of BALB/c mice at a dose of 100 渭 g each time for 2 weeks after the last immunization. The expression of chlamydia pneumoniae MOMP gene in mouse tissues was detected by immunofluorescence histochemistry. Mouse spleen cells were isolated without bacteria. After stimulation with specific recombinant protein antigen, the level of IFN- 纬 in culture supernatant was measured by ELISA double antibody sandwich method. The levels of anti MOMP and anti MOMP-IL-2 specific antibodies in serum of BALB/c mice were determined by ELISA indirect method. MTT assay and lymphocyte transformation test were used to determine the specific proliferation of splenic lymphocytes. Results: 1. The prokaryotic expression vector and eukaryotic expression vector of MOMP,IL-2 and MOMP-IL-2 fusion gene were successfully constructed. 2. PcDNA3.1 ()-MOMP,pcDNA3.1 ()-MOMP-IL-2 fusion gene nucleic acid vaccine was successfully constructed. 3. After inoculation, specific antibody was produced in mice, and the highest titer of antibody was 1: 12804 after 6 weeks. The splenic lymphocytes in MOMP single gene vaccine group and MOMP-IL-2 fusion gene nucleic acid vaccine group were stimulated by specific antigen, and the IFN- 纬 in the culture supernatant was significantly increased (up to 586 卤42.3 PG / mL 695 卤43.7pg/mL, respectively). 5. The stimulation index of spleen lymphocytes in MOMP single gene vaccine group and MOMP-IL-2 fusion gene nucleic acid vaccine group was (1.508 卤0.010 10) 1.53 卤0.012 (P < 0.05). 6. MOMP gene could be detected by PCR in mouse muscle tissue. Conclusion: 1. Both Cpn MOMP single gene nucleic acid vaccine and MOMP-IL-2 fusion gene nucleic acid vaccine can stimulate strong cellular and humoral immune responses. 2. The efficiency of inducing specific antibody by double gene nucleic acid vaccine was significantly different from that of single gene nucleic acid vaccine, and it could induce a stronger and more lasting cellular immune response.
【学位授予单位】：南华大学
【学位级别】：硕士
【学位授予年份】：2008
【分类号】：R392.1
[Abstract]:Aim: to construct recombinant single gene vaccine and double gene fusion vaccine of Chlamydia pneumoniae (Chlamydophila pneumoniae, Cpn) and to establish animal infection model and compare the immune effect of two nucleic acid vaccines against Cpn infection. For the development of high-efficiency, new Cpn nucleic acid vaccine to lay the experimental foundation. Methods: the sequences of Cpn outer membrane protein MOMP and human IL-2 gene were analyzed by Prime software. Specific primers were designed to amplify the whole MOMP gene and human IL-2 gene by PCR and RT-PCR, respectively. At the same time, 18 oligonucleotides encoding 6 amino acids were used as the junction, the MOMP gene was fused with IL-2 gene by recombinant PCR method, and the PCR product was purified by enzyme digestion and cloned into pcDNA3.1 () eukaryotic expression vector. The positive clones were digested by double enzyme and sequenced, and then purified as nucleic acid vaccine. The recombinant plasmids pcDNA3.1 ()-MOMP,pcDNA3.1 ()-IL-2, were prepared respectively. PcDNA3.1 ()-MOMP-IL-2 and control plasmid pcDNA3.1 () were injected into the quadriceps femoris of the hind leg of BALB/c mice at a dose of 100 渭 g each time for 2 weeks after the last immunization. The expression of chlamydia pneumoniae MOMP gene in mouse tissues was detected by immunofluorescence histochemistry. Mouse spleen cells were isolated without bacteria. After stimulation with specific recombinant protein antigen, the level of IFN- 纬 in culture supernatant was measured by ELISA double antibody sandwich method. The levels of anti MOMP and anti MOMP-IL-2 specific antibodies in serum of BALB/c mice were determined by ELISA indirect method. MTT assay and lymphocyte transformation test were used to determine the specific proliferation of splenic lymphocytes. Results: 1. The prokaryotic expression vector and eukaryotic expression vector of MOMP,IL-2 and MOMP-IL-2 fusion gene were successfully constructed. 2. PcDNA3.1 ()-MOMP,pcDNA3.1 ()-MOMP-IL-2 fusion gene nucleic acid vaccine was successfully constructed. 3. After inoculation, specific antibody was produced in mice, and the highest titer of antibody was 1: 12804 after 6 weeks. The splenic lymphocytes in MOMP single gene vaccine group and MOMP-IL-2 fusion gene nucleic acid vaccine group were stimulated by specific antigen, and the IFN- 纬 in the culture supernatant was significantly increased (up to 586 卤42.3 PG / mL 695 卤43.7pg/mL, respectively). 5. The stimulation index of spleen lymphocytes in MOMP single gene vaccine group and MOMP-IL-2 fusion gene nucleic acid vaccine group was (1.508 卤0.010 10) 1.53 卤0.012 (P < 0.05). 6. MOMP gene could be detected by PCR in mouse muscle tissue. Conclusion: 1. Both Cpn MOMP single gene nucleic acid vaccine and MOMP-IL-2 fusion gene nucleic acid vaccine can stimulate strong cellular and humoral immune responses. 2. The efficiency of inducing specific antibody by double gene nucleic acid vaccine was significantly different from that of single gene nucleic acid vaccine, and it could induce a stronger and more lasting cellular immune response.
【学位授予单位】：南华大学
【学位级别】：硕士
【学位授予年份】：2008
【分类号】：R392.1
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