炭疽杆菌保护性抗原重组腺病毒疫苗的构建及气溶胶免疫探索性研究

发布时间：2018-01-12 06:42

本文关键词：炭疽杆菌保护性抗原重组腺病毒疫苗的构建及气溶胶免疫探索性研究　出处：《中国人民解放军军事医学科学院》2006年硕士论文　论文类型：学位论文

【摘要】：目的：探讨利用腺病毒载体作为炭疽杆菌基因工程疫苗载体的可行性；探索重组腺病毒对雾化的耐受能力；探讨腺病毒活载体疫苗雾化吸入免疫途径的可行性。方法： 1．PCR扩增PA基因后克隆至质粒pAdTrack-CMV，经与pAdeasy-1同源重组，构建重组腺病毒vAd-PA，转染293细胞，经Western-blot检测表明PA在293细胞中得到表达。 2．重组腺病毒肌肉注射和滴鼻免疫途径免疫BALB／c小鼠，通过ELISA和MTT等实验对免疫鼠的特异性抗体、黏膜免疫反应及细胞免疫应答进行分析。 3．优化重组腺病毒雾化条件，选择最优条件雾化免疫小鼠，通过ELISA和MTT等实验对免疫鼠的特异性抗体、黏膜免疫反应及细胞免疫应答进行分析。结果： 1．通过酶切鉴定的方法验证，成功构建了重组腺病毒vAd-PA，并用Western-blot的方法验证了PA蛋白在细胞内的表达。 2．重组腺病毒通过肌肉注射和滴鼻的途径免疫小鼠，均能诱导特异的体液、黏膜和细胞免疫应答。表现为血清中IgG升高，肺灌洗液中的IgA升高，以及脾细胞培养上清中的细胞因子浓度有所升高，MMT实验结果显示T细胞增殖程度高于对照组。 3．重组腺病毒雾化途径免疫小鼠，可诱导特异的体液、黏膜和细胞免疫应答。表现为血清中IgG升高，肺灌洗液中的IgA升高，MMT实验结果显示T细胞增殖程度高于对照组。结论：炭疽杆菌重组腺病毒基因工程疫苗有望发展成为一种新型的基因工程疫苗；雾化吸入免疫途径可考虑作为炭疽杆菌腺病毒活载体疫苗的免疫方式。
[Abstract]:Objective: to explore the feasibility of using adenovirus vector as gene engineering vaccine vector of Bacillus anthracis. To explore the tolerance of recombinant adenovirus to atomization; To investigate the feasibility of aerosol inhalation of adenovirus live vector vaccine. Methods: 1. The PA gene was amplified by PCR and cloned into plasmid pAdTrack-CMV. After homologous recombination with pAdeasy-1, the recombinant adenovirus vAd-PA was constructed. After transfection into 293 cells, Western-blot analysis showed that PA was expressed in 293 cells. 2. BALB/c mice were immunized by recombinant adenovirus intramuscular injection and nasal drip immunization. Specific antibodies were obtained by ELISA and MTT. Mucosal immune response and cellular immune response were analyzed. 3. Optimize the nebulization conditions of recombinant adenovirus and select the optimal conditions to immunize mice. The specific antibodies of immunized mice were obtained by ELISA and MTT experiments. Mucosal immune response and cellular immune response were analyzed. Results: 1. The recombinant adenovirus vAd-PAwas successfully constructed by restriction endonuclease assay, and the expression of PA protein in the cells was verified by Western-blot. 2. The recombinant adenovirus could induce specific humoral, mucosal and cellular immune responses in mice by intramuscular injection and nasal drip. IgA in lung lavage fluid and cytokine concentration in the supernatant of spleen cell culture were increased. The results showed that the proliferation of T cells was higher than that of control group. 3. The specific humoral, mucosal and cellular immune responses were induced by recombinant adenovirus nebulization pathway in mice. The results showed that IgG in serum and IgA in lung lavage fluid were increased. MMT results showed that T cell proliferation was higher than that in control group. Conclusion: the recombinant adenovirus gene engineering vaccine of Bacillus anthracis is expected to be a new type of genetic engineering vaccine. Nebulized inhalation can be considered as a vaccine against anthrax adenovirus.
【学位授予单位】：中国人民解放军军事医学科学院
【学位级别】：硕士
【学位授予年份】：2006
【分类号】：R392.1

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