异种IL-5 DNA疫苗真核表达载体的构建、鉴定及表达

发布时间：2018-05-22 07:32

本文选题：支气管哮喘 + IL-5　；参考：《山西医科大学》2006年硕士论文

【摘要】：背景：支气管哮喘是临床内、儿科常见的，严重危害健康的慢性疾病，近年其发病率、死亡率逐年增高，严重威胁人类健康，因此日益受到世界各国的重视。对其发病机制已有多方面的探讨，但仍有许多尚未明了。文献研究表明，淋巴细胞中Th1／Th2细胞的比例失衡，如Th1减少，Th2增加是支气管哮喘的重要发病机制，这一失衡可引起Th2类细胞因子如：IL-5等产生过多，Th1类细胞因子如IFN-γ产生减少。越来越多的证据表明，IL-5可激活嗜酸性粒细胞(EOS)并诱导EOS肺部浸润，从而引发受累器官(肺、支气管)的慢性炎症。因此，以IL-5为靶点的抗哮喘主动免疫基因治疗具有良好的应用前景。目的：制备人IL-5 DNA疫苗(hIL-5)，鉴定其在真核细胞有效表达后，用该疫苗免疫小鼠，通过hIL-5诱导抗小鼠IL-5的交叉反应来打破小鼠免疫系统对自身IL-5的免疫耐受，产生抗自身IL-5抗体。方法：我们从Invivogen公司购回分别含有人和小鼠IL-5全段基因的克隆质粒载体PORF9-hIL5和PORF9-mIL5，用Premier Primer 5.0软件辅助设计PCR引物，通过PCR技术将人和小鼠IL-5基因扩增。然后利用基因工程技术，通过酶切、连接反应，将目的基因构建于真核表达质粒pcDNA3.1(+)中，在体外转染真核细胞(COS-1)，，通过Western blot的检测方法其证实有效表达后，在大肠杆菌中扩增并回收大量质粒，用这些真核表达质粒作为DNA疫苗，肌肉注射免疫小鼠，利用Western blot方法检测是否产生抗小鼠自身IL-5的体液免疫反应。结果：通过酶切、测序鉴定证实构建了正确的含人IL-5(hIL-5)和小鼠IL-5(mIL-5)的真核表达重组质粒。利用Western blot方法鉴定均能够在真核细胞COS-1中有效表达相应的蛋白质分子。并且大量制备和纯化了这二个DNA质粒和相应的空对照质粒，用这三种质粒及生理盐水分别免疫小鼠，在第四次免疫后一周收集外周血，Western blot检测发现hIL-5质粒免疫小鼠血清中有抗小鼠IL-5的特异性抗体存在，而mIL-5质粒、空质粒及生理盐水对照组免疫的小鼠血清中没有检测到抗小鼠IL-5的特异性抗体存在。结论：利用与哮喘相关的异种同源基因疫苗(人IL-5 DNA疫苗)来打破免疫耐受，诱导产生种与种之间的交叉免疫反应，为将来研究哮喘的防治奠定坚实的理论基础。
[Abstract]:Background: bronchial asthma is a common and serious chronic disease in pediatrics. In recent years, the incidence and mortality rate of bronchial asthma are increasing year by year, which is a serious threat to human health, so it has been paid more and more attention to all over the world. Many aspects of its pathogenesis have been discussed, but many are still unclear. Literature studies have shown that the imbalance in the proportion of Th1/Th2 cells in lymphocytes, such as the decrease of Th1 and the increase of Th2, is an important pathogenesis of bronchial asthma. This imbalance can cause the production of excessive Th1 cytokines such as Th2 cytokines such as: IL-5, such as IFN- 纬, and decrease the production of Th1 type cytokines such as IFN- 纬. There is increasing evidence that IL-5 activates eosinophil eosinophils and induces pulmonary infiltration in EOS, leading to chronic inflammation in the affected organs (lungs, bronchi). Therefore, anti-asthma active immunotherapy with IL-5 as target has a good application prospect. Aim: to prepare human IL-5 DNA vaccine hIL-5 and identify its expression in eukaryotic cells. Mice were immunized with this vaccine. The cross-reaction of anti-mouse IL-5 was induced by hIL-5 to break the immune tolerance of mouse immune system to autogenic IL-5 and to produce anti-IL-5 antibody. Methods: the cloned plasmids PORF9-hIL5 and PORF9-mIL5 containing human and mouse IL-5 gene were purchased from Invivogen Company respectively. PCR primers were designed with Premier Primer 5.0 software, and the IL-5 gene of human and mouse were amplified by PCR technique. Then the target gene was constructed in eukaryotic expression plasmid pcDNA3.1 () by restriction endonuclease digestion and ligation reaction, and transfected into eukaryotic cells in vitro. The expression of the target gene was confirmed by Western blot assay. A large number of plasmids were amplified and recovered from Escherichia coli. These eukaryotic expression plasmids were used as DNA vaccine to immunize mice intramuscularly. The Western blot method was used to detect whether humoral immune reaction against IL-5 in mice was produced. Results: the recombinant plasmid containing human IL-5 (hIL-5) and mouse IL-5mIL-5 (mIL-5) was constructed by restriction endonuclease digestion and sequencing. Western blot method was used to identify the effective expression of protein molecules in eukaryotic COS-1. The two DNA plasmids and the corresponding blank control plasmids were prepared and purified in large quantities. Mice were immunized with these three plasmids and normal saline respectively. A week after the fourth immunization, the peripheral blood samples were collected to detect the presence of specific antibodies against mouse IL-5 in the sera of mice immunized with hIL-5 plasmid, while the mIL-5 plasmid was found to exist in the sera of mice immunized with hIL-5 plasmids. No specific antibodies against IL-5 were detected in serum of mice immunized with empty plasmids and saline control groups. Conclusion: the heterologous gene vaccine related to asthma (human IL-5 DNA vaccine) was used to break the immune tolerance and induce cross-immunoreaction between species, which laid a solid theoretical foundation for the future study on the prevention and treatment of asthma.
【学位授予单位】：山西医科大学
【学位级别】：硕士
【学位授予年份】：2006
【分类号】：R392

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