幽门螺杆菌HP0596的结构和功能关系的研究

发布时间：2018-04-18 06:26

本文选题：幽门螺杆菌 + 二聚体　；参考：《西南大学》2010年硕士论文

【摘要】： 幽门螺杆菌(Helicobacter pylori, H.pylori)是一种微需氧的革兰氏阴性菌,主要寄生在人胃粘膜表面的黏液层,可以导致慢性胃炎、胃溃疡、十二指肠溃疡、胃腺癌和胃淋巴瘤等胃部疾病,被世界卫生组织列为Ⅰ类致癌因子(Type I carcinogen)。目前临床上的治疗主要采用抗生素的手段,但是又存在着诸多的缺点,因此疫苗的开发也就有着重要的意义。而现在研究中的抗原比如幽门螺杆菌的全菌破碎物、尿素酶、过氧化氢酶等都不是十分理想,所以寻找出新的具有保护性的候选抗原极具意义。我们之前通过生物信息学的分析等手段筛选出候选基因hpO596,分析其具有脂蛋白框,是一种可能的膜蛋白。然后我们通过血清学检测出HP0596与胃炎、胃癌呈现出相关性,是H.pylori导致胃部疾病的相关致病因子。同时先前的研究初步证实HP0596可以通过NF-κB途径刺激巨噬细胞分泌肿瘤坏死因子α(TNF-α)。因此,本研究旨在通过对HP0596蛋白的定位的研究和细胞因子变化等方面来阐述其相关的功能。首先,我们由H.pylori26695基因组中扩增hpO596及其信号肽的序列,并且将信号肽序列连接到增强型绿色荧光蛋白(EGFP)的序列,将其克隆至表达载体pET22b(+)上,转化入感受态细胞E.coli BL21(DE3)中,在经过IPTG的诱导后均实现了表达。通过荧光共聚焦显微镜观察EGFP激发出的荧光显示目的重组蛋白定位在膜上。我们通过提取重组菌pET-22b/HP0596的周质蛋白和培养基上清蛋白进行western blot分析,证实了之前分析的准确性。通过构建去掉信号肽序列的HP0596N,和将HP0596N的两个半胱氨酸(Cys)突变成极性相近,结构相似的丝氨酸(Ser),命名为HP0596NT,随后克隆至pET22b(+)然后转化到BL21(DE3)中,经过IPTG的诱导后均得到了表达,然后采用镍柱亲和层析的方法来纯化目的蛋白,获得目的蛋白的纯度在90%以上。最后应用所纯化的HP0596N蛋白免疫大耳白兔获得了高效价的多抗血清。最后将获得的纯化后的两重组蛋白刺激小鼠巨噬细胞系RAW264.7细胞,通过检测所产生的相应的TNF-a、IL-6、IFN-γ和IL-1β因子,得出HP0596NT并未丧失促炎症因子的活性,而且HP0596二聚体可增强蛋白的生理活性,对其生理活性有积极的意义。这一初步研究的结果,为我们对其进行深入的研究打了下基础。
[Abstract]:Helicobacter pylori, a microaerobic gram-negative bacterium that parasitizes the mucous layer on the surface of the human gastric mucosa, can lead to chronic gastritis, gastric ulcers, duodenal ulcers, gastric adenocarcinoma and gastric lymphoma.It was classified by the World Health Organization (WHO) as the carcinogen type I.At present, antibiotics are mainly used in clinical treatment, but there are many shortcomings, so the development of vaccine is of great significance.But the antigens of Helicobacter pylori, such as the whole bacteria fragment, urease and catalase, are not very ideal, so it is very important to find a new protective candidate antigen.We previously screened the candidate gene hpO596 by bioinformatics analysis, and analyzed its lipoprotein frame, which is a possible membrane protein.Then we detected the correlation between HP0596 and gastritis and gastric cancer through serology, which is the related pathogenic factor of gastric disease caused by H.pylori.At the same time, previous studies have preliminarily confirmed that HP0596 can stimulate macrophages to secrete tumor necrosis factor 伪-TNF- 伪 via NF- 魏 B pathway.Therefore, the purpose of this study was to elucidate the function of HP0596 protein by studying its localization and cytokine changes.First, we amplified the sequence of hpO596 and its signal peptide from the H.pylori26695 genome, ligated the signal peptide sequence to the sequence of enhanced green fluorescent protein (EGFP), cloned it into the expression vector pET22b () and transformed it into the receptive cell E.coli BL21DE3).After induction by IPTG, the expression was achieved.A fluorescent confocal microscope was used to observe the localization of the recombinant protein stimulated by EGFP on the membrane.The accuracy of the previous analysis was confirmed by western blot analysis of the peripheral protein extracted from the recombinant strain pET-22b/HP0596 and the supernatant protein of the culture medium.By constructing HP0596N, which removed the signal peptide sequence, and mutating the two cysteine cysteine cysts of HP0596N into a serine serine with similar polarity and similar structure, it was named HP0596NT.Then it was cloned into pET22b () and then transformed into BL21DE3). After the induction of IPTG, the expression of HP0596N was obtained.The target protein was purified by nickel column affinity chromatography and the purity of the target protein was over 90%.Finally, the purified HP0596N protein was used to immunize rabbits with high titer polyantibodies.Finally, the purified two recombinant proteins were used to stimulate murine macrophage cell line RAW264.7. By detecting the corresponding TNF-ahl-6 IFN- 纬 and IL-1 尾, it was concluded that HP0596NT did not lose the activity of pro-inflammatory factor.Moreover, HP0596 dimer can enhance the physiological activity of protein, and has positive significance to its physiological activity.The results of this preliminary study lay the foundation for our further study.
【学位授予单位】：西南大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R378

【参考文献】