幽门螺杆菌napA基因在乳酸菌中的表达及免疫原性分析

发布时间：2018-03-05 00:25

本文选题：幽门　切入点：杆菌　出处：《中国协和医科大学》2008年硕士论文　论文类型：学位论文

【摘要】： 幽门螺杆菌(Helicobacter pylori,H pylori,Hp)是一种微需氧革兰氏阴性螺旋杆菌,在全球人口中的感染率超过50%。H.pylori感染是慢性胃炎和消化性溃疡的主要病因,与胃癌、胃粘膜相关性淋巴样组织(Mucosa-associatedlymphoid tissue,MALT)淋巴瘤的发生密切相关,被WHO确定为Ⅰ类致癌因子。研制疫苗是控制H.pylori感染及相关疾病经济有效的途径。幽门螺杆菌中性粒细胞激活蛋白(H.pylori neutropphil-activatingprotein,HP-NAP)位于细菌细胞质中,可通过细菌自溶而释放,并附着在细菌外膜表面,介导了H.pylori与细胞表面的结合,能够趋化和激活中性粒细胞,导致炎症。而最新实验表明,HP-NAP在酸性条件下,能够结合甚至浓缩DNA,以保护DNA免受宿主环境的损伤。可见,HP-NAP在H.pylori的粘附、定植,以及引起炎症反应中都起到重要作用,是H.pylori重要的致病因子之一。乳酸菌是人和动物肠胃中正常存在的菌群,被公认为安全级微生物,已广泛地应用于食品及饮料加工业。并且乳酸菌进入肠道后能发挥佐剂的活性刺激机体的粘膜免疫反应。乳酸菌和可降解的生物颗粒大小差不多,可以被粘膜M细胞摄取,故可作为一种有效的口服疫苗载体。本实验利用分子克隆技术扩增了H.pylori的napA基因,并构建携带napA基因的重组原核表达质粒pNICE:sec-nap,将重组质粒转化乳酸菌NZ9000株,优化表达条件后,NAP在乳酸菌中获得表达,表达量占菌体蛋白总量的9.5%。表达的蛋白能与兔抗H.pylori血清特异性反应,具有良好的免疫原性。用重组表达NAP蛋白的乳酸菌口服免疫小鼠后能刺激小鼠产生特异性的IgG和IgA。实验结果说明以乳酸菌作为递送载体的口服疫苗可以刺激机体产生局部的粘膜免疫反应和全身的系统免疫反应。这对于H.pylori的治疗和预防都有重要的意义,将会是一种具有很好发展前景的新型疫苗。本实验对乳酸菌作为抗原递送载体进行了初步尝试,为H.pylori口服疫苗的开发提供一定的实验依据。
[Abstract]:Helicobacter pylori (Helicobacter pylori) is a microaerobic gram-negative helicobacter pylori. The infection rate in the global population is more than 50. H. pylori infection is the main cause of chronic gastritis and peptic ulcer, and gastric cancer. Mucosa-associated lymphoid tissue (Mucosa-associated lymphoid tissue malt) lymphoma is closely related to the occurrence of Mucosa-associated lymphoid tissue lymphoma, which is identified by WHO as a class I carcinogen. The development of vaccine is a cost-effective way to control H. pylori infection and related diseases. Helicobacter pylori neutropphil-activating protein (HP-NAPs), which is located in the cytoplasm of Helicobacter pylori, can be released by bacterial autolysis and attached to the surface of the bacterial outer membrane, which mediates the binding of H. pylori to the cell surface, and can chemoattractant and activate neutrophil. Recent experiments have shown that HP-NAP can bind or even condense DNA to protect DNA from host environment in acidic conditions. HP-NAP plays an important role in H. pylori adhesion, colonization, and inflammatory response. H.pylori is one of the important pathogenic factors. Lactic acid bacteria (Lactic acid bacteria) are normal bacteria in human and animal intestines and stomachs, which are recognized as safe microbes. It has been widely used in food and beverage processing industries. And when lactic acid bacteria enter the gut, they can act as adjuvants to stimulate the mucosal immune response of the body. Lactic acid bacteria are the same size as biodegradable organisms and can be ingested by mucosal M cells. In this experiment, the napA gene of H.pylori was amplified by molecular cloning technique, and the recombinant prokaryotic expression plasmid pNICE: sec-napus was constructed. The recombinant plasmid was transformed into NZ9000 strain of lactic acid bacteria. After optimized expression conditions, naps were expressed in lactic acid bacteria, which accounted for 9.5% of the total bacterial protein. The expressed protein could react specifically with rabbit anti-H.pylori serum. Lactic acid bacteria which expressed NAP protein by oral administration could stimulate mice to produce specific IgG and IgA. The results show that oral vaccine with lactic acid bacteria as delivery vector can stimulate local mucosal immune response and systemic immune response, which is of great significance for the treatment and prevention of H.pylori. Lactic acid bacteria (Lactic acid bacteria) as a carrier of antigen delivery were preliminarily attempted to provide some experimental basis for the development of H. pylori oral vaccine.
【学位授予单位】：中国协和医科大学
【学位级别】：硕士
【学位授予年份】：2008
【分类号】：R392

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