本文選題：幽門　切入點(diǎn)：桿菌　出處：《中國協(xié)和醫(yī)科大學(xué)》2008年碩士論文　論文類型：學(xué)位論文

【摘要】： 幽門螺桿菌(Helicobacter pylori,H pylori,Hp)是一種微需氧革蘭氏陰性螺旋桿菌,在全球人口中的感染率超過50%。H.pylori感染是慢性胃炎和消化性潰瘍的主要病因,與胃癌、胃粘膜相關(guān)性淋巴樣組織(Mucosa-associatedlymphoid tissue,MALT)淋巴瘤的發(fā)生密切相關(guān),被WHO確定為Ⅰ類致癌因子。研制疫苗是控制H.pylori感染及相關(guān)疾病經(jīng)濟(jì)有效的途徑。 幽門螺桿菌中性粒細(xì)胞激活蛋白(H.pylori neutropphil-activatingprotein,HP-NAP)位于細(xì)菌細(xì)胞質(zhì)中,可通過細(xì)菌自溶而釋放,并附著在細(xì)菌外膜表面,介導(dǎo)了H.pylori與細(xì)胞表面的結(jié)合,能夠趨化和激活中性粒細(xì)胞,導(dǎo)致炎癥。而最新實(shí)驗(yàn)表明,HP-NAP在酸性條件下,能夠結(jié)合甚至濃縮DNA,以保護(hù)DNA免受宿主環(huán)境的損傷。可見,HP-NAP在H.pylori的粘附、定植,以及引起炎癥反應(yīng)中都起到重要作用,是H.pylori重要的致病因子之一。 乳酸菌是人和動(dòng)物腸胃中正常存在的菌群,被公認(rèn)為安全級(jí)微生物,已廣泛地應(yīng)用于食品及飲料加工業(yè)。并且乳酸菌進(jìn)入腸道后能發(fā)揮佐劑的活性刺激機(jī)體的粘膜免疫反應(yīng)。乳酸菌和可降解的生物顆粒大小差不多,可以被粘膜M細(xì)胞攝取,故可作為一種有效的口服疫苗載體。本實(shí)驗(yàn)利用分子克隆技術(shù)擴(kuò)增了H.pylori的napA基因,并構(gòu)建攜帶napA基因的重組原核表達(dá)質(zhì)粒pNICE:sec-nap,將重組質(zhì)粒轉(zhuǎn)化乳酸菌NZ9000株,優(yōu)化表達(dá)條件后,NAP在乳酸菌中獲得表達(dá),表達(dá)量占菌體蛋白總量的9.5%。表達(dá)的蛋白能與兔抗H.pylori血清特異性反應(yīng),具有良好的免疫原性。用重組表達(dá)NAP蛋白的乳酸菌口服免疫小鼠后能刺激小鼠產(chǎn)生特異性的IgG和IgA。 實(shí)驗(yàn)結(jié)果說明以乳酸菌作為遞送載體的口服疫苗可以刺激機(jī)體產(chǎn)生局部的粘膜免疫反應(yīng)和全身的系統(tǒng)免疫反應(yīng)。這對(duì)于H.pylori的治療和預(yù)防都有重要的意義,將會(huì)是一種具有很好發(fā)展前景的新型疫苗。本實(shí)驗(yàn)對(duì)乳酸菌作為抗原遞送載體進(jìn)行了初步嘗試,為H.pylori口服疫苗的開發(fā)提供一定的實(shí)驗(yàn)依據(jù)。
[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.
【學(xué)位授予單位】：中國協(xié)和醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2008
【分類號(hào)】：R392

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