本文選題：幽門螺桿菌 + 二聚體　； 參考：《西南大學(xué)》2010年碩士論文

【摘要】： 幽門螺桿菌(Helicobacter pylori, H.pylori)是一種微需氧的革蘭氏陰性菌,主要寄生在人胃粘膜表面的黏液層,可以導(dǎo)致慢性胃炎、胃潰瘍、十二指腸潰瘍、胃腺癌和胃淋巴瘤等胃部疾病,被世界衛(wèi)生組織列為Ⅰ類致癌因子(Type I carcinogen)。目前臨床上的治療主要采用抗生素的手段,但是又存在著諸多的缺點(diǎn),因此疫苗的開發(fā)也就有著重要的意義。而現(xiàn)在研究中的抗原比如幽門螺桿菌的全菌破碎物、尿素酶、過氧化氫酶等都不是十分理想,所以尋找出新的具有保護(hù)性的候選抗原極具意義。 我們之前通過生物信息學(xué)的分析等手段篩選出候選基因hpO596,分析其具有脂蛋白框,是一種可能的膜蛋白。然后我們通過血清學(xué)檢測出HP0596與胃炎、胃癌呈現(xiàn)出相關(guān)性,是H.pylori導(dǎo)致胃部疾病的相關(guān)致病因子。同時先前的研究初步證實(shí)HP0596可以通過NF-κB途徑刺激巨噬細(xì)胞分泌腫瘤壞死因子α(TNF-α)。因此,本研究旨在通過對HP0596蛋白的定位的研究和細(xì)胞因子變化等方面來闡述其相關(guān)的功能。 首先,我們由H.pylori26695基因組中擴(kuò)增hpO596及其信號肽的序列,并且將信號肽序列連接到增強(qiáng)型綠色熒光蛋白(EGFP)的序列,將其克隆至表達(dá)載體pET22b(+)上,轉(zhuǎn)化入感受態(tài)細(xì)胞E.coli BL21(DE3)中,在經(jīng)過IPTG的誘導(dǎo)后均實(shí)現(xiàn)了表達(dá)。通過熒光共聚焦顯微鏡觀察EGFP激發(fā)出的熒光顯示目的重組蛋白定位在膜上。我們通過提取重組菌pET-22b/HP0596的周質(zhì)蛋白和培養(yǎng)基上清蛋白進(jìn)行western blot分析,證實(shí)了之前分析的準(zhǔn)確性。 通過構(gòu)建去掉信號肽序列的HP0596N,和將HP0596N的兩個半胱氨酸(Cys)突變成極性相近,結(jié)構(gòu)相似的絲氨酸(Ser),命名為HP0596NT,隨后克隆至pET22b(+)然后轉(zhuǎn)化到BL21(DE3)中,經(jīng)過IPTG的誘導(dǎo)后均得到了表達(dá),然后采用鎳柱親和層析的方法來純化目的蛋白,獲得目的蛋白的純度在90%以上。最后應(yīng)用所純化的HP0596N蛋白免疫大耳白兔獲得了高效價的多抗血清。 最后將獲得的純化后的兩重組蛋白刺激小鼠巨噬細(xì)胞系RAW264.7細(xì)胞,通過檢測所產(chǎn)生的相應(yīng)的TNF-a、IL-6、IFN-γ和IL-1β因子,得出HP0596NT并未喪失促炎癥因子的活性,而且HP0596二聚體可增強(qiáng)蛋白的生理活性,對其生理活性有積極的意義。這一初步研究的結(jié)果,為我們對其進(jìn)行深入的研究打了下基礎(chǔ)。
[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 偽-TNF- 偽 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- 緯 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.
【學(xué)位授予單位】：西南大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2010
【分類號】：R378

【參考文獻(xiàn)】

相關(guān)期刊論文 前6條

1 葉方寅;信號肽假說的提出及證實(shí)——1999年諾貝爾生理學(xué)醫(yī)學(xué)獎獲得者Günter Blobel簡介[J];國外醫(yī)學(xué)(分子生物學(xué)分冊);1999年06期

2 龐智 ,蕭樹東;幽門螺桿菌的免疫外膜蛋白質(zhì)組學(xué)及其與胃部疾病的關(guān)系[J];臨床腫瘤學(xué)雜志;2005年02期

3 王濤;陶好霞;王令春;袁盛凌;展德文;王們;王艷春;張微;姜娜;張兆山;劉純杰;;應(yīng)用生物信息學(xué)方法篩選幽門螺桿菌疫苗候選抗原[J];生物技術(shù)通訊;2009年02期

4 郭濤,錢家鳴;幽門螺桿菌感染相關(guān)的炎癥反應(yīng)和胃酸分泌[J];胃腸病學(xué)和肝病學(xué)雜志;2004年02期

5 黃映芳,彭孝緯;IL-1B及IL-1RN基因多態(tài)性與幽門螺桿菌相關(guān)的胃十二指腸疾病[J];胃腸病學(xué)和肝病學(xué)雜志;2005年04期

6 李洪臻;幽門螺桿菌相關(guān)性消化性潰瘍的治療進(jìn)展[J];醫(yī)學(xué)理論與實(shí)踐;2004年07期

，

本文編號：1767180