本文選題：幽門螺桿菌 + 微孔蛋白　； 參考：《江蘇大學(xué)》2008年碩士論文

【摘要】： 目的:克隆幽門螺桿菌(Helicobacter pylori,Hp)微孔蛋白(porin)HopX編碼基因;原核表達(dá)并純化HopX融合蛋白;制備針對HopX融合蛋白的兔源性多抗;通過體外實(shí)驗(yàn)觀察微孔蛋白HopX誘導(dǎo)人胃癌細(xì)胞株SGC7901表達(dá)分泌IL-8的能力,并初步探討MAPK信號通路在HopX誘導(dǎo)SGC7901細(xì)胞表達(dá)分泌IL-8中的作用;探討HopX對人胃癌細(xì)胞株SGC7901增殖能力的影響。為進(jìn)一步了解幽門螺桿菌微孔蛋白HopX的生物學(xué)功能奠定基礎(chǔ)。 方法: 1)應(yīng)用PCR技術(shù)從Hp DNA基因組擴(kuò)增微孔蛋白HopX編碼基因片段,將其TA克隆和測序,并與GenBank公布的其他Hp菌株的基因序列比較,用信息學(xué)軟件分析其物理化學(xué)特性; 2)以重組質(zhì)粒pQE30-hopX轉(zhuǎn)化E.coli M15宿主菌,IPTG誘導(dǎo)陽性E.coli M15菌株,用NTA-Ni~(2+)樹脂分離純化所表達(dá)的HopX融合蛋白,純化后蛋白經(jīng)SDS-PAGE電泳及Western Blot鑒定; 3)將融合蛋白HopX與弗氏佐劑充分乳化后,免疫家兔,制備抗HopX抗血清,酶聯(lián)免疫吸附試驗(yàn)(enzyme-linked immunosorbentassay,ELISA)測其效價(jià); 4)不同濃度蛋白HopX與SGC7901細(xì)胞共培養(yǎng)不同時(shí)間,設(shè)陽性對照和陰性對照;收集培養(yǎng)上清,ELISA法檢測分泌IL-8蛋白的量;提取細(xì)胞總RNA后,熒光定量RT-PCR檢測IL-8mRNA表達(dá)水平;用MAPK信號通路ERK1/ERK2特異性抑制劑PD-98059預(yù)先處理細(xì)胞,再加入HopX與細(xì)胞作用,提取細(xì)胞總RNA,熒光定量RT-PCR檢測IL-8mRNA表達(dá)水平的變化; 5)應(yīng)用細(xì)胞計(jì)數(shù)、細(xì)胞活力、MTT方法觀察HopX對細(xì)胞增殖的影響;通過細(xì)胞周期和DNA倍體分析、DNA凝膠電泳,Hoechest33258熒光染色等方法分析HopX誘導(dǎo)SGC7901細(xì)胞凋亡作用。 結(jié)果: 1)擴(kuò)增hopX基因全長為1284bp,(申請GenBank的登錄號為EF208122)與GenBank公布的ATCC43504及J99同源性為97%,與26695同源性為96%。軟件預(yù)測表達(dá)的蛋白的相對分子質(zhì)量(Mr)約為47kDa,并顯示具有良好的抗原性。 2)成功構(gòu)建pQE30-hopX原核表達(dá)重組質(zhì)粒。通過NTA-Ni~(2+)樹脂分離純化目的蛋白,獲得原核表達(dá)的HopX融合蛋白,SDS-PAGE及Western Blot鑒定大小約為47KDa,與預(yù)測一致。 3)HopX融合蛋白免疫家兔獲得多克隆抗體,效價(jià)為1:8×10~4。 4)HopX可誘導(dǎo)胃癌細(xì)胞SGC7901表達(dá)和分泌IL-8,該效應(yīng)呈濃度依賴性;經(jīng)PD-98059處理后,細(xì)胞IL-8mRNA表達(dá)水平與未處理前相比明顯下降。 5)HopX可抑制SGC7901細(xì)胞的增殖及活力,呈劑量-時(shí)間依賴關(guān)系。未出現(xiàn)典型的凋亡形態(tài)改變。 結(jié)論:本研究首次克隆NCTC11637 hopX基因,成功構(gòu)建pQE30-hopX載體并獲得原核表達(dá)的HopX融合蛋白;HopX可誘導(dǎo)胃癌細(xì)胞株SGC7901表達(dá)和分泌IL-8,該作用在一定程度上依賴MAPK信號通路;純化的HopX蛋白對胃癌細(xì)胞SCG7901的增殖和細(xì)胞活力都有一定的抑制作用。以上的研究為更進(jìn)一步探討Hp外膜蛋白生物學(xué)功能、致病機(jī)制及Hp疫苗的研究奠定了基礎(chǔ)。
[Abstract]:Objective: to clone Helicobacter pylori (Helicobacter pylori) porin HopX gene, prokaryotic express and purify HopX fusion protein, and prepare rabbit polyclonal antibody against HopX fusion protein. To observe the ability of human gastric cancer cell line SGC7901 to express and secrete IL-8 induced by HopX in vitro, and to explore the role of MAPK signal pathway in the expression and secretion of IL-8 in SGC7901 cells induced by HopX, and to explore the effect of HopX on the proliferation of human gastric cancer cell line SGC7901. It lays a foundation for further understanding the biological function of Helicobacter pylori microporin HopX. Methods: 1) the micropore protein HopX coding gene fragment was amplified by PCR from the HP DNA genome, cloned and sequenced, and compared with the gene sequence of other HP strains published by GenBank. The physical and chemical properties of the recombinant plasmid pQE30-hopX were analyzed by the software of informatics. 2) the recombinant plasmid pQE30-hopX was transformed into E. coli M15 to induce the positive E. coli M15 strain by IPTG, and the expressed HopX fusion protein was isolated and purified by NTA-NiAX) resin. The purified protein was identified by SDS-PAGE electrophoresis and Western Blot. 3) the fusion protein HopX was emulsified with Freund's adjuvant and then the rabbit was immunized to prepare anti-HopX antiserum. Enzyme linked immunosorbent assay (Elisa) was used to determine its titer. 4) different concentrations of protein HopX and SGC7901 cells were co-cultured for different time, positive control and negative control were set up, and the amount of IL-8 secreted by Elisa in culture supernatant was detected by Elisa. After total RNA was extracted, IL-8 mRNA expression was detected by fluorescence quantitative RT-PCR, and PD-98059, a specific inhibitor of ERK1 / ERK2, was pre-treated with MAPK signaling pathway, and then HopX was added to the cells. Total RNAs were extracted and the expression of IL-8 mRNA was detected by fluorescence quantitative RT-PCR. 5) the effect of HopX on cell proliferation was observed by cell count and MTT assay. Cell cycle and DNA ploidy analysis were used to analyze the apoptosis of SGC7901 cells induced by HopX by Hoechest33258 fluorescence staining. Results: 1) the total length of hopX gene was 1284 BP (accession number of GenBank was EF208122) and the homology of ATCC 43504 and J99 published by GenBank was 97 and 96 with 26695 respectively. The software predicted the relative molecular weight of the expressed protein was about 47 kDa and showed good antigenicity. 2) the prokaryotic expression plasmid pQE30-hopX was successfully constructed. The target protein was isolated and purified by NTA-NiHX (2) resin. The expressed HopX fusion protein was identified by SDS-PAGE and Western Blot to be about 47 KDa. it was consistent with the prediction that the fusion protein was used to immunize rabbits to obtain polyclonal antibodies. The titer was 1:8 脳 104.4HopX could induce the expression and secretion of IL-8 in SGC7901 cells in a concentration-dependent manner, and was treated with PD-98059. The expression of IL-8 mRNA in SGC7901 cells was significantly lower than that before treatment. 5. HopX inhibited the proliferation and activity of SGC7901 cells in a dose-time dependent manner. No typical morphological changes of apoptosis were observed. Conclusion: in this study, NCTC11637 hopX gene was cloned for the first time, and pQE30-hopX vector was successfully constructed, and the prokaryotic expression of HopX fusion protein HopX could induce the expression and secretion of IL-8 in gastric cancer cell line SGC7901, which depends on MAPK signaling pathway to some extent. The purified HopX protein inhibited the proliferation and viability of gastric cancer cell line SCG7901. The above studies have laid a foundation for further study on the biological function, pathogenesis and HP vaccine of HP outer membrane protein.
【學(xué)位授予單位】：江蘇大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2008
【分類號】：R378.3

【參考文獻(xiàn)】

相關(guān)期刊論文 前1條

1 姜政,黃愛龍,陶小紅,王丕龍,蒲丹;幽門螺桿菌Mr26000外膜蛋白編碼基因的克隆及表達(dá)[J];細(xì)胞與分子免疫學(xué)雜志;2002年04期

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本文編號：2048680