本文選題：雙歧桿菌　切入點(diǎn)：Serpin　出處：《南昌大學(xué)》2010年碩士論文

【摘要】： 雙歧桿菌是人體胃腸道的原籍菌和優(yōu)勢菌,通過與乳酸桿菌及其它厭氧菌一起共同占據(jù)腸粘膜表面,形成生物屏障,在維持腸道微生態(tài)平衡和促進(jìn)宿主健康方面發(fā)揮了重要作用。黏附和免疫是雙歧桿菌發(fā)揮重要生理作用的基礎(chǔ),然而,有關(guān)其黏附腸上皮細(xì)胞的作用機(jī)制和參與免疫作用的物質(zhì)基礎(chǔ)仍不甚清楚。目前有關(guān)黏附和免疫的研究主要集中在雙歧桿菌的外膜蛋白上,而其中之一的Serine protease inhibitor (Serpin)蛋白,近三年引起了關(guān)注。 Serpin是一類多肽類的絲氨酸蛋白酶抑制劑的總稱,由350至500個氨基酸組成。在真核生物中,Serpin通過調(diào)節(jié)絲氨酸蛋白酶和半胱氨酸蛋白酶的活性,參與纖溶、炎癥反應(yīng)、細(xì)胞遷移、分化以及凋亡等許多重要的生命活動。然而,有關(guān)原核生物中Serpin的功能作用的報(bào)道甚少。2006年瑞士雀巢研究中心發(fā)現(xiàn)長雙歧桿菌的Serpin蛋白可以有效抑制外源蛋白酶的活性,為雙歧桿菌黏附于腸道提供條件。為研究Serpin在不同種屬的雙歧桿菌中的分布,從18株分屬兩歧雙歧桿菌、嬰兒雙歧桿菌、短雙歧桿菌、長雙歧桿菌、青春雙歧桿菌、乳酸雙歧桿菌、動物雙歧桿菌的7個種的雙歧桿菌中克隆到3個serpin基因,分別來源于B. infantis WBAN07、B. bifidum WBBI02和B. longum NCC2705,序列同源性為99.9%。序列分析表明這3個serpin基因存在8個堿基差異,其中B. infantis WBAN07中的149位和770位的序列改變導(dǎo)致了在B. longum NCC2705中組氨酸和谷氨酰胺均變成了精氨酸,而B. bifidum WBBI02中只有149位的堿基改變使得組氨酸轉(zhuǎn)變?yōu)榫彼帷?鑒于B. bifidum WBBI02的高粘附力,本實(shí)驗(yàn)重點(diǎn)對其Serpin的表達(dá)和生物學(xué)功能進(jìn)行了初步研究,即從B. bifidum WBBI02克隆到去除兩端疏水區(qū)域的serpin基因,構(gòu)建原核表達(dá)載體pBX2-WBBI02。在25℃,0.2mM IPTG誘導(dǎo)3-4小時的條件下獲得約32 kD的可溶性Serpin蛋白,使用NTA His-Bind樹脂柱獲得純化的Serpin蛋白�；钚匝芯康某醪浇Y(jié)果表明：Serpin在體外能有效地抑制糜蛋白酶和胰彈性蛋白酶的活性,最高抑制率分別為90%和97%,顯微觀察結(jié)果證實(shí)Serpin能促進(jìn)雙歧桿菌對HT-29細(xì)胞的粘附。 Serpin克隆表達(dá)及其功能研究將為探討Serpin在不同益生菌(雙歧桿菌)中的功能和作用機(jī)制,奠定良好的試驗(yàn)基礎(chǔ),也必將為分析雙歧桿菌其它細(xì)胞成分的黏附和免疫機(jī)理提供理論和方法學(xué)參考。
[Abstract]:Bifidobacterium is the original and dominant bacteria in the gastrointestinal tract of the human body. Bifidobacteria occupy the intestinal mucosal surface together with Lactobacillus and other anaerobic bacteria to form a biological barrier. It plays an important role in maintaining intestinal microecological balance and promoting host health. Adhesion and immunity are the basis for bifidobacterium to play an important physiological role, however, The mechanism of its adhesion to intestinal epithelial cells and the material basis involved in immune action are still unclear. At present, the researches on adhesion and immunity mainly focus on the outer membrane protein of Bifidobacterium, one of which is Serine protease inhibitor serpinprotein. In the last three years, attention has been drawn. Serpin is a class of polypeptide serine protease inhibitors, consisting of 350 to 500 amino acids. In eukaryotes, Serpin participates in fibrinolysis, inflammation and cell migration by regulating the activities of serine proteases and cysteine proteases. Differentiation and apoptosis are many important biological activities. However, there are few reports about the function of Serpin in prokaryotes. In 2006, the Swiss Nestle Research Center found that the Serpin protein of Bifidobacterium longifera can effectively inhibit the activity of exogenous protease. In order to study the distribution of Serpin in different species of Bifidobacterium, 18 strains belong to Bifidobacterium, including Bifidobacterium infantis, Bifidobacterium brevis, Bifidobacterium longifera, Bifidobacterium adolescence. Three serpin genes were cloned from 7 species of Bifidobacterium lactic acid and bifidobacterium in animals, which were derived from B. infantis WBAN07B. Bifidum WBBI02 and B. longum NCC2705, respectively. The sequence homology was 99.9. The results of sequence analysis showed that the three serpin genes had 8 bases difference. The sequence changes of 149th and 770 positions in B. infantis WBAN07 resulted in the transformation of histidine and glutamine into arginine in B. longum NCC2705, while only 149in B. bifidum WBBI02 resulted in the transformation of histidine to arginine. In view of the high adhesion of B. bifidum WBBI02, the expression and biological function of Serpin were studied primarily, namely, the serpin gene was cloned from B. bifidum WBBI02 to remove the hydrophobic region at both ends. The prokaryotic expression vector pBX2-WBBI02was constructed. The soluble Serpin protein was obtained at 25 鈩，

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