双歧杆菌丝氨酸蛋白酶抑制剂的克隆表达及功能的初步研究

发布时间：2018-03-23 13:21

  本文选题：双歧杆菌　切入点：Serpin　出处：《南昌大学》2010年硕士论文

【摘要】： 双歧杆菌是人体胃肠道的原籍菌和优势菌,通过与乳酸杆菌及其它厌氧菌一起共同占据肠粘膜表面,形成生物屏障,在维持肠道微生态平衡和促进宿主健康方面发挥了重要作用。黏附和免疫是双歧杆菌发挥重要生理作用的基础,然而,有关其黏附肠上皮细胞的作用机制和参与免疫作用的物质基础仍不甚清楚。目前有关黏附和免疫的研究主要集中在双歧杆菌的外膜蛋白上,而其中之一的Serine protease inhibitor (Serpin)蛋白,近三年引起了关注。 Serpin是一类多肽类的丝氨酸蛋白酶抑制剂的总称,由350至500个氨基酸组成。在真核生物中,Serpin通过调节丝氨酸蛋白酶和半胱氨酸蛋白酶的活性,参与纤溶、炎症反应、细胞迁移、分化以及凋亡等许多重要的生命活动。然而,有关原核生物中Serpin的功能作用的报道甚少。2006年瑞士雀巢研究中心发现长双歧杆菌的Serpin蛋白可以有效抑制外源蛋白酶的活性,为双歧杆菌黏附于肠道提供条件。为研究Serpin在不同种属的双歧杆菌中的分布,从18株分属两歧双歧杆菌、婴儿双歧杆菌、短双歧杆菌、长双歧杆菌、青春双歧杆菌、乳酸双歧杆菌、动物双歧杆菌的7个种的双歧杆菌中克隆到3个serpin基因,分别来源于B. infantis WBAN07、B. bifidum WBBI02和B. longum NCC2705,序列同源性为99.9%。序列分析表明这3个serpin基因存在8个碱基差异,其中B. infantis WBAN07中的149位和770位的序列改变导致了在B. longum NCC2705中组氨酸和谷氨酰胺均变成了精氨酸,而B. bifidum WBBI02中只有149位的碱基改变使得组氨酸转变为精氨酸。 鉴于B. bifidum WBBI02的高粘附力,本实验重点对其Serpin的表达和生物学功能进行了初步研究,即从B. bifidum WBBI02克隆到去除两端疏水区域的serpin基因,构建原核表达载体pBX2-WBBI02。在25℃,0.2mM IPTG诱导3-4小时的条件下获得约32 kD的可溶性Serpin蛋白,使用NTA His-Bind树脂柱获得纯化的Serpin蛋白。活性研究的初步结果表明：Serpin在体外能有效地抑制糜蛋白酶和胰弹性蛋白酶的活性,最高抑制率分别为90%和97%,显微观察结果证实Serpin能促进双歧杆菌对HT-29细胞的粘附。 Serpin克隆表达及其功能研究将为探讨Serpin在不同益生菌(双歧杆菌)中的功能和作用机制,奠定良好的试验基础,也必将为分析双歧杆菌其它细胞成分的黏附和免疫机理提供理论和方法学参考。
[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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