双歧杆菌黏附素的提纯及鉴定

双歧杆菌资料

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Email: wcjd@ World Chin J Digestol, 2002 October;10(10):1149-1151世界华人消化杂志　ISSN 1009-3079 CN 14-1260/R

２００２　年版权归世界胃肠病学杂志社

基础研究　ＢＡＳＩＣ　ＲＥＳＥＡＲＣＨ　

双歧杆菌黏附素的提纯及鉴定

郑跃杰，　潘令嘉，　王立生，　周殿元，　郭立安，　闫　　　哲

郑跃杰，深圳市儿童医院内科　　广东省深圳市　５１８０２６

潘令嘉，　王立生，　周殿元，全军消化疾病研究所　广东省广州市　５１０５１５郭立安，　闫哲，　第四军医大学基础部中心实验室，陕西省西安市　７１００３２

郑跃杰，　男，１９６３－０９－１６生，　山西省稷山县人，汉族．博士，副教授，副主任医师．中华预防医学会微生态学分会儿科学组副组长．发表论文５０篇．

全军医药卫生科研基金Ｎｏ．９８Ｑ０６２

项目负责人　　郑跃杰，　５１８０２６，　广东省深圳市，　深圳市儿童医院内科．

电话：０７５５－８３９３６１１９

收稿日期　　２００２－０４－０１　　　　接收日期　　２００２－０６－０６

RESULTS: Bifidobacterial soluble adhesin was isolated withammonium sulfate precipitation. The resulting precipitatewas further purified by the Superdex 75 gel filtration andthe Q-Sepharose FF anion exchange chromatography. Theadhesin did not bind to Q-Sepharose and could be readilywashed from the column. The protein profile by SDS-PAGE re-vealed that the adhesin eluted from Q-Sepharose FF columnwas essentially pure. The molecular weight was about 16 ku.CONCLUSION:The adhesin of Bifidobacterium may be a16ku protein that presents in the spent culture supernatant.

Zheng YJ, Pan LJ, Wang LS, Zhou DY, Guo LA, Yan Z. Purification andcharacterization of adhesin from bifidobacterium. Shijie Huaren XiaohuaZazhi 2002;10(10):1149-1151

Purification and characterization ofadhesin from bifidobacterium

Yue-Jie Zheng, Ling-Jia Pan, Li-Sheng Wang,Dian-Yuan Zhou,Li-An Guo, Zhe Yan

Yue-Jie Zheng, Shenzhen Children’s Hospital, Shenzhen 518026,Guangdong Province, China

Ling-Jia Pan, Li-Sheng Wang, Dian-Yuan Zhou, Institute of Gastro-enterology of Chinese PLA, First Military Medical University, Guang,zhou 510515, Guangdong Province,China

Li-An Guo, Zhe Yan, Central Lab,Faculty of Preclinical Medicine, FourthMilitary Medical University ,Xi,an 710033,Shanxi Province, ChinaSupported by the Medical Science Foundation of Chinese PLA, No. 98Q062Correspondence to: Dr.Yue-Jie Zheng, Shenzhen Children’s Hospital,Shenzhen, 518026, Guangdong Province, China. djikui 20@Received 2002-04-01 Accepted 2002-06-06

摘要

目的：　双歧杆菌已广泛应用于益生菌及发酵乳的研制．由于双歧杆菌黏附于肠黏膜表面是其发挥作用的首要条件，开展对其黏附机制的研究有着重要意义．但目前有关双歧杆菌与肠黏膜上皮细胞的黏附机制所知不多，我们从青春型双歧杆菌中分离及纯化其黏附素．

方法　：黏附试验为检测青春型双歧杆菌与人肠上皮细胞系Ｌｏｖｏ细胞间的黏附．收集双歧杆菌培养上清并用３００　ｇ／Ｌ硫酸铵沉淀，在４　℃下８　０００　ｒ／ｍｉｎ离心３０　ｍｉｎ收集沉淀物．沉淀物溶解于０．０１　ｍｏｌ／Ｌ　ｐＨ７．４　ＰＢＳ，加入至０．０２　ｍｏｌ／ＬｐＨ７．０　ＰＢＳ平衡的Ｓｕｐｅｒｄｅｘ　７５柱中进行洗脱，速度为０．７ｍＬ／ｍｉｎ，２２６　ｎｍ波长检测光密度ＡＢＳ值．收集合并活性组分，　冷冻干燥．干燥物溶解后加入至０．０５　ｍｏｌ／Ｌ　ｐＨ８．０ＰＢ平衡的Ｑ－Ｓｅｐｈａｒｏｓｅ　ＦＦ柱中，　先用同一缓冲液洗脱，　然后分别用含０．１　ｍｏｌ／Ｌ、０．３　ｍｏｌ／Ｌ、０．５　ｍｏｌ／Ｌ及１．０　ｍｏｌ／Ｌ　氯化钠的０．０５　ｍｏｌ／Ｌ　ＰＢ进行阶段洗脱，　速度为１．５　ｍＬ／ｍｉｎ，检测各峰黏附活性，　活性组分用ＳＤＳ－ＰＡＧＥ进行分析．结果：使用硫酸铵沉淀对双歧杆菌黏附素进行了分离，沉淀物进一步经Ｓｕｐｅｒｄｅｘ　７５凝胶过滤和Ｑ－ＳｅｐｈａｒｏｓｅＦＦ离子交换色谱纯化．结果黏附素不能结合与Ｑ－Ｓｅｐｈａｒｏｓｅ柱，经ＳＤＳ－ＰＡＧＥ分析，证实纯化的黏附素成分基本单一，其Ｍｒ　　约为１６　ｋｕ．

结论：双歧杆菌黏附素可能为一种Ｍｒ　１６　ｋｕ的蛋白质，并且存在于培养液上清中．

郑跃杰，　潘令嘉，　王立生，　周殿元，　郭立安，　闫哲．　双歧杆菌黏附素的提纯及鉴定．世界华人消化杂志　　　２００２；１０（１０）：１１４９－１１５１

Abstract

AIM:Bifidobacteria is extensively used as the species ofprobiotics and fermented milk. As adherence to the mu-cosal surfaces is an important prerequisite for probiotic ef-fects in the gastrointestinal tract, it has an important signifi-cance to study the mechanism of the adhesion ofbifidobacteria. To data little is known about adherence ofbifidobacteria to the intestinal epithelial cells. This study isto isolate and purify the adhesin from the humanBifidobacterium adolescentis.

METHODS:The Bifidobacterium adolescetis was tested forits adherence to human intestinal cell line Lovo cells. Thespent culture supernatant of Bifidobacterium adolescentiswas collected and precipitated with 300 g/L ammoniumsulfate, and the resulting precipitate was collected by cen-trifugation at 8000 r/min for 30 min at 4℃. Then the pelletwas resuspended in 0.01mol/L pH 7.4 PBS and applied to aSuperdex 75 column equilibrated with 0.02 mol/L pH 7.0PBS. The column was eluted at a flow rate of 0.7 ml/min.A226 was monitored. The adhesion activity of fractions wasdetermined by adherence assay. The fractions containingthe adhesin were pooled and lyophilized. The lyophilizedmaterial was then applied to a Q-Sepharose FF column equili-brated with 0.05 mol/L pH8.0 PB. The column was washedwith 2 column volumes of the same buffer, and then elutedwith a gradient of 0.1 to 1.0 mol/L NaCl in 0.05 mol/L pH8.0PB at 1.5 ml/min. The fractions were evaluated by adher-ence assay and further analyzed by sodium dodecyl sul-fate-polyacrylamide gel electrophoresis (SDS-PAGE).

双歧杆菌资料

０　　　引言

双歧杆菌是人体肠道最重要的生理性细菌，对宿主发挥生物屏障、营养、免疫、控制内毒素血症、延缓衰老、抗肿瘤等生理作用，　已广泛应用于益生菌制剂（ｐｒｏｂｉｏｔｉｃｓ）及发酵乳的研制［１－３］　．由于双歧杆菌黏附于肠黏膜上皮细胞是其发挥作用的首要条件，开展对其黏附机制的研究有 …… 此处隐藏：3571字，全部文档内容请下载后查看。喜欢就下载吧 ……