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@http://www.77cn.com.cn http://www.77cn.com.cnWorld 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@http://www.77cn.com.cnReceived 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着重要意义．在液体培养下，　双歧杆菌的黏附素是一种分泌性蛋白质，　　存在于细菌耗尽培养液上清中［４］　．但是尚未见到双歧杆菌黏附素纯化的研究报道．我们对双歧杆菌的黏附素进行了分离纯化及鉴定．１　　　材料和方法

１．１材料　收集青春型双歧杆菌１０２７株培养上清２　５００　ｍＬ，加入饱和硫酸铵溶液至终浓度为３００　ｇ／Ｌ，４　℃搅拌３　　ｈ，４　℃　８　０００　ｒ／ｍｉｎ　离心３０　ｍｉｎ．沉淀物溶解于０．０１　ｍｏｌ／ＬｐＨ７．４　ＰＢＳ中，－２０　℃保存备用．

１．２　方法　　Ｓｕｐｅｒｄｅｘ　７５　层析采用ＬＫＢ　Ｂｒｏｍｍａ快速蛋白液相色谱系统（ＦＰＬＣ）．用０．０２　ｍｏｌ／Ｌ　ｐＨ７．０　ＰＢＳ平衡Ｓｕｐｅｒｄｅｘ　７５（Ｐｈａｒｍａｃｉａ．）柱（１．６×５５　ｃｍ）后，加入硫酸铵沉淀物１　ｍＬ，０．０２　ｍｏｌ／Ｌ　ｐＨ７．０　ＰＢＳ进行洗脱，洗脱速度为０．７　ｍＬ／ｍｉｎ，２２６　ｎｍ波长检测光密度ＡＢＳ值．收集各组分，　检测黏附活性，　合并活性组分，　冷冻干燥浓缩．Ｑ－Ｓｅｐｈａｒｏｓｅ　ＦＦ层析用０．０５　ｍｏｌ／Ｌ　ｐＨ８．０　ＰＢ平衡Ｑ－Ｓｅｐｈａｒｏｓｅ　ＦＦ（Ｐｈａｒｍａｃｉａ．）柱（１．２×１０　ｃｍ）后，加入Ｓｕｐｅｒｄｅｘ　７５柱活性组分，　先用同一缓冲液洗脱，　然后分别用含０．１ｍｏｌ／Ｌ、０．３ｍｏｌ／Ｌ、０．５ｍｏｌ／Ｌ及１．０ｍｏｌ／Ｌ　氯化钠的０．０５　ｍｏｌ／Ｌ　ＰＢ进行阶段洗脱，　洗脱速度为１．５　ｍＬ／ｍｉｎ，２２６　ｎｍ波长检测．收集各峰检测黏附活性，　活性峰产品冷冻干燥，　纯度使用ＳＤＳ－ＰＡＧＥ进行分析．双歧杆菌黏附活性检测按革兰染色法进行．青春型双歧杆菌１０２７株菌悬液离心后，沉淀菌体分别用待检测样品和原细菌培养液上清悬浮，调整密度为１×１０１１　／Ｌ．加入至Ｌｏｖｏ细胞玻片上，３７　℃湿盒温育２　ｈ．ｐＨ７．４ＰＢＳ漂洗６次，自然干燥，甲醇固定，Ｇｒａｍ染色，高倍镜下计数３０个细胞黏附的细菌数，计算平均数，　与阳性对照比较．阳性对照为用原细菌培养液上清悬浮的双歧杆菌悬液．ＳＤＳ－ＰＡＧＥ分析采用不连续ＳＤＳ－ＰＡＧＥ　系统．浓缩胶３０　ｇ／Ｌ；分离胶１２０　ｇ／Ｌ．ｐＨ８．３　Ｔｒｉｓ－甘氨酸电泳缓冲液．上样前样品于ＳＤＳ加样缓冲液中１００　℃加热５　ｍｉｎ．　凝胶用硝酸银染色．

２　　　结果

２．１　双歧杆菌黏附素的提纯　　３００　ｇ／Ｌ硫酸铵沉淀物经Ｓｕｐｅｒｄｅｘ　７５凝胶过滤，分段收集样品进行黏附活性测定，　结果活性部分位于第二峰的起始部，　初步判断该物质的分子质量在１４－２３　ｋｕ　（图１）．对Ｓｕｐｅｒｄｅｘ　７５层析得到的活性组分进一步进行Ｑ－Ｓｅｐｈａｒｏｓｅ　ＦＦ离子交换层析，　共得６个洗脱峰，　经黏附活性测定，　第１峰为对应活性峰（图２）．

２．２双歧杆菌黏附素的鉴定　将硫酸铵沉淀物经Ｓｕｐｅｒｄｅｘ７５凝胶过滤和Ｑ－Ｓｅｐｈａｒｏｓｅ　ＦＦ离子交换层析后，　得到一单一条带蛋白质，　Ｍｒ　１６　ｋｕ（图３）．

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图　１　　双歧杆菌黏附素在Ｓｕｐｅｒｄｅｘ　７５柱分离色谱图．阴影部分含黏附素活性组分．

1.00.50.30.10

图　２　　双歧杆菌黏附素在Ｑ－Ｓｅｐｈａｒｏｓｅ　ＦＦ柱分离色谱图．第一峰含黏附素活性组分．

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31ku20 ku14 ku

１　　分子质量标准；２　　硫酸铵沉淀物；

３　　Ｓｕｐｅｒｄｅｘ７５凝胶过滤活性组分；

４　　Ｑ－Ｓｅｐｈａｒｏｓｅ　ＦＦ离子交换层析活性组分图　３　　双歧杆菌黏附素ＳＤＳ－ＰＡＧＥ分析．

３　　　讨论

双歧杆菌对肠上皮细胞及其分泌的黏液的黏附能力已作为评定其益生作用的主要指标之一，但有关双歧杆菌黏附机制的研究报道甚少［５－１２］　．黏附是细菌黏附素与宿主细胞相应受体之间的特异性结合过程．革兰阳性细菌细胞壁中含有丰富的磷壁酸，因此对革兰阳性细菌黏附素的研究绝大多数集中在磷壁酸，大量的研究已

双歧杆菌资料

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证实，链球菌及葡萄球菌的黏附素为脂磷壁酸（ＬＴＡ）．传统认为双歧杆菌也是通过其表面的ＬＴＡ介导其黏附，但近年的一些研究表明，双歧杆菌的黏附素可能是一种细胞表面相关的或分泌到培养液上清中的蛋白质［４，１３，１４］　．Ｆｕｊｉｗａｒａ在研究双歧杆菌对大肠杆菌黏附抑制作用时，从双歧杆菌培养上清中纯化出一种Ｍｒ　　５２　ｋｕ的蛋白质，发现其能抑制大肠杆菌对ＧＡ１的黏附，可能也是双歧杆菌的黏附素［１５，１６］　．我们在以往研究的基础上，采用硫酸铵沉淀，　凝胶过滤和离子交换色谱法，　从双歧杆菌耗尽培养液上清中纯化出一种Ｍｒ　　１６　ｋｕ的蛋白质，　经证实为双歧杆菌黏附素．这一结果为进一步研究双歧杆菌黏附的基因调控提供了物质基础．细菌在肠道中黏附与定植是一个非常复杂的过程，　往往有多种因素的参与．黏附不仅发生在细菌与宿主细胞之间；而且还涉及到细菌与黏液层之间的相互作用．另外，　有些细菌的黏附可能需要多种黏附素及其受体的参与．我们首次发现，１６　ｋｕ的蛋白质能介导双歧杆菌对体外培养细胞系Ｌｏｖｏ细胞的黏附．但在体内，　双歧杆菌与肠道上皮细胞间的黏附是否还有其他黏附素的参与以及黏液层在黏附中的作用如何，　尚待进一步的研究．４　　　　　　参考文献

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