[1]黄茜,黄璐,潘道东,等.植物乳杆菌Lactobacillu plantarumY1菌株胆盐水解酶基因(bsh)的克隆及重组表达[J].南京师大学报(自然科学版),2010,33(03):91-96.
　Huang Qian,Huang Lu,Pan Daodong,et al.Molecular Cloning and Expression of the Bile Salt Hydrolase Gene(bsh) From Lactobacillus plantarum Y1[J].Journal of Nanjing Normal University(Natural Science Edition),2010,33(03):91-96.
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植物乳杆菌Lactobacillu plantarumY1菌株胆盐水解酶基因(bsh)的克隆及重组表达()
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《南京师大学报（自然科学版）》[ISSN:1001-4616/CN:32-1239/N]

卷:
第33卷
期数:
2010年03期
页码:
91-96
栏目:
生命科学
出版日期:
2010-09-20

文章信息/Info

Title:
Molecular Cloning and Expression of the Bile Salt Hydrolase Gene(bsh) From Lactobacillus plantarum Y1
作者:
黄茜;黄璐;潘道东;杨瑶;
国家乳品加工技术研发分中心, 南京师范大学食品科学与营养系, 江苏南京210097
Author(s):
Huang Qian; Huang Lu; Pan Daodong; Yang Yao
Branch Center of National Dairy Products Processing Technology Development,Department of Food Science and Nutrition,Nanjing Normal University,Nanjing 210097,China
关键词:
植物乳杆菌;  胆盐水解酶;  原核表达;  融合标签技术
Keywords:
Lactobacillus p lantarum;  B ile sa lt hydro lase;  prokaryo tic expression;  fus ion tags techno logy
分类号:
Q78
摘要:
经同源蛋白比对分析设计引物,PCR扩增获得植物乳杆菌Y1菌株bsh基因(975 bp),首先克隆至表达载体pET-28a,转化E.coli BL21(DE3)菌株,IPTG诱导表达,SDS-PAGE分析结果显示表达的重组蛋白为包涵体.选择能为大肠杆菌稀有密码子提供额外tRNA的E.coli Rosetta(DE3)作为宿主菌,仍旧没有改善表达产物的可溶性.但是,选择含IF2融合蛋白标签的pLS-IF2质粒构建表达载体,SDS-PAGE分析及Western blot鉴定结果显示表达的融合重组蛋白IF2-BS
Abstract:
Based on the hom o logous prote in BLAST result, the bsh gene o fLactobacillus p lantarum Y1( 975 bp) was amp lified by PCR and subcloned in to expression vecto r pET-28a. The constructed recomb inant plasm id pET-28a-BSH w as transform ed toE scherichia coli BL21( DE3) fo r express ion under induc tion o f IPTG. SDS-PAGE pro file ind icated that the expressed prote in ma in ly ex isted in a form of inclusion body. The so lubility of the expressed prote in w as not improved when choo sing E. coli Rose tta ( DE3) as the expression host that cou ld supply additional tRNA for rare codons. H ow eve r, w hen pLS-IF2 was chosen to construct the expression p lasm id that conta ined the fusion pro te in tag  IF2, SDSPAGE profile andW estern b lo t results showed that the expressed product IF2-BSH junction to improve the so lub ility eff-i c iency, respectively. The re fo re, on basis of the fusion tags techno logy, the b sh gene ofL. p lan tarum Y1 was successfu-l ly c loned and expressed in E. coli. , wh ich la id a foundation of study on b ioactiv ity and re lation betw een struc ture and functions o f B ile Sa ltH ydrolase from Lactobacillu s sp.

参考文献/References:

[ 1]G illiland S E, Ne lson C R, M axwe ll C. Assim ilation o f cholestero l by Lactobacillus acidoph ilus[ J]. App l Env ironM icrob io,l 1985, 49: 377-381.
[ 2]Má ire B, Co lin H, Corm ac G M. B ile sa lt hydro lase activ ity in prob io tics[ J] . App l Env iron M icrob io ,l 2006, 72: 1 729- 1 738.
[ 3]DeanM, Ce rve llati C, Casanova E, et a .l Character iza tion o f cho ly lglyc ine hydro lase from a bile- adapted strain o f Xanthom onas m altoph ilia and its app lication for quan tita tive hydro lysis o f con jugated b ile sa lts [ J]. Appl Env iron M icrob iol,2002, 68: 3 126-3 128.
[ 4]Knarreborg A, Engberg R M, Jensen S K, et a .l Quantitative determ ination o f b ile sa lt hydro lase activ ity in bacter ia iso la ted from the sm a ll intestine of ch ickens[ J]. Appl Env ironM icrob io ,l 2002, 68: 6 425-6 428.
[ 5]Ste llwag E J, H y lem on P B. Pur ification and characteriza tion of b ile sa lt hydrolase from Bactero ides frag ilis subsp. frag ilis [ J]. B iochim B iophys Acta, 1976, 452: 165-176.
[ 6]Christiaens H, LeerR J, Pouw e ls P H, et a .l C lon ing and expression o f a conjuga ted b ile acid hydrolase gene from Lac tobac illus planta rum by using a direct plate assay[ J]. Appl Env ironM icrob io,l 1992, 58: 3 792-3 798.
[ 7]K leerebezem M, Boekhorst J, K ranenburg R V, et a.l Com plete genom e sequence of Lactobac illus p lantarum WCFS1[ J]. Pro cNa tlAcad Sc iUSA, 2003, 100: 1 990-1 995.
[ 8]Lambert JM, Bongers R S, K lee rebe zem M. C re- lox-based system form ultiple gene deletions and se lectab le-m arker rem oval in Lactobac illus plan tarum [ J]. Appl Env ironM icrob io ,l 2007, 73: 1 126-1 135.
[ 9]E lk ins C A, M oser S A, Savage D C. Genes encod ing bile salt hydrolases and conjugated b ile salt transporters in Lactobac i-l lus johnson ii 100-100 and other Lac tobac illus species[ J]. M ic robio logy, 2001, 147: 3 403-3 412.
[ 10]Oh, H ae-Keun, Lee J Y, et a.l M o lecu lar cloning and character ization o f a bile sa lt hydro lase from lactobacillus ac idophilus PF01[ J]. JM icrobio,l 2008, 18: 449-456.
[ 11]ZhangW Y, Wu R N, Sun ZH, et a.l M o lecu la r c lon ing and character iza tion o f bile sa lt hydro lase in Lactobac illus casei Zhang [ J]. Anna ls ofM icrob io logy, 2009, 59( 4): 721-726.
[ 12]战媛媛, 王长远, 于长青. 植物乳杆菌中胆盐水解酶基因的原核表达及纯化[ J]. 中国生物制品学杂志, 2009, 22 ( 9): 876-879.
[ 13]S??rensen H P, Petersen H U, M ortensen K K. A favorab le solub ility partne r for the recom b inant expression of strep tav id in [ J]. Prote in Express and Puri,f 2003, 32: 252-259.
[ 14]萨姆布鲁克J, 拉W. 分子克隆实验指南[M ]. 2版. 黄培堂, 译. 北京: 科学出版社, 1999.
[ 15]李永进, 陈媛媛, 毕利军. 融合标签技术及其应用[ J]. 生物工程学报, 2006, 22( 4) : 523-527.
[ 16]Terpe K. Overv iew of tag pro te in fusions: from mo lecu lar and bio chem ica l fundam entals to comm erc ia l system s[ J]. Appl M icrobio l B io techno,l 2003, 60: 523-533.
[ 17]H earnM T W, Acosta D. Applica tions of nov el affinity cassettem ethods: use of peptide fusion handles fo r the pur ification o f recomb inant prote ins[ J] . JM ol Recogn it, 2001, 14: 323-369.
[ 18]Uh lenM, Fo rsberg G, M oks T, et a.l Fus ion prote ins in b iotechno logy[ J]. CurrOp in B io tech, 1992, 3( 4): 363-369.

备注/Memo

备注/Memo:
基金项目: 江苏省高校自然科学基金( 09KJB550003) . 通讯联系人: 杨瑶, 博士, 讲师, 研究方向: 功能乳酸菌的筛选及分子生物学. E-mail:cranny-yang@ hotmail.com
更新日期/Last Update: 2013-04-08