王欣莹,范洪学,严杰
WANG Xin-ying~△,FAN Hong-xue,YAN Jie ( ~△School of Public Health

• 1:吉林大学公共卫生学院
• 2:吉林大学公共卫生学院
• 3:浙江大学医学院病原生物学教研室 长春130021
• 4:浙江大学医学院病原生物学教研室

摘要(Abstract)：

目的克隆问号钩端螺旋体鞭毛相关基因flhA、flhB2和fliR,并构建其原核表达载体。方法按常规苯酚-氯仿法提取问号钩端螺旋体黄疸出血群赖型56601株基因组DNA,高保真PCR扩增flhA、flhB2和fliR基因片段,T-A克隆后测序,构建原核表达载体,采用SDS-PAGE和免疫印迹法检测目的融合蛋白的表达。结果问号钩体56601株flhA、flhB2和fliR基因扩增片段的核苷酸序列与报道的相应序列同源性分别为100%、99·9%和99·9%,氨基酸序列同源性分别为100%、99·8%和100%。所构建的重组原核表达系统在IPTG的诱导下,能有效地表达目的融合蛋白Trx-FlhA、Trx-FlhB2和Trx-FliR,产量约为细菌总蛋白的10%。结论已成功构建了问号钩端螺旋体鞭毛相关基因flhA、flhB2和fliR原核表达系统。
Objective To clone the flagellar biosynthesis genes flhA,flhB_2 and fliR of Leptospira interrogans and construct their prokaryotic expression system.Methods Extract genomic DNA from Leptospira interrogans serogroup Icterohaemorrhagiae serovar Lai strain 56601 by phenol-chloroform method and amplify flhA,flhB_2 and fliR gene fragments by high fidelity PCR.After T-A cloning,the amplified genes were identified by sequencing and used for the construction of prokaryotic expression vector.Identify the expressed fusion protein by SDS-PAGE and Western blot.Results The homologies of nucleotide sequences of amplified flhA,flhB_2 and fliR genes to those reported were 100%,99.9% and 99.9%,and those of the deduced amino acid sequences were 100%,100% and 99.8%,respectively.Fusion proteins Trx-FlhA,Trx-FlhB_2 and Trx-FliR were effectively expressed in the constructed prokaryotic expression system under induction of IPTG.The expressed product contained about 10% of total somatic protein.Conclusion The prokaryotic expression system of flagellar biosynthesis genes flhA,flhB_2 and fliR of Leptospira interrogans was successfully constructed.

关键词(KeyWords)： 问号钩端螺旋体;鞭毛相关基因;克隆;表达
Leptospira interrogans;Flagellar biosynthesis gene;Cloning;Expression

Abstract:

Keywords:

基金项目(Foundation): 国家自然科学基金资助项目(30370072)

作者(Author): 王欣莹,范洪学,严杰
WANG Xin-ying~△,FAN Hong-xue,YAN Jie ( ~△School of Public Health

DOI: 10.13200/j.cjb.2006.01.10.wangxy.002

参考文献(References)：

• [1]Levett PN.Leptospirosis.Clin Microbiol Rev,2001,14(2):296-326.
• [2]De La Pena-Moctezuma A,Bulach DM,Adler B.Genetic differences among the LPS biosynthetic Loci of serovars ofLeptospir interrogans andLeptospira borgpetersenii.FEMS Immununol Med Microbiol,2001,31(1):73-81.
• [3]Faine S,Adler B,Bolin C,et al.Leptospira and leptospirosis.Second edition.Melbourne:MediSci,1999.
• [4]Lee SH,Kim S,Park SC,et al.Cytotoxic activities ofLeptospira inter-roganshemolysin SphH as a pore-forming protein on mammalian cells.Infect Immun,2002,70(1):315-322.
• [5]Hueck CJ.TypeⅢprotein secretion systems in bacterial pathogens of animals and plants.Microbiol Mol Bio Rev,1998,62(2):379-433.
• [6]Young GM,Schmiel DH,Miller VL.A newpathway for the secretion of virulence factors by bacteria:the flagellar export apparatus func-tions as a protein-secretion system.Proc Natl Acad Sci USA,1999,96(11):6456-6461.
• [7]Sambrook J,Fritsch EF,Maniatis T.Molecular cloning,a laboratory manual.New York:Gold Spring Harbor Laboratory Press,1989.
• [8]Ren SX,Fu G,Jiang XG,et al.Unique physiological and pathogenic features ofLeptospira interrogansrevealed by whole-genome sequen-cing.Nature,2003,422(6934):888-893.
• [9]李立伟,严杰,毛亚飞,等.不同毒力的问号钩端螺旋体对Vero及J774A·1细胞的黏附和内化.中华微生物学和免疫学杂志,2004,24(2):98~102.
• [10]Li LW,Liu YY,Yan J,et al.Apoptosis and ultrastructural lesions in Vero and J774A·1cells induced byLeptospira interrogans.J Zhe-jiang Uni:Med Sci,2005,34(1):4-8.
• [11]Fleiszig SMJ,Arora SK,Van R,et al.FlhA,a component of the fla-gellum assembly apparatus ofPseudomonas aeruginosa,plays a role in internalization by corneal epithelial cells.Infect Immun,2001,69(8):4931-4937.
• [12]Carpenter PB,Zuberi AR,Ordal GW.Bacillus subtilis flagellar pro-teins FliP,FliQ,FliR and FlhB are related toShigella flexneriviru-lence factors.Gene,1993,137(2):243-245.
• [13]Fraser GM,Hirano T,Ferris HU,et al.Substrate specificity of typeⅢflagellar protein export inSalmonellais controlled by subdomain interactions in FlhB.Mol Microbiol,2003,48(4):1043-1057.
• [14]Fan F,Ohnishi K,Francis NR,et al.The FliP and FliR proteins of Salmonella typhimurium,putative components of the typeⅢflagellar export apparatus,are located in the flagellar basal body.Mol Microbi-ol,1997,26(5):1035-1046.