人小肠三叶因子在乳酸菌中的表达
摘要
肠三叶因子(intestinal trefoil factor,ITF)是1991年发现的特异分布于胃肠道黏膜表面的小分子蛋白,它由59个氨基酸残基构成,分子量约6×10~3,属于三叶肽家族。ITF包含1个由38-39个氨基酸残基组成的特殊保守序列,其中的6个半胱氨酸按1～5,2～4,3～6的顺序依次形成3个二硫键,从而产生特异而稳定的三叶草型结构。该结构可确保ITF不受消化酶和pH变化的影响,有利于其在胃肠道中发挥生物效应,即维护肠黏膜的完整性,促进受损肠道修复。由于ITF具有特殊的空间结构,不仅能促进胃肠黏膜细胞增殖,还能同胃肠黏液中的糖蛋白结合,稳定胃肠黏液,维护胃肠黏液屏障,因而在减轻胃肠道损害,促进胃肠道修复方面具有重要意义。
乳酸菌是人和动物肠胃中正常存在的菌群,被公认为安全级微生物,已广泛地应用于食品及饮料加工业。乳酸菌作为基因工程表达菌株具有无内毒素安全性好、遗传背景清楚、表达效率高、表达蛋白不用纯化等优点。因此,乳酸菌表达系统逐渐受到重视和广泛应用。已有利用乳酸菌获得了医用的疫苗、抗原、酶、必需氨基酸等的高效表达的研究报道。如人干扰素α-2b(IFNα-2b)已成功的在德氏乳杆菌DM890表达,并且具有很好的抗病毒活性,用表达hCGβ的重组乳酸杆菌活疫苗经阴道黏膜途径免疫不同品系小鼠产生抗hCGβ免疫应答。
ITF对受损胃肠黏膜的疗效得到了肯定,大量动物实验证明,它是一种具有潜在药用价值的短肽,目前多采用基因工程的方法获取重组ITF。1999年国内有肠三叶因子在大肠杆菌中表达的报道,目前国内外也有酵母表达的报道。鉴于采用基因工程表达ITF产量低、活性差,ITF作为小肽存在纯化困难的问题,为此本研究利用乳酸乳球菌表达系统表达ITF。首先克隆hITFcDNA,构建hITF乳酸乳球菌分泌型表达载体,再转化乳酸乳球菌,获得稳定整合菌株,实现hITF在乳酸乳球菌中的表达,克服了纯化的困难,为乳酸乳球菌做为口服性药物的递送载体和ITF的深入研究奠定基础。
Intestinal trefoil factor(TFF3/ITF),a member of trefoil factor family(TFF) domain peptides,is normally expressed by goblet cells and secreted to he lumen of the intestinal tract.TFF3/ITF plays an important role in the maintenance and repair of the intestinal mucosal barrier.The distinct three-looped secondary structure contributes to the remarkable resistance of trefoil factors to acid and proteolytic digestion,enables them to function in the harsh environment of the gastrointestinal tract lumen,and allows them to remain biologically active after enteral administration.
Lactococcus lactis is a Gram-Positive food-grade bacterium and has a long history of widespread use in the food industry for the production of fermented milk products.And it can stimulate a mucosal immune response as a adjuvant in the intestinal tract. Lactococcus lactis is approximately the same size as biodegradable microparticles that are known to be taken up by M cells and have been shown to be capable of acting as effective oral vaccine vehicles.In this study,the hITF gene was amplified and clone into the prokaryotic expressive vector pNICE:sec.The recombinant vector was transformated into Lactococcus lactis strain NZ9000.The fusion protein(5.9kDa) was expressed in L.lactis by the induction of the nisin.The quantity of expression accounted for 5%of the total bacterial protein.Western bolt analysis confirmed that fusion protein could be recognized specifically by the monoclonal anti-human TFF3.The recombinant L.lactis expressing hITF protein was used to administrate to rabbits.
Rabbits are divided into blank control group,preparation group and therapeutic group which are respectively administrated wih PBS and pNICE:sec-ITF lactococcus lactis.By grades of ulcer test whether administrated pNICE:sec-ITF lactococcus lactis protects against HCL-induced gastric injury in rabbits.
These results suggest that Preparetion groups and therapeutic groups do good than control group.Prove that administrated pNICE:sec-ITF lactococcus lactis is biologically active in an HCL-induced rabbit gastric mucosal injury model.
乳酸菌是人和动物肠胃中正常存在的菌群,被公认为安全级微生物,已广泛地应用于食品及饮料加工业。乳酸菌作为基因工程表达菌株具有无内毒素安全性好、遗传背景清楚、表达效率高、表达蛋白不用纯化等优点。因此,乳酸菌表达系统逐渐受到重视和广泛应用。已有利用乳酸菌获得了医用的疫苗、抗原、酶、必需氨基酸等的高效表达的研究报道。如人干扰素α-2b(IFNα-2b)已成功的在德氏乳杆菌DM890表达,并且具有很好的抗病毒活性,用表达hCGβ的重组乳酸杆菌活疫苗经阴道黏膜途径免疫不同品系小鼠产生抗hCGβ免疫应答。
ITF对受损胃肠黏膜的疗效得到了肯定,大量动物实验证明,它是一种具有潜在药用价值的短肽,目前多采用基因工程的方法获取重组ITF。1999年国内有肠三叶因子在大肠杆菌中表达的报道,目前国内外也有酵母表达的报道。鉴于采用基因工程表达ITF产量低、活性差,ITF作为小肽存在纯化困难的问题,为此本研究利用乳酸乳球菌表达系统表达ITF。首先克隆hITFcDNA,构建hITF乳酸乳球菌分泌型表达载体,再转化乳酸乳球菌,获得稳定整合菌株,实现hITF在乳酸乳球菌中的表达,克服了纯化的困难,为乳酸乳球菌做为口服性药物的递送载体和ITF的深入研究奠定基础。
Intestinal trefoil factor(TFF3/ITF),a member of trefoil factor family(TFF) domain peptides,is normally expressed by goblet cells and secreted to he lumen of the intestinal tract.TFF3/ITF plays an important role in the maintenance and repair of the intestinal mucosal barrier.The distinct three-looped secondary structure contributes to the remarkable resistance of trefoil factors to acid and proteolytic digestion,enables them to function in the harsh environment of the gastrointestinal tract lumen,and allows them to remain biologically active after enteral administration.
Lactococcus lactis is a Gram-Positive food-grade bacterium and has a long history of widespread use in the food industry for the production of fermented milk products.And it can stimulate a mucosal immune response as a adjuvant in the intestinal tract. Lactococcus lactis is approximately the same size as biodegradable microparticles that are known to be taken up by M cells and have been shown to be capable of acting as effective oral vaccine vehicles.In this study,the hITF gene was amplified and clone into the prokaryotic expressive vector pNICE:sec.The recombinant vector was transformated into Lactococcus lactis strain NZ9000.The fusion protein(5.9kDa) was expressed in L.lactis by the induction of the nisin.The quantity of expression accounted for 5%of the total bacterial protein.Western bolt analysis confirmed that fusion protein could be recognized specifically by the monoclonal anti-human TFF3.The recombinant L.lactis expressing hITF protein was used to administrate to rabbits.
Rabbits are divided into blank control group,preparation group and therapeutic group which are respectively administrated wih PBS and pNICE:sec-ITF lactococcus lactis.By grades of ulcer test whether administrated pNICE:sec-ITF lactococcus lactis protects against HCL-induced gastric injury in rabbits.
These results suggest that Preparetion groups and therapeutic groups do good than control group.Prove that administrated pNICE:sec-ITF lactococcus lactis is biologically active in an HCL-induced rabbit gastric mucosal injury model.
引文
[1] SUEMOR I S, LY NCH2 DEVANEY K, PODOLSKYD K,et al. Identification and characterization of rat intestinal trefoil factor, tissue and cell specific member of the trefoil pr otein family[J]. Proc Natl Acad Sci USA. 1991,88(24):11017-11021.
[2] Thim L. A new family of growth factor21ike pep tides:'trefoil'disulphide loop structures as a common feature in breast cancer as a sociated pep tide ( pS2 ), pancreatic spasmolytic polypep tide ( PSP) and frog skin pep tides ( spasmolysins) [J].
[3] Gajhede M et al.Structure,1993;l(4);253~262. [4] May FEB , Westley BR. CI ose physical linkage of the genes encoding the pnr22 / pS2 pr otein and human spasmolytic protein (hsp) [ J ]. HumGenet, 1997, 99: 303~ 307
[5] Seib T,Blin N, Hilgert K, etal . The three human trefoil genes tff1,tff2 and tff3 are located within a region of 55kb on chromosome 21q22.3 [ J ]. Genomics, 1997, 40: 200-202.
[6] Taupin D, Pedersen J, FamilariM, etal . Augmented intestinal trefoil factor ( TFF3 ) and loss of pS2 ( TFFI ) expression precedes metaplastic differentiation of gastric ep ithelium[J]. Lab Invest, 2001, 81 (3): 397~408
[7] Pouls om R, Hanby AM, Lalani EN, etal. Intestinal trefoil factor ( TFF3) and pS2 ( TFFI), but not spasmolytic polypeptide ( TFF2) mRNAs are coexpressed in normal, hyperplastic, and neoplastic human breast epithelium[ J ]. J Pathol, 1997, 183 (1): 30~38
[8] dos Santos Silva E, Ulrich M, Doring G, etal. Trefoil factor family domain peptides in the human respiratory tract [ J ]. J Pathol, 2000,190(2): 133~ 142
[9] Ogata H, Podolsky DK . Trefoil peptide expression and secretion is regulated by neuropeptides and acetylcholine[ J ]. Am J Physiol, 1997, 273 (2Pt 1): G348~354
[10] Mashimo H. Podolsky DK and Fishman MC. Structure and expression of murine intestinal trefoil factor: high evolutionary conservation and postnatal expressi on[ J ]. Biochem Biophys Res Commun, 1995, 210 (1): 31—37
[11] Otto WR, Patel K . Trefoil factor family ( TFF) domain peptides in the mouse: embryonic gastr ointestinal expression and wounding response [J]. Anat Embryol Berl, 1999,199 (6): 499-508
[12] Lin J, Holzman 1 R, Jiang P, et al . Expression of intestinal trefoil factor in developing rat intestine[J]. Biol Neonate, 1999, 76 (2): 92-97
[13] Hauser F, Pouls om R, Chinery R, etal . hP1. B, a human P2 domain peptide homologous with rat intestinal trefoil factor is expressed also in the ulcer-associated cell lineage and the uterus[ J ]. Proc Natl Acad Sci USA, 1993, 90(15): 6961—6965
[14]Lien GS, Chen CN, Cheng YS, etal. Early colonic carcinoma with extensive lymph node metastases : case report and review of literature[ J ].Int J Col orectal Dis, 2001, 16 (4): 262-266
[15]Tran CP, FamilariM, Parker LM, etal . Short chain fatty acids inhibit intestinal trefoil factor gene expression in colon cancer cells[ J ]. Am J Physiol, 1998, 275 (1 Pt 1): G85-94
[16]LoncarMB,A12 azzeh ED, Sommer PS, etal . Tumour necrosis factor alpha and nuclear factor kappa B inhibit transcription of human TFF3 encoding a gastrointestinal healing peptide [ J ]. Gut, 2003, 52 (9): 1297~ 1303
[17] Mark B et al- Gastroenterology, 1996:110(2): 489~ 497.
[18] Beck PL, Wong JF,Li Y, et al. Chemotherapy and radiotherapy induced intestinal damage is regulated by intestinal trefoil factor[ J ] . Gastroenterology, 2004, 126 (3): 796-808.
[19] Dong X, Zhou L,Lin S, et al. Effect of recombinant human trefoil factor on protection of gastric mucosa and healing of chronic gastric ulcer in rats [ J ]. Beijing Da Xue Xue Bao, 2003, 35 ( 6) :639-641.
[20]Kidon H etal. Gastroenterology, 1995; 109(2): 516~523.
[21] Mashimo H et al. Science,1996: 274(11):262~265.
[22]Dignass A etal. Science 1996; 274(11): 262 ~265.
[23] Kang Yu, Song-Fu Jiang1,Mao-Fang Lin, etal. Extraction and purification of biologically active intestinal trefoil factor from human meconium Laboratory Investigation,2004 84,390-392.
[24]王蔚,口如琴,李今媛,等.人小肠三叶因子在大肠杆菌中的表达[J].中国生物化学与分子生物学报,1999,15(5):724-728.
[25]孙勇,彭曦,张勇,吕尚军,等.hITF毕赤酵母表达载体的构建及分泌表达.第三军医大学学报,2006 28(6):523-527.
[26]TH I M L,WOLD IKE H F,NIELSEN P F,etal.Characterization of human and rat intestinal trefoil fact or produced in yeast[J].Biochemistry,1995,34(14):4757-4764.
[27]左晓峰,张晓钰,单龙,等.人小肠三叶因子基因在生菜中整合和表达.植物学报,2001 43(10):1047-1051.
[28]徐晓晶,张竞,李曦,等.人小肠三叶因子在糙皮侧耳中的整合.表达及检测.微生物学报,2004 44(5).
[29]向华,张亦清,卫文仲,等.人谷胱甘肽硫转移酶Al在乳酸乳球菌中的表达及活性研究.[J].微生物学报,2000,4(2):132-134
[30]Bolotin A,Wncker P,Mauger S,et al.The complete genome sequence of the lactic acid bacterium Lactococcus lactis ssp.Lactis L1403[J].Genome Res,2001,11(5):731 753
[31]W.M.de Vos.Gene expression systems for lactic acid bacteria.Curr Opin Microbiol.1999,2(3):289-295.
[32]Enouf,V.,Langella,E,Commissaire,J.,Cohen,J.and Corthier,G..Bovine rotavirus nonstructural protein 4 produced by Lactococcus lactis is antigenic and immunogenic.Environ.Microbiol.2001,(67):1423-1328.
[33]Bermudez-Humaran,L.G.,Langella,P.,Commissaire,J.,Gibert,S.,Le Loir,Y.,L'Haridon,R.and Cortheir,G.Controlled intra-or extracellular production of staphylococcal nuclease and ovine omega interferion in Lactococcus lactis.FEMS Microbiol.Lett.2003,224:307-313.
[34]陈辉,贾润清,康白,等.人干扰素α-2b基因在德氏乳杆菌的表达与鉴定[J].中国微生态学杂志,2004,16(3):129-131.
[35]姚晓英,李大金,袁敏敏.表达hCG β的重组乳酸杆菌经阴道免疫小鼠后 的体液免疫应答[J].中华微生物学和免疫学杂志,2006,26(7):659-664.
[36]Guth PH,Aures D,Paulsen G,etal.Topic aspirin plus HCI gastric lesion in the rat.Gastroenterology,1979;76:88-91.
[37]张守仁,邵金莺,於毓文,等.呋喃唑酮和一些常用抗溃疡药对四种大鼠胃溃疡模型的影响.药学学报,1984;91:5-11.
[38]Chamberlain,L.;Wells,J M.;Robinson,K.;Schofield,K.;Le Page,R W F.Mucosal immunization with recombinant Lactococcus lactis.In:Pozzi G,Wells J M.,editors.Gram-positive bacteria as vaccine vehicles for mucosal immunization.Austin,Tex:Landes Bioscience;1997:83-106.
[39]Schagger H,von Jagow G.Tricine2sodium dodecyl sulfate-polyacrylamid gel electroporesis for the separation of proteins in the range from 1 to 100kDa.Analytical Biochemistry,1987,166:368-379
[40]曹佐武.小分子肽的Tricine-SDS-PAGE分离方法.生物学通报,2003,38(3:55-56.
[41]Sambrook J,Fritsch E F,Maniatis T.Molecular Cloning.2~(nd) edition.New York:Cold Spring Harbor Laboratory Press,1989,1847-1848.
[1]Thim L.Trefoil peptides:from structure to function[J].CellMol Life Sci,1997,53(11-12):888-903.
[2]May FEB,Westley BR.Close physical linkage of the genes encoding the pnr22/pS2 protein and human spasmolytic protein(hsp)[J].Hum Genet,1997,99:303-307.
[3]Seib T,Blin N,Hilgert K,etal.The three human trefoil genes tff1,tff2 and tff3 are located within a region of 55kb on chromosome 21q22.3[J].Genomics,1997,40:200-202.
[4]dosSantos Silva E etal JPathol,2000:190(2):133-142.
[5]Hauser F et al Proc Nail Acad Sci USA,1993:90(15):6961-6965.
[6]May FE et al J Pathot,1997:182(4) 404-413.
[7]Podolsky DK etal J Biot Chem、1993;268(9):6694-6702.
[8]Suemori S et al.Proc Natl Acad Sci USA,1991 88(24):11017-11021.
[9]Mashimo H et al.biochem Biophys Res Commun.1995;210(1):31-37.
[10]Ogata H et al Am J Physiol,1997:273(2pt1):G348-G354.
[11]Chambon C etal.Gastroenterology.1991 100(2):375-379.
[12]Wright NA et al.Nature,1990:343(6253).82-85.
[13]Hauser F et al Proc Natl Acad Sci USA,1993:90(15):6961-6965.
[14]Alison M R et al.J Patho,1995:175(4):405-414.
[15]Uehino H et al.Gastroenterolgy,2000,118(1):60-69.
[16]May FE et al J Pathot,1997:182(4) 404-413.
[17]Mark WB et al.Gastroenterology,1996;110(2):489-497.
[18]Kindon H etal.Gastroenterology,1995;109(2):516-523.
[19]Mashimo H et al Science 1996:274(11):262-265.
[20]Digasis A et al.J Clin Invest.1994:94(1):376-383.
[21]Emami S,Le Fl och N,Bruyneel E,et al.Induction of scattering andcellular invasion by trefoil peptides in sac and RhoA-transformed kidney and colonic epithelial cells[J].FASEB J,2001,15(2):351-361
[22]Tanaka S,PodolskyDK,Engel E,et al.Human s pasmolytic polypep tide decreases proton permeation through gastric mucus in vivo and in vitro[J].Am J Physiol,1997,272:1473-1480.
[23]Mc Kenzie C,Marchbank T,Playford RJ,et al.Pancreatic pasmolytic polypeptide protects the gastric mucosa but does not inhibit acid secre tion ormotility[J]. Am J Physiol, 1997, 273: 112-117
[24]Babyatsky MW, deBeaumont M, Thim L, et al . Oral trefoil peptides protect against ethanol and indomethacin-induced gastric injury in rats [J]. Gastroenterology, 1996, 110: 489-497.
[25]DignassA,Lynch2 Devaney K, Kindon H, et al . Trefoil pep tides pr omote epithelialmigration through a trensforming growth factor beta-independent pathway[J]. J Clin Invest, 1994, 94 (1):376~383.
[26]Liu D, el-Hariry 1, Karayiannakis AJ, et al . Phos phorylation of betacatenin and epidermal growth factor receptor by intestinal trefoil factor [J]. Lab Invest, 1997, 77 (6): 557-563.
[27]Nie SN , Qian XM, Wu XH , et al . Role of TFF in healing of stress-in duced gastric lesions[J], World J Gastroenterol, 2003, 9(8): 1772-1776.
[28]Andoh A, Kinoshita K, Rosenbergl, et al . Intestinal trefoil fact or induces decay-accelerating factor expression and enhances the p rotective activities against complement activation in intestinal ep ithelial cells [J]. J Immunol, 2001, 167 (7): 3887-3893.
[29]]Chen YH, Lu Y, De Plaen IG, et al . Transcription fact or NF-kappaB signals antianoikic function of trefoil factor 3 on intestinal ep ithelial cells[J]. Biochem Biophys Res Commun, 2000, 274(3): 576-582.
[30]Tan XD, Chen YH, Liu QP, et al . Prostanoids mediate the p rotective effect of trefoil factor 3 in oxidant2induced intestinal ep ithelial cell injury : role of cycl ooxygenase[J]. J Immunol, 2001, 167(7): 3887-3893.
[31}KanaiM, Mullen C, PodolskyDK . Intestinal trefoil factor induces acti vation of extracellular signal-regulated protein kinase in intestinal epihelial cells . Proc Natl Acad Sci USA, 1998,9:178.
[32]Kinoshida K, Taup in DR, Itoh H, et al. Distinct pathways of cellmigration and antiapoptotic response to epithilial injury: structure - functi on analysis of human intestinal trefoil factor [J] . Mol Cell Biol,2000,20(13):4680-4690.
[33]Ogata H,Podolsky DK.Am J Physiol,1997:273(2 part 1):G348-G354.
[34]Poulsom R,Chinery R,Sarraf C,J Clin Gstroenterol,1993;17(suppl 1):S78-S91.
[35]Tran.CP,Familari M,Parker LM et al.Am J Physiol,1998;275(1 part 1):G85-G94.
[36]Chinery r,Playford RJ.Olin Sci Colch,1995;88(4):401-403.
[37]Chinery R,Cox HM.Br J Pharmacol,1995;115(1):77-80.
[38]左晓峰,张晓钰,单龙,等.人小肠三叶因子基因在生菜中整合和表达.植物学报,2001 43(10):1047-1051.
[39]徐晓晶,张竞,李曦,等.人小肠三叶因子在糙皮侧耳中的整合.表达及检测.微生物学报,2004 44(5).
[40]王蔚,口如琴,李今媛,等.人小肠三叶因子在大肠杆菌中的表达[J].中国生物化学与分子生物学报,1999,15(5):724-728.
[41]孙勇,彭曦,张勇,吕尚军,等.hITF毕赤酵母表达载体的构建及分泌表达.第三军医大学学报,2006 28(6):523-527.
[42]TH I M L,WOLD IKE H F,N IELSEN P F,etal.Characterization of human and rat intestinal trefoil fact or produced in yeast[J].Biochemistry,1995,34(14):4757-4764.
[43]向华,张亦清,卫文仲,等.人谷胱甘肽硫转移酶Al在乳酸乳球菌中的表达及活性研究.[J].微生物学报,2000,4(2):132-134
[44]Bolotin A,Wncker P,Mauger S,et al.The complete genome sequence of the lactic acid bacterium Lactococcus lactis ssp.Lactis L1403[J].Genome Res,2001,11(5):731 753
[45]W.M.de Vos.Gene expression systems for lactic acid bacteria.Curr Opin Microbiol.1999,2(3):289-295.
[46]Enouf,V.,Langella,P.,Commissaire,J.,Cohen,J.and Corthier,G..Bovine rotavirus nonstructural protein 4 produced by Lactococcus lactis is antigenic and immunogenic.Environ.Microbiol.2001,(67):1423-1328.
[47]Bermudez-Humaran,L.G.,Langella,P.,Commissaire,J.,Gibert,S.,Le Loir,Y.,L'Haridon,R.and Cortheir,G.Controlled intra-or extracellular production of staphylococcal nuclease and ovine omega interferion in Lactococcus lactis.FEMS Microbiol.Lett.2003,224:307-313.
[48]陈辉,贾润清,康白,等.人干扰素α-2b基因在德氏乳杆菌的表达与鉴定[J].中国微生态学杂志,2004,16(3):129-131.
[49]姚晓英,李大金,袁敏敏.表达hCG β的重组乳酸杆菌经阴道免疫小鼠后的体液免疫应答[J].中华微生物学和免疫学杂志,2006,26(7):659-664.
[50]Chamberlain,L.;Wells,J M.;Robinson,K.;Schofield,K.;Le Page,R W F.Mucosal immunization with recombinant Lactococcus lactis.In:Pozzi G,Wells J M.,editors.Gram-positive bacteria as vaccine vehicles for mucosal immunization.Austin,Tex:Landes Bioscience;1997:83-106.
[51]DharDK,Wang TC,Tabara H,et al.Expression of trefoil factor family members correlates with patient prognosis and neoangiogenesis[J].Clin Cancer Res,2005,15,11(18):6472-6478.
[52]ChanMW,Chart VY,LeungWK,et al.Anti-sense trefoil factor family-3(intestinal trefoil fact or) inhibits cell gr owth and induces chemosen-sitivity to adriamycin in human gastric cancer cells[J].Life Sci,2005,15,76(22):2581-2592.
[53]Beck PL,Wong JF,Li Y,et al.Chemotherapy-and radi otherapy-induced intestinal damage is regulated by intestinal trefoil fact or[J].Gastroenterology,2004,126(3):796-808.
[54]Vestergaard EM,BorreM,Poulsen SS,et al.Plasma levels of trefoil factors are increased in patients with advanced prostate cancer[J].Clin Cancer Res,2006,12(3Pt 1):807-812.
[55]Khoury T,Chadha K,JavleM,et al.Exp ressi on of intestinal trefoil factor (TFF-3) in hepatcellular carcinoma[J].I nt J Gastrointest Cancer,2005,35(3):171-177.
[56]Yang T,Zou K,Qian W.Effects of intestinal trefoil,factor on colonic mucsa in experimental colitis of rats[J].Huazhong Univ Sci Technolog Med Sci,2005,25(3): 300-302
[2] Thim L. A new family of growth factor21ike pep tides:'trefoil'disulphide loop structures as a common feature in breast cancer as a sociated pep tide ( pS2 ), pancreatic spasmolytic polypep tide ( PSP) and frog skin pep tides ( spasmolysins) [J].
[3] Gajhede M et al.Structure,1993;l(4);253~262. [4] May FEB , Westley BR. CI ose physical linkage of the genes encoding the pnr22 / pS2 pr otein and human spasmolytic protein (hsp) [ J ]. HumGenet, 1997, 99: 303~ 307
[5] Seib T,Blin N, Hilgert K, etal . The three human trefoil genes tff1,tff2 and tff3 are located within a region of 55kb on chromosome 21q22.3 [ J ]. Genomics, 1997, 40: 200-202.
[6] Taupin D, Pedersen J, FamilariM, etal . Augmented intestinal trefoil factor ( TFF3 ) and loss of pS2 ( TFFI ) expression precedes metaplastic differentiation of gastric ep ithelium[J]. Lab Invest, 2001, 81 (3): 397~408
[7] Pouls om R, Hanby AM, Lalani EN, etal. Intestinal trefoil factor ( TFF3) and pS2 ( TFFI), but not spasmolytic polypeptide ( TFF2) mRNAs are coexpressed in normal, hyperplastic, and neoplastic human breast epithelium[ J ]. J Pathol, 1997, 183 (1): 30~38
[8] dos Santos Silva E, Ulrich M, Doring G, etal. Trefoil factor family domain peptides in the human respiratory tract [ J ]. J Pathol, 2000,190(2): 133~ 142
[9] Ogata H, Podolsky DK . Trefoil peptide expression and secretion is regulated by neuropeptides and acetylcholine[ J ]. Am J Physiol, 1997, 273 (2Pt 1): G348~354
[10] Mashimo H. Podolsky DK and Fishman MC. Structure and expression of murine intestinal trefoil factor: high evolutionary conservation and postnatal expressi on[ J ]. Biochem Biophys Res Commun, 1995, 210 (1): 31—37
[11] Otto WR, Patel K . Trefoil factor family ( TFF) domain peptides in the mouse: embryonic gastr ointestinal expression and wounding response [J]. Anat Embryol Berl, 1999,199 (6): 499-508
[12] Lin J, Holzman 1 R, Jiang P, et al . Expression of intestinal trefoil factor in developing rat intestine[J]. Biol Neonate, 1999, 76 (2): 92-97
[13] Hauser F, Pouls om R, Chinery R, etal . hP1. B, a human P2 domain peptide homologous with rat intestinal trefoil factor is expressed also in the ulcer-associated cell lineage and the uterus[ J ]. Proc Natl Acad Sci USA, 1993, 90(15): 6961—6965
[14]Lien GS, Chen CN, Cheng YS, etal. Early colonic carcinoma with extensive lymph node metastases : case report and review of literature[ J ].Int J Col orectal Dis, 2001, 16 (4): 262-266
[15]Tran CP, FamilariM, Parker LM, etal . Short chain fatty acids inhibit intestinal trefoil factor gene expression in colon cancer cells[ J ]. Am J Physiol, 1998, 275 (1 Pt 1): G85-94
[16]LoncarMB,A12 azzeh ED, Sommer PS, etal . Tumour necrosis factor alpha and nuclear factor kappa B inhibit transcription of human TFF3 encoding a gastrointestinal healing peptide [ J ]. Gut, 2003, 52 (9): 1297~ 1303
[17] Mark B et al- Gastroenterology, 1996:110(2): 489~ 497.
[18] Beck PL, Wong JF,Li Y, et al. Chemotherapy and radiotherapy induced intestinal damage is regulated by intestinal trefoil factor[ J ] . Gastroenterology, 2004, 126 (3): 796-808.
[19] Dong X, Zhou L,Lin S, et al. Effect of recombinant human trefoil factor on protection of gastric mucosa and healing of chronic gastric ulcer in rats [ J ]. Beijing Da Xue Xue Bao, 2003, 35 ( 6) :639-641.
[20]Kidon H etal. Gastroenterology, 1995; 109(2): 516~523.
[21] Mashimo H et al. Science,1996: 274(11):262~265.
[22]Dignass A etal. Science 1996; 274(11): 262 ~265.
[23] Kang Yu, Song-Fu Jiang1,Mao-Fang Lin, etal. Extraction and purification of biologically active intestinal trefoil factor from human meconium Laboratory Investigation,2004 84,390-392.
[24]王蔚,口如琴,李今媛,等.人小肠三叶因子在大肠杆菌中的表达[J].中国生物化学与分子生物学报,1999,15(5):724-728.
[25]孙勇,彭曦,张勇,吕尚军,等.hITF毕赤酵母表达载体的构建及分泌表达.第三军医大学学报,2006 28(6):523-527.
[26]TH I M L,WOLD IKE H F,NIELSEN P F,etal.Characterization of human and rat intestinal trefoil fact or produced in yeast[J].Biochemistry,1995,34(14):4757-4764.
[27]左晓峰,张晓钰,单龙,等.人小肠三叶因子基因在生菜中整合和表达.植物学报,2001 43(10):1047-1051.
[28]徐晓晶,张竞,李曦,等.人小肠三叶因子在糙皮侧耳中的整合.表达及检测.微生物学报,2004 44(5).
[29]向华,张亦清,卫文仲,等.人谷胱甘肽硫转移酶Al在乳酸乳球菌中的表达及活性研究.[J].微生物学报,2000,4(2):132-134
[30]Bolotin A,Wncker P,Mauger S,et al.The complete genome sequence of the lactic acid bacterium Lactococcus lactis ssp.Lactis L1403[J].Genome Res,2001,11(5):731 753
[31]W.M.de Vos.Gene expression systems for lactic acid bacteria.Curr Opin Microbiol.1999,2(3):289-295.
[32]Enouf,V.,Langella,E,Commissaire,J.,Cohen,J.and Corthier,G..Bovine rotavirus nonstructural protein 4 produced by Lactococcus lactis is antigenic and immunogenic.Environ.Microbiol.2001,(67):1423-1328.
[33]Bermudez-Humaran,L.G.,Langella,P.,Commissaire,J.,Gibert,S.,Le Loir,Y.,L'Haridon,R.and Cortheir,G.Controlled intra-or extracellular production of staphylococcal nuclease and ovine omega interferion in Lactococcus lactis.FEMS Microbiol.Lett.2003,224:307-313.
[34]陈辉,贾润清,康白,等.人干扰素α-2b基因在德氏乳杆菌的表达与鉴定[J].中国微生态学杂志,2004,16(3):129-131.
[35]姚晓英,李大金,袁敏敏.表达hCG β的重组乳酸杆菌经阴道免疫小鼠后 的体液免疫应答[J].中华微生物学和免疫学杂志,2006,26(7):659-664.
[36]Guth PH,Aures D,Paulsen G,etal.Topic aspirin plus HCI gastric lesion in the rat.Gastroenterology,1979;76:88-91.
[37]张守仁,邵金莺,於毓文,等.呋喃唑酮和一些常用抗溃疡药对四种大鼠胃溃疡模型的影响.药学学报,1984;91:5-11.
[38]Chamberlain,L.;Wells,J M.;Robinson,K.;Schofield,K.;Le Page,R W F.Mucosal immunization with recombinant Lactococcus lactis.In:Pozzi G,Wells J M.,editors.Gram-positive bacteria as vaccine vehicles for mucosal immunization.Austin,Tex:Landes Bioscience;1997:83-106.
[39]Schagger H,von Jagow G.Tricine2sodium dodecyl sulfate-polyacrylamid gel electroporesis for the separation of proteins in the range from 1 to 100kDa.Analytical Biochemistry,1987,166:368-379
[40]曹佐武.小分子肽的Tricine-SDS-PAGE分离方法.生物学通报,2003,38(3:55-56.
[41]Sambrook J,Fritsch E F,Maniatis T.Molecular Cloning.2~(nd) edition.New York:Cold Spring Harbor Laboratory Press,1989,1847-1848.
[1]Thim L.Trefoil peptides:from structure to function[J].CellMol Life Sci,1997,53(11-12):888-903.
[2]May FEB,Westley BR.Close physical linkage of the genes encoding the pnr22/pS2 protein and human spasmolytic protein(hsp)[J].Hum Genet,1997,99:303-307.
[3]Seib T,Blin N,Hilgert K,etal.The three human trefoil genes tff1,tff2 and tff3 are located within a region of 55kb on chromosome 21q22.3[J].Genomics,1997,40:200-202.
[4]dosSantos Silva E etal JPathol,2000:190(2):133-142.
[5]Hauser F et al Proc Nail Acad Sci USA,1993:90(15):6961-6965.
[6]May FE et al J Pathot,1997:182(4) 404-413.
[7]Podolsky DK etal J Biot Chem、1993;268(9):6694-6702.
[8]Suemori S et al.Proc Natl Acad Sci USA,1991 88(24):11017-11021.
[9]Mashimo H et al.biochem Biophys Res Commun.1995;210(1):31-37.
[10]Ogata H et al Am J Physiol,1997:273(2pt1):G348-G354.
[11]Chambon C etal.Gastroenterology.1991 100(2):375-379.
[12]Wright NA et al.Nature,1990:343(6253).82-85.
[13]Hauser F et al Proc Natl Acad Sci USA,1993:90(15):6961-6965.
[14]Alison M R et al.J Patho,1995:175(4):405-414.
[15]Uehino H et al.Gastroenterolgy,2000,118(1):60-69.
[16]May FE et al J Pathot,1997:182(4) 404-413.
[17]Mark WB et al.Gastroenterology,1996;110(2):489-497.
[18]Kindon H etal.Gastroenterology,1995;109(2):516-523.
[19]Mashimo H et al Science 1996:274(11):262-265.
[20]Digasis A et al.J Clin Invest.1994:94(1):376-383.
[21]Emami S,Le Fl och N,Bruyneel E,et al.Induction of scattering andcellular invasion by trefoil peptides in sac and RhoA-transformed kidney and colonic epithelial cells[J].FASEB J,2001,15(2):351-361
[22]Tanaka S,PodolskyDK,Engel E,et al.Human s pasmolytic polypep tide decreases proton permeation through gastric mucus in vivo and in vitro[J].Am J Physiol,1997,272:1473-1480.
[23]Mc Kenzie C,Marchbank T,Playford RJ,et al.Pancreatic pasmolytic polypeptide protects the gastric mucosa but does not inhibit acid secre tion ormotility[J]. Am J Physiol, 1997, 273: 112-117
[24]Babyatsky MW, deBeaumont M, Thim L, et al . Oral trefoil peptides protect against ethanol and indomethacin-induced gastric injury in rats [J]. Gastroenterology, 1996, 110: 489-497.
[25]DignassA,Lynch2 Devaney K, Kindon H, et al . Trefoil pep tides pr omote epithelialmigration through a trensforming growth factor beta-independent pathway[J]. J Clin Invest, 1994, 94 (1):376~383.
[26]Liu D, el-Hariry 1, Karayiannakis AJ, et al . Phos phorylation of betacatenin and epidermal growth factor receptor by intestinal trefoil factor [J]. Lab Invest, 1997, 77 (6): 557-563.
[27]Nie SN , Qian XM, Wu XH , et al . Role of TFF in healing of stress-in duced gastric lesions[J], World J Gastroenterol, 2003, 9(8): 1772-1776.
[28]Andoh A, Kinoshita K, Rosenbergl, et al . Intestinal trefoil fact or induces decay-accelerating factor expression and enhances the p rotective activities against complement activation in intestinal ep ithelial cells [J]. J Immunol, 2001, 167 (7): 3887-3893.
[29]]Chen YH, Lu Y, De Plaen IG, et al . Transcription fact or NF-kappaB signals antianoikic function of trefoil factor 3 on intestinal ep ithelial cells[J]. Biochem Biophys Res Commun, 2000, 274(3): 576-582.
[30]Tan XD, Chen YH, Liu QP, et al . Prostanoids mediate the p rotective effect of trefoil factor 3 in oxidant2induced intestinal ep ithelial cell injury : role of cycl ooxygenase[J]. J Immunol, 2001, 167(7): 3887-3893.
[31}KanaiM, Mullen C, PodolskyDK . Intestinal trefoil factor induces acti vation of extracellular signal-regulated protein kinase in intestinal epihelial cells . Proc Natl Acad Sci USA, 1998,9:178.
[32]Kinoshida K, Taup in DR, Itoh H, et al. Distinct pathways of cellmigration and antiapoptotic response to epithilial injury: structure - functi on analysis of human intestinal trefoil factor [J] . Mol Cell Biol,2000,20(13):4680-4690.
[33]Ogata H,Podolsky DK.Am J Physiol,1997:273(2 part 1):G348-G354.
[34]Poulsom R,Chinery R,Sarraf C,J Clin Gstroenterol,1993;17(suppl 1):S78-S91.
[35]Tran.CP,Familari M,Parker LM et al.Am J Physiol,1998;275(1 part 1):G85-G94.
[36]Chinery r,Playford RJ.Olin Sci Colch,1995;88(4):401-403.
[37]Chinery R,Cox HM.Br J Pharmacol,1995;115(1):77-80.
[38]左晓峰,张晓钰,单龙,等.人小肠三叶因子基因在生菜中整合和表达.植物学报,2001 43(10):1047-1051.
[39]徐晓晶,张竞,李曦,等.人小肠三叶因子在糙皮侧耳中的整合.表达及检测.微生物学报,2004 44(5).
[40]王蔚,口如琴,李今媛,等.人小肠三叶因子在大肠杆菌中的表达[J].中国生物化学与分子生物学报,1999,15(5):724-728.
[41]孙勇,彭曦,张勇,吕尚军,等.hITF毕赤酵母表达载体的构建及分泌表达.第三军医大学学报,2006 28(6):523-527.
[42]TH I M L,WOLD IKE H F,N IELSEN P F,etal.Characterization of human and rat intestinal trefoil fact or produced in yeast[J].Biochemistry,1995,34(14):4757-4764.
[43]向华,张亦清,卫文仲,等.人谷胱甘肽硫转移酶Al在乳酸乳球菌中的表达及活性研究.[J].微生物学报,2000,4(2):132-134
[44]Bolotin A,Wncker P,Mauger S,et al.The complete genome sequence of the lactic acid bacterium Lactococcus lactis ssp.Lactis L1403[J].Genome Res,2001,11(5):731 753
[45]W.M.de Vos.Gene expression systems for lactic acid bacteria.Curr Opin Microbiol.1999,2(3):289-295.
[46]Enouf,V.,Langella,P.,Commissaire,J.,Cohen,J.and Corthier,G..Bovine rotavirus nonstructural protein 4 produced by Lactococcus lactis is antigenic and immunogenic.Environ.Microbiol.2001,(67):1423-1328.
[47]Bermudez-Humaran,L.G.,Langella,P.,Commissaire,J.,Gibert,S.,Le Loir,Y.,L'Haridon,R.and Cortheir,G.Controlled intra-or extracellular production of staphylococcal nuclease and ovine omega interferion in Lactococcus lactis.FEMS Microbiol.Lett.2003,224:307-313.
[48]陈辉,贾润清,康白,等.人干扰素α-2b基因在德氏乳杆菌的表达与鉴定[J].中国微生态学杂志,2004,16(3):129-131.
[49]姚晓英,李大金,袁敏敏.表达hCG β的重组乳酸杆菌经阴道免疫小鼠后的体液免疫应答[J].中华微生物学和免疫学杂志,2006,26(7):659-664.
[50]Chamberlain,L.;Wells,J M.;Robinson,K.;Schofield,K.;Le Page,R W F.Mucosal immunization with recombinant Lactococcus lactis.In:Pozzi G,Wells J M.,editors.Gram-positive bacteria as vaccine vehicles for mucosal immunization.Austin,Tex:Landes Bioscience;1997:83-106.
[51]DharDK,Wang TC,Tabara H,et al.Expression of trefoil factor family members correlates with patient prognosis and neoangiogenesis[J].Clin Cancer Res,2005,15,11(18):6472-6478.
[52]ChanMW,Chart VY,LeungWK,et al.Anti-sense trefoil factor family-3(intestinal trefoil fact or) inhibits cell gr owth and induces chemosen-sitivity to adriamycin in human gastric cancer cells[J].Life Sci,2005,15,76(22):2581-2592.
[53]Beck PL,Wong JF,Li Y,et al.Chemotherapy-and radi otherapy-induced intestinal damage is regulated by intestinal trefoil fact or[J].Gastroenterology,2004,126(3):796-808.
[54]Vestergaard EM,BorreM,Poulsen SS,et al.Plasma levels of trefoil factors are increased in patients with advanced prostate cancer[J].Clin Cancer Res,2006,12(3Pt 1):807-812.
[55]Khoury T,Chadha K,JavleM,et al.Exp ressi on of intestinal trefoil factor (TFF-3) in hepatcellular carcinoma[J].I nt J Gastrointest Cancer,2005,35(3):171-177.
[56]Yang T,Zou K,Qian W.Effects of intestinal trefoil,factor on colonic mucsa in experimental colitis of rats[J].Huazhong Univ Sci Technolog Med Sci,2005,25(3): 300-302