大肠杆菌不耐热肠毒素B亚单位构建表达与毒性实验
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摘要
第一部分:pBV220-LTB基因克隆与表达
目的:采用基因组提取方法从野生型产毒性大肠杆菌E.coli H 44815中提取LTB基因,利用基因工程技术将其重组于表达载体pBV220,然后对含有pBV220-LTB的工程菌进行表达,蛋白质分析,为研究该蛋白质的生物学特性提供基础。
方法:用PCR方法扩增LTB目的基因片段,克隆至pBV220质粒中,构建pBV220-LTB的表达质粒,转化E.coli DH5α,经温度诱导表达重组蛋白。
结果:经测序基因片段由374bp组成,为编码124个氨基酸残基的多肽。经SDS-PAGE分析相对分子量(Mr)约为30000D。结论: pBV220- LTB的原核表达质粒构建成功,并在大肠杆菌DH5α中高效的表达。
第二部分:LTB蛋白质的家兔毒性实验
目的:LTB的粘膜免疫作用已比较明确,但是任何疫苗的开发,安全性是必须考虑的问题,重组表达载体pBV220–LTB表达蛋白有无毒性,是其能否用于疫苗研究的前提,因此探讨重组LTB蛋白质对家兔的毒性作用,评价感染的可行性,为后续的双歧杆菌研究做准备,
PARTⅠCloning and Expression of pBV220-LTB
Objective: To construct and express pBV220-LTB,we distill Heat-labile enterotoxin B subunit from E.coli H 44815 by gene-engineering, which would lay a foundation for the biological idiosyncrasy.
Methods: Heat-labile enterotoxin B subunit gene was amplified by PCR and cloned into plasmid pBV220. The recombinant plamsid was identified by sequencing ,then transformed into E.coli DH5a.. The transformant colony was induced with IPTG. Purify the expressed protein by Ni~(2+)-NTA column chromatography. Expression of the protein was analyzed by SDS-PAGE .
Results: The gene fragment at length of 396 bp was amplified and successfully cloned into plasmid pBV220.SDS-PAGE showed a protein band with relative molecular weight of 30 000,which was consistent with the expectation.
Conclusion: The recombinant plasmid of pBV220-LTB was successfully constructed and Heat-labile enterotoxin B subunit gene was highly expressed in E.coli DH5a.
PARTⅡThe rabbit`s toxicity experimentation of the protein of Heat-labile enterotoxin B subunit
目的:采用基因组提取方法从野生型产毒性大肠杆菌E.coli H 44815中提取LTB基因,利用基因工程技术将其重组于表达载体pBV220,然后对含有pBV220-LTB的工程菌进行表达,蛋白质分析,为研究该蛋白质的生物学特性提供基础。
方法:用PCR方法扩增LTB目的基因片段,克隆至pBV220质粒中,构建pBV220-LTB的表达质粒,转化E.coli DH5α,经温度诱导表达重组蛋白。
结果:经测序基因片段由374bp组成,为编码124个氨基酸残基的多肽。经SDS-PAGE分析相对分子量(Mr)约为30000D。结论: pBV220- LTB的原核表达质粒构建成功,并在大肠杆菌DH5α中高效的表达。
第二部分:LTB蛋白质的家兔毒性实验
目的:LTB的粘膜免疫作用已比较明确,但是任何疫苗的开发,安全性是必须考虑的问题,重组表达载体pBV220–LTB表达蛋白有无毒性,是其能否用于疫苗研究的前提,因此探讨重组LTB蛋白质对家兔的毒性作用,评价感染的可行性,为后续的双歧杆菌研究做准备,
PARTⅠCloning and Expression of pBV220-LTB
Objective: To construct and express pBV220-LTB,we distill Heat-labile enterotoxin B subunit from E.coli H 44815 by gene-engineering, which would lay a foundation for the biological idiosyncrasy.
Methods: Heat-labile enterotoxin B subunit gene was amplified by PCR and cloned into plasmid pBV220. The recombinant plamsid was identified by sequencing ,then transformed into E.coli DH5a.. The transformant colony was induced with IPTG. Purify the expressed protein by Ni~(2+)-NTA column chromatography. Expression of the protein was analyzed by SDS-PAGE .
Results: The gene fragment at length of 396 bp was amplified and successfully cloned into plasmid pBV220.SDS-PAGE showed a protein band with relative molecular weight of 30 000,which was consistent with the expectation.
Conclusion: The recombinant plasmid of pBV220-LTB was successfully constructed and Heat-labile enterotoxin B subunit gene was highly expressed in E.coli DH5a.
PARTⅡThe rabbit`s toxicity experimentation of the protein of Heat-labile enterotoxin B subunit
引文
[1] 郭学青,邹全明 霍乱毒素及大肠杆菌不耐热肠毒素生物学特性的研究.[J]世界华人消化杂志 2000 年 3 月;8(3):325-326
[2] Holmgren J,czerkiIlsky C,Edksson K,et a1.mucosal immunisation and adjuvsnts:a blief overview of recent advances and challenges.[J]Vaccine,2003,21(suppl2): 89-95
[3] Haan L,Verweij WR,HoltropM et a1.Nasal or intramuscular immunization of mice with influenza subunit antigen and the B subunit of Escherichia coli heat-labile toxin induces IgA orIgGmediated protective mucosal immunity.[J]Vaccine,2001,19(20-22):2898-2907.
[4] Millar DG,Hirst TR,Snider DP.Escherichia coli heat-labile enterotoxin B subunit is a more potent mucosal adjuvant than its closely related homologue,the B subunit of cholera toxin.[J]Infect lmmun,2001,69(5):3476-3482.
[5] Gozaka S,YasudaY,IsakaM,et a1.efficient extracellular production of recombinant Escherichia coli heat-labile enterotoxin B subunit by using the expression /secretion system of bacillus brevis and its mucosal immunoadjuvanticity. [J]Vaccine,2OO0,18(17):1730-1737.
[6] Nashar TO,Betteridge ZE,Mitchell RN.Evidence for a role of ganglioside GM1 in antigen presenation : binding enhances presenation of Escherichia coli enterotoxin Bsubunit(EtxB)to CD4(+)T cells.[J]Int Immunol,2001,13(4):541-551.
[7] DeHaanL,Verweij WR,Feil Ⅸ et a1.Role of GM1 binding in the mucosal immunogenicity and adjuvant aetivity of the Escherichia coli heat-labile enterotoxin and its B subunit. [J] Immunology,1998,94:424-430.
[8] 房海主编.[M]大肠埃希氏菌.河北:河北科学技术出版社,1997,133-143
[1] Bourget N,Simonet Jm,Decaris B.Analysis of the genome of the five Bifidobacterium breve strain:plasmid content pulsed-field gel electrophoresis genome size estimation and rrn loci number[J].FEMS microbiol lett,1993 ,110:11.
[2] Matteuzzi D,Brigidip,Rossim,et al.Characterization and molecular clonging of Bifidobacterium longum cryptic plasmid pMB1[J].Let inAppli Microbiol,1990,11:220.
[3] Rossi M,Brigidi P,Gonzalez A,el al.Characterization of the plasmid pMB1 from Bifidobacterium longum and its use of Shuttle vector construction[J].Res microbiol,1999,147:133.
[4] Nathalie corneau,Eric Emond, Gisele Lapointe.molecular characterization of three plasmids from Bifidobacterium longum [J] .plasmid,2004,51:87.
[5] Myeony S.Park,Dong W.Shin,el al.Sequence anolysis of plasmid pKJ50 from Bifidobacterium longum[J].Microbiology,1999,145(3):585.
[6] PARK,MYEONG.SOO,HYE WON MOON,et al.molecular characterization of plasmid from Bifidobacterium longum.J microbiol Biotechnol,2003,13(3):457.
[7] MissichR,SgorbatiB,Donald JL.Transformation of Bifidobacterium longum with PRMZ,a constructed Escherichia coli-B.longum shuttle vector[J]. plasmid,1994,32:208.
[8] Park,M.S;Shin,D.W;Lee,K.H;et al.Sequence analysis of plasmid pKJ50 from Bifidobacterium longum[J].Microbiology,1999,145:585.
[9] Matsumura,H ; Takeuchi,A ; Kano,Y.construction of Escherichia coli-Bifidobacterium longum shuttle vector transforming Bifidobacterium longum 105-A and 108-A[J].Biosci Biotechnol Biochem,1997,61:1211.
[10] Ming-Ni Hung,Byog H.Lee.Cloning and expression of β-galactosidase gene from Bifidobacterium infantis into Escherichia coli.Biotecbnology letters,1998,20(7):659.
[11] Myeony S.park,Dong W.Shin,Ke H.Lee,et al.Ji.Sequence analysis of plasmid pKJ50 from Bifidobacterium longum[J].Microbiology,1999,145,585.
[12] Rochet V,Rigottier-Gois L,Beguet F,el al.composition of human intestinal flora analysis by fluorescent in situ hybridisation using group-specific 16s rRNA-targeted oligonucleotide probes[J].Genetics selection evolution, 2001,33(1): s339.
[13] Kaufmann P,Pfefferkorn A,Teuber M,et al.Identification and quantification of Bifidobacterium species isolated from food with genus-specific 16s rRNA-targeted probes by colony hybridization and PCR[J].Appl EnvironMicrobiol,1997,63(4):1268.
[14] ROY D,WARD P,CHAMPAGNE G.Differentiation of Bifidobacterium by use of plused-field gel electrophoresis and polymerase chain reaction[J].In J food Microbiol,1996,29(1):11.
[15] ERJA MALINEN,JAANA MARIO,MERJA SALMITIE,et al.Analysis of Bifidobacterium populations in human faecal samples from a comsumption trial with Bifidobacterium lactis Bb-12 and a galacto-oligasacc-haride preparation[J]. System Appl Microbiol,2002,25:249.
[16] Mitsuo Sakamoto,Hidenori Hayashi,and Yoshimi Benno.Terminal Restriction Fragment Length Polymorphism Analysis for Human Fecal Microbiota and Its Application for Analysis of complex Bifidobacterium communities[J].Microbiol Immunol,2003,47(2):133.
[17] V.DELCENSERIE,N.BECHOUX,T.LEONARO,el al.Discrimination between Bifidobacterium species from Human and Animal origin by PCR-Restriction Fragment Length Porlymorphism[J].Journal of Food protection,2004,67(6): 1284.
[18] Harmsen HJ,Wildeboer-Veloo AC,Raang GC,el al.Analysis of intestinal flora development in breast-fed and formula-fed infants by using molecular identification and detection methods[J].J Pediatr Gastroenterol Nutr,2000, 30(1):61.
[19] SEAN M,CUSICK AND DANIEL J,O'SULLIVAN.Use of a single Triplicate Arbitrarily Primed-PCR procedure for Molecular Fingerprinting of Lactic Acid Bacteria[J].APPLIED AND ENVIRONMENTAL NICROBIOLOGY,2000,P:2227.
[20] 董明盛,江汉湖,张磊.DNA 指纹图谱鉴别双歧杆菌的研究[J].微生物学杂志,2001,21(3).
[21] Koen Venema, Annet JH,Maathuis. A PCR-based method for identification of Bifidobacterium from the Human alimentary tract at the species level[J].FEMS Microbiology Letters,2003,224:143.
[22] Li Sheng Wang,Hui Ming Zhu,Dian Yuan Zhou,el al.Influence of whole peptidoglycan of Bifidobacterium on cytotoxic effectors produced by mouseperitoneal macrophages[J].World J Gastroenterol,2001,7(3):440.
[23] Roberfroid MB.Prebiotics and probiotics:are they functional foods? [J]. Am J Clin Nutr,2000,71(suppl 6):1682.
[24] Tejada-Simon MV,Prstka JJ.poinflammatory cytokine and nitric oxidc induction in murine macrophages by cell wall and cytoplasmic extracts of lactic acid bacteria[J].J Food Prot,1999,62:1435.
[25] Rhee YK,Bae EA, Kim SY,el al.Antitumor activity of Bifidobacterium spp isolated from a health Korean[J].Arch Pharm Res,2000,23:482.
[26] 蒋虹,胡宏.双歧杆菌表面分子诱导大肠癌细胞分化的研究[J].中华微生物学和免疫学杂志,2001,21(2).
1 Roy D.Media for the isolation and enumeration of Bifidobacteria in dairy products.Int J Food Microbiol,2001,69:167-182
2 Payne J F,Morris A E J,Beers P .Note:Evaluation of selective media for the enumeration of Bifidobacteria sp.J Appl Microbiol,1999,86:353-358
3 Renata G.K ,Jan Bew,Paul Simpson et al.A collaborative study of a method forthe enumeration of probiotic Bifidobacteria in animal feed.Int J Food Microbiol,2003,83:161-170
4 Juha H.A.Apajalahti,Anu Kettunen,Paivi H.Nurminen et al.Selective plating underestimates abundance and shows differential recovery of Bifidobacteria species from human feces.App Envion Microbiol,2003,69(9):5731-5735
5 Tharmaraj N,Shah NP.Selective enumeration of lactobacillus, delbrueckii ssp. Bulgaricus Streptococcus,thermophilus,Lactobacillus,acidophilus,bifidobacteria,Lactobacillus casei,Lactobacillus rhamnosus and propionibacteria.J Dairy Sci,2003,86(7):2288-96
6 Simpson P.J,fitzgerald G F,Stanton C et al.The evaluation of a mupirocin-based selective medium for the enumeration of Bifidobacteria from probiotic animal feed.J Microbiol Methods,2004, 57:9-16
[2] Holmgren J,czerkiIlsky C,Edksson K,et a1.mucosal immunisation and adjuvsnts:a blief overview of recent advances and challenges.[J]Vaccine,2003,21(suppl2): 89-95
[3] Haan L,Verweij WR,HoltropM et a1.Nasal or intramuscular immunization of mice with influenza subunit antigen and the B subunit of Escherichia coli heat-labile toxin induces IgA orIgGmediated protective mucosal immunity.[J]Vaccine,2001,19(20-22):2898-2907.
[4] Millar DG,Hirst TR,Snider DP.Escherichia coli heat-labile enterotoxin B subunit is a more potent mucosal adjuvant than its closely related homologue,the B subunit of cholera toxin.[J]Infect lmmun,2001,69(5):3476-3482.
[5] Gozaka S,YasudaY,IsakaM,et a1.efficient extracellular production of recombinant Escherichia coli heat-labile enterotoxin B subunit by using the expression /secretion system of bacillus brevis and its mucosal immunoadjuvanticity. [J]Vaccine,2OO0,18(17):1730-1737.
[6] Nashar TO,Betteridge ZE,Mitchell RN.Evidence for a role of ganglioside GM1 in antigen presenation : binding enhances presenation of Escherichia coli enterotoxin Bsubunit(EtxB)to CD4(+)T cells.[J]Int Immunol,2001,13(4):541-551.
[7] DeHaanL,Verweij WR,Feil Ⅸ et a1.Role of GM1 binding in the mucosal immunogenicity and adjuvant aetivity of the Escherichia coli heat-labile enterotoxin and its B subunit. [J] Immunology,1998,94:424-430.
[8] 房海主编.[M]大肠埃希氏菌.河北:河北科学技术出版社,1997,133-143
[1] Bourget N,Simonet Jm,Decaris B.Analysis of the genome of the five Bifidobacterium breve strain:plasmid content pulsed-field gel electrophoresis genome size estimation and rrn loci number[J].FEMS microbiol lett,1993 ,110:11.
[2] Matteuzzi D,Brigidip,Rossim,et al.Characterization and molecular clonging of Bifidobacterium longum cryptic plasmid pMB1[J].Let inAppli Microbiol,1990,11:220.
[3] Rossi M,Brigidi P,Gonzalez A,el al.Characterization of the plasmid pMB1 from Bifidobacterium longum and its use of Shuttle vector construction[J].Res microbiol,1999,147:133.
[4] Nathalie corneau,Eric Emond, Gisele Lapointe.molecular characterization of three plasmids from Bifidobacterium longum [J] .plasmid,2004,51:87.
[5] Myeony S.Park,Dong W.Shin,el al.Sequence anolysis of plasmid pKJ50 from Bifidobacterium longum[J].Microbiology,1999,145(3):585.
[6] PARK,MYEONG.SOO,HYE WON MOON,et al.molecular characterization of plasmid from Bifidobacterium longum.J microbiol Biotechnol,2003,13(3):457.
[7] MissichR,SgorbatiB,Donald JL.Transformation of Bifidobacterium longum with PRMZ,a constructed Escherichia coli-B.longum shuttle vector[J]. plasmid,1994,32:208.
[8] Park,M.S;Shin,D.W;Lee,K.H;et al.Sequence analysis of plasmid pKJ50 from Bifidobacterium longum[J].Microbiology,1999,145:585.
[9] Matsumura,H ; Takeuchi,A ; Kano,Y.construction of Escherichia coli-Bifidobacterium longum shuttle vector transforming Bifidobacterium longum 105-A and 108-A[J].Biosci Biotechnol Biochem,1997,61:1211.
[10] Ming-Ni Hung,Byog H.Lee.Cloning and expression of β-galactosidase gene from Bifidobacterium infantis into Escherichia coli.Biotecbnology letters,1998,20(7):659.
[11] Myeony S.park,Dong W.Shin,Ke H.Lee,et al.Ji.Sequence analysis of plasmid pKJ50 from Bifidobacterium longum[J].Microbiology,1999,145,585.
[12] Rochet V,Rigottier-Gois L,Beguet F,el al.composition of human intestinal flora analysis by fluorescent in situ hybridisation using group-specific 16s rRNA-targeted oligonucleotide probes[J].Genetics selection evolution, 2001,33(1): s339.
[13] Kaufmann P,Pfefferkorn A,Teuber M,et al.Identification and quantification of Bifidobacterium species isolated from food with genus-specific 16s rRNA-targeted probes by colony hybridization and PCR[J].Appl EnvironMicrobiol,1997,63(4):1268.
[14] ROY D,WARD P,CHAMPAGNE G.Differentiation of Bifidobacterium by use of plused-field gel electrophoresis and polymerase chain reaction[J].In J food Microbiol,1996,29(1):11.
[15] ERJA MALINEN,JAANA MARIO,MERJA SALMITIE,et al.Analysis of Bifidobacterium populations in human faecal samples from a comsumption trial with Bifidobacterium lactis Bb-12 and a galacto-oligasacc-haride preparation[J]. System Appl Microbiol,2002,25:249.
[16] Mitsuo Sakamoto,Hidenori Hayashi,and Yoshimi Benno.Terminal Restriction Fragment Length Polymorphism Analysis for Human Fecal Microbiota and Its Application for Analysis of complex Bifidobacterium communities[J].Microbiol Immunol,2003,47(2):133.
[17] V.DELCENSERIE,N.BECHOUX,T.LEONARO,el al.Discrimination between Bifidobacterium species from Human and Animal origin by PCR-Restriction Fragment Length Porlymorphism[J].Journal of Food protection,2004,67(6): 1284.
[18] Harmsen HJ,Wildeboer-Veloo AC,Raang GC,el al.Analysis of intestinal flora development in breast-fed and formula-fed infants by using molecular identification and detection methods[J].J Pediatr Gastroenterol Nutr,2000, 30(1):61.
[19] SEAN M,CUSICK AND DANIEL J,O'SULLIVAN.Use of a single Triplicate Arbitrarily Primed-PCR procedure for Molecular Fingerprinting of Lactic Acid Bacteria[J].APPLIED AND ENVIRONMENTAL NICROBIOLOGY,2000,P:2227.
[20] 董明盛,江汉湖,张磊.DNA 指纹图谱鉴别双歧杆菌的研究[J].微生物学杂志,2001,21(3).
[21] Koen Venema, Annet JH,Maathuis. A PCR-based method for identification of Bifidobacterium from the Human alimentary tract at the species level[J].FEMS Microbiology Letters,2003,224:143.
[22] Li Sheng Wang,Hui Ming Zhu,Dian Yuan Zhou,el al.Influence of whole peptidoglycan of Bifidobacterium on cytotoxic effectors produced by mouseperitoneal macrophages[J].World J Gastroenterol,2001,7(3):440.
[23] Roberfroid MB.Prebiotics and probiotics:are they functional foods? [J]. Am J Clin Nutr,2000,71(suppl 6):1682.
[24] Tejada-Simon MV,Prstka JJ.poinflammatory cytokine and nitric oxidc induction in murine macrophages by cell wall and cytoplasmic extracts of lactic acid bacteria[J].J Food Prot,1999,62:1435.
[25] Rhee YK,Bae EA, Kim SY,el al.Antitumor activity of Bifidobacterium spp isolated from a health Korean[J].Arch Pharm Res,2000,23:482.
[26] 蒋虹,胡宏.双歧杆菌表面分子诱导大肠癌细胞分化的研究[J].中华微生物学和免疫学杂志,2001,21(2).
1 Roy D.Media for the isolation and enumeration of Bifidobacteria in dairy products.Int J Food Microbiol,2001,69:167-182
2 Payne J F,Morris A E J,Beers P .Note:Evaluation of selective media for the enumeration of Bifidobacteria sp.J Appl Microbiol,1999,86:353-358
3 Renata G.K ,Jan Bew,Paul Simpson et al.A collaborative study of a method forthe enumeration of probiotic Bifidobacteria in animal feed.Int J Food Microbiol,2003,83:161-170
4 Juha H.A.Apajalahti,Anu Kettunen,Paivi H.Nurminen et al.Selective plating underestimates abundance and shows differential recovery of Bifidobacteria species from human feces.App Envion Microbiol,2003,69(9):5731-5735
5 Tharmaraj N,Shah NP.Selective enumeration of lactobacillus, delbrueckii ssp. Bulgaricus Streptococcus,thermophilus,Lactobacillus,acidophilus,bifidobacteria,Lactobacillus casei,Lactobacillus rhamnosus and propionibacteria.J Dairy Sci,2003,86(7):2288-96
6 Simpson P.J,fitzgerald G F,Stanton C et al.The evaluation of a mupirocin-based selective medium for the enumeration of Bifidobacteria from probiotic animal feed.J Microbiol Methods,2004, 57:9-16