预防幽门螺杆菌感染的蛋黄抗体的研制
摘要
目的:采用基因工程技术大量表达重组幽门螺杆菌(Helicobac ter pylori,H.pylori)鞭毛粘附素(HpaA),用此重组蛋白作为抗原,制备高效价的抗HpaA的鸡蛋黄抗体IgY,为研制H.pylori预防性抗体制剂奠定基础。
方法:
1.诱导重组质粒pQE30-hpaA在大肠杆菌DH5a中表达rHpaA,SDS-PAGE分析其表达形式。
2.重组蛋白经Ni~(2+)-NTA树脂纯化后,采用Bradford法测定蛋白浓度。
3.用rHpaA免疫30周龄立克体鸡,以水稀释法联合盐析法提取HpaA-IgY,纯化浓缩后用Bradford法测定其含量,SDS-PAGE电泳检测纯度,Western blot鉴定抗原特异性。
4. ELISA测定效价和巴氏消毒后的活性。
结果:
1. SDS-PAGE电泳显示pQE30-hpaA-DH5a表达产物相对分子量为30 000。
2. SDS-PAGE分析表达形式,rHpaA主要存在于细菌裂解液上清中,约占70%,部分存在于包涵体中,经Ni~(2+)-NTA树脂对上清进行纯化后,纯度约为90%,Bradford法测得蛋白质含量为0.95mg/ml。
3. HpaA-IgY提纯后,Bradford法测得含量为24.6mg/ml,纯度约为90%,Western blot显示在相对分子量30 000处出现单一条带,ELISA效价为1:12800。
Objective: To observe anti-rHpaA IgY from eggs yolk of hens immunized with recombinant Helicobacter pylori HpaA protein by gene-engineering.
Methods: Recombinant Helicobacter pylori HpaA protein were aquired by gene-engineering. Laying hens were immunized with recombinant. rHpaA.IgY was isolated by using the water dilution method (WD) ,and their concentration and purity were measured respectively with Bradford method and SDS-PAGE. The specificity of IgY and titer of IgY was assayed with Western blot and ELISA respectively. The titer of post-pasteurized was assayed with ELISA.
Results: The concentration and purity of IgY were 24.6mg/ml and 90 % respectively. The titer of IgY was 1:12800,and the titer of post-pasteurized did not decrease. Its specificity was confirmed by Western blot.
Conclusion: rHpaA-IgY with high concentration, high purity, high titer, pasteurized-stability and specificity was successfully preparated.
方法:
1.诱导重组质粒pQE30-hpaA在大肠杆菌DH5a中表达rHpaA,SDS-PAGE分析其表达形式。
2.重组蛋白经Ni~(2+)-NTA树脂纯化后,采用Bradford法测定蛋白浓度。
3.用rHpaA免疫30周龄立克体鸡,以水稀释法联合盐析法提取HpaA-IgY,纯化浓缩后用Bradford法测定其含量,SDS-PAGE电泳检测纯度,Western blot鉴定抗原特异性。
4. ELISA测定效价和巴氏消毒后的活性。
结果:
1. SDS-PAGE电泳显示pQE30-hpaA-DH5a表达产物相对分子量为30 000。
2. SDS-PAGE分析表达形式,rHpaA主要存在于细菌裂解液上清中,约占70%,部分存在于包涵体中,经Ni~(2+)-NTA树脂对上清进行纯化后,纯度约为90%,Bradford法测得蛋白质含量为0.95mg/ml。
3. HpaA-IgY提纯后,Bradford法测得含量为24.6mg/ml,纯度约为90%,Western blot显示在相对分子量30 000处出现单一条带,ELISA效价为1:12800。
Objective: To observe anti-rHpaA IgY from eggs yolk of hens immunized with recombinant Helicobacter pylori HpaA protein by gene-engineering.
Methods: Recombinant Helicobacter pylori HpaA protein were aquired by gene-engineering. Laying hens were immunized with recombinant. rHpaA.IgY was isolated by using the water dilution method (WD) ,and their concentration and purity were measured respectively with Bradford method and SDS-PAGE. The specificity of IgY and titer of IgY was assayed with Western blot and ELISA respectively. The titer of post-pasteurized was assayed with ELISA.
Results: The concentration and purity of IgY were 24.6mg/ml and 90 % respectively. The titer of IgY was 1:12800,and the titer of post-pasteurized did not decrease. Its specificity was confirmed by Western blot.
Conclusion: rHpaA-IgY with high concentration, high purity, high titer, pasteurized-stability and specificity was successfully preparated.
引文
[1] Vandenplas Y. Helicobacter pylori infection[J].World J Gastroenterol. 2000,6:20- 31
[2] Yang Y, Deng CS, Yao XJ,et al. Effect of Helicobacter pylori on morphology and growth of gastric epithelial cells[J].Shijie Huaren Xiaohua Zazhi. 2000,8(5):500- 504
[3] Liu HF,Liu WW,Fang DC,et al. Gastric epithelial apoptosis induced by Helicobacter pylori and its relationship with Bax protein expression[J].Shijie Huaren Xiaohua Zazhi. 2000,8(8):860-862
[4] Chen SY,Wang JY,Ji Y,et al. Effects of Helicobacter pylori and protein kinase C on gene mutation in gastric cancer and precancerous lesions[J].Shijie Huaren Xiaohua Zazhi. 2001,9(3):302-307
[5] International agency for reseach on cancer. Schistosomes, liver fluker and Helicobacter Pylori.IARC monographs on the evalition on carcinogenic risks to human[J]. Lyon: IASC. Vo161,1994
[6] Pounder RE, Ng D. The prevalence of Helicobacter Pylori infection in different countries[J].Science. 1995, 267:17
[7] Evans DG, Evans DJ Jr, Lampert HC,et al. Restriction fragment length polymorphism in the adhesin gene HpaA of Helicobacter pylori[J]. Am J Gastroenterol. 1995,90(8):1282-1288.
[8] Mao YF,Yan J,Li LW,et al.Construction of HpaA gene from a clinical isolate of Helicobacter pylori and identification of fusion protein[J]. World J Gastroenterol 2003,9(7):1529-1536
[9] Mao YF, Yan J, Li LW.Cloning, expression and identification of HpaA gene from a clinical isolate of Helicobacter pylori[J].Zhejiang Da Xue Xue Bao Yi Xue Ban. 2003, 32(1):9-12.
[10]Voland P, Hafsi N, Zeitner M,et al.Antigenic properties of HpaA and Omp18, two outer membrane proteins of Helicobacter pylori[J]. Infect Immun. 2003, 71(7): 3837-43
[11]Lundstrom AM, Bolin I, Bystrom M,et al.Recombinant HpaA purified from Escherichia coli has biological properties similar to those of native Helicobacterpylori HpaA[J].APMIS. 2003 ,111(3):389-97.
[12]Zhu S, Chen M, Chen J,et al. Construction of HpaA gene-engineered attenuated Salmonella typhimurium vaccine strain[J]. Wei Sheng Wu Xue Bao. 2002, 42(1): 27-32
[13]Mine Y,Kovacs-Nolan J.Chicken egg yolk antibodies as therapeutics in enteric infectious disease:a review[J].Med Food. 2002 Fall;5(3):159-69
[14]Shin Ji-Hyun,Mierha Yand,Seung Woo Nam,et al.Use of egg yolk-derived immunoglobulin as an alternative to antibiotic treatment for control of Helicobacter pylori infection[J].Clin Diagn Lab Immunol. 2002 Sep;9(5):1061-6
[15]J.萨姆布鲁克,E.F.弗里奇,T.曼尼阿蒂斯著,金冬雁,黎孟枫等译.分子克隆实验指南[M]. 第 2 版,北京:科学出版社,1999 年,p22-23.
[16]杨廷彬,尹学念.《实用免疫学》[M].长春出版社,1992 年, p443-453, p365-378.
[17]Mine Y,Kovacs N J. Chiken egg yolk antibodies as therapeutics in enteric infections disease[J].J Med Food. 2002;5(3):159-169.
[18]郭立君,李春晖,赵锋等.抗轮状病毒 IgY 的研制[J].中国生物制品学杂志. 2001,14(1):24-26.
[19]Sarker SA,Casswall TH,Juneja LR, et al.Randomized,placebo-contrlled,clinical trial of hyperimmunized chicken egg yolk immunoglobulin in children with rotavirus diarrhea[J].J Pediatr Gastroenterol Nutr. 2001Jan:32(1):19-25.
[20]王忠泽,侯小军,荫俊等.抗大肠杆菌 O157:H7 鸡蛋黄抗体的制备及其被动保护作用的研究[J].中国人兽共患病杂志. 2002,18(2):17-19.
[21]许扬,熊勇华,魏华等. 幽门螺杆菌全菌抗原对母鸡的免疫原性[J].中国生物制品学杂志. 2000;13(3):147-150.
[22]刘立华,杨贵珍,齐名等. IgY 抗体对幽门螺杆菌菌体抗原细胞毒抗原的中和作用[J]. 中国生物制品学杂志. 1999;12(3):139-141.
[23]陈翠萍,杨朝晖,王永谦等. IgY 抗体在体外和体内对幽门螺杆菌作用的研究. 中华微生物学和免疫学杂志. 2002;22(1):37-40.
[24] Ji-Hyun Shin,Mierha Yang,Seung Woo Nam, et al.Use of Egg Yolk-Derived Immunoglobulin as an Alternative to Antibiotic Treatment for Control of Helicobacter pylori infection.2002;9(5):1061-1066.
[25]Sturmer AM,Driscoll DP,Jackson-Matthews DE. A quantitative immunoassay usingchicken antibodies for detection of native and recombinant alpha-amidating enzyme[J].Immunol Methods. 1992,146(1):105-110
[26]Sasse M,Kruger M,Schade R, et al.Herstellung und Charakterisierung aviarer vitelliner Antikorper gegen Lipopolysaccharide(IgY) gegen Endotoxine[J].Berl Munch Tierarztl Wochenschr. 1998,111(4):121-126
[27]Svendsen L,Crowley A,Ostergaard LH, et al.Development and comparison of purification strategies for chicken antibodies from egg yolk[J].Lab Anim Sci. 1995,45(1):89-93
[1] Mine Y,Kovacs-Nolan J,et al.chicken egg yolk antibodies as therapeutics in enteric infectious disease:a review[J].J Med Food. 2002 Fall,5(3):159-66
[2] de Alemeida CM,Quintana-Flores VM,Medina-Acosta E, et al.Egg yolk anti-BfpA antibodies as a tool for recognizing and identifying enteropathogenic Escherichia coli[J].Scand J Immunol. 2003 Jun,57(6):573-82
[3] DI Lonardo AD,Marcante ML,Poggiali F et al.Egg yolk antibodies against the E7 oncogenic protein of human papillomavirus type 16[J].Arch Virol. 2001, 146(1): 117-25
[4] Camenisch G,Tini M,Chilov D,et al.General applicability of chicken egg yolk antibodies:the performance of IgY immunoglobulins raised against the hypoxia-inducible factor lalpha[J].FASEB J. 1999 Jan,13(1):81-8
[5] Ohnishi T,Kakimoto K,Hashida S et al. Development of highly sensitive enzyme-linked immunosorbent assays for hepatocyte growth factor/scatter factor (HGF/SF): determination of HGF/SF in serum and urine from normal human subjects[J].J Immunol Methods. 2000 Oct 20,244(1-2):163-73
[6] Lemamy GJ,Roger P,Mani JC, et al.High-affinity antibodies from hen’s-egg yolks against human mannose-6-phosphate/insulin-like growth factor-Ⅱ recepter (M6p/EGFⅡ-R):characterization and potential use in clinical cancer studies[J].Int J Cancer. 1999 Mar 15,80(6):896-902
[7] Sturegard E,Sjunnesson H,Ho B,et al.Severe gastritis in guinea-pigs infacted with Helicobacter pylori[J].J Med Microbiol. 1998,47(12):1123-1129.
[8] Rokitan K.Role of heat ahock proteins in gastric mucosal protection[J].J Castroenterol Hepatol. 2000,15:12-19.
[9] Sutton P,Wilson J,Lee A.Further development of the Helicobacter pylori mouse vaccination model[J].Vaccne. 2000,18(24):2677-2685
[10]Ruiz-Bustos-E,Sierra-Beltran-A,Romero MJ,et al.Protection of BALB/c mice against experimental Helicobacter pylori infection by oral immumization with H.pylori heparan sulphate-binding proteins coupled to cholera toxin beta-subunit[J].J Med Microbiol. 2000,49(6):535-541.
[11] Kyong Ae Lee, Sung Keun Chang, Yoon Jin Lee,et al. Acid Stability of Anti –Helic.obacter pyroli IgY in Aqueous Polyol Solution[J]. Journal of Biochemistry and Molecular Biology. 2002,35(5): 488-493.
[12] Cho, Y. and Song, K. B. Effect of g-irradiation on the molecular properties of bovine serum albumin and b-lactoglobulin[J].J. Biochem. Mol. Biol.2000,33:133-137.
(1) Yang Zhibang, Zhang BIXia,Lu Qishan et al. Large-Scale Outbreak of Infection with Mycobacterium chelonae subsp.abscessus after Penicillin Infection. J-Clin-Microbiol,2002,40(7):2626-2628
(2) 李良成,扈庆华,刘军等。一起由龟分支杆菌脓肿亚种引起院内术后暴发流行感染的病原学研究。中国防痨杂志,1998,20(4):198
(3) Tejpratap S.P.Tiwari, Beverly Ray, Kenneth C. Jost et al. Forty Years of Disinfectant Failure: Outbreak of Postinjection Mycobacterium abscessus Infection Caused by Contamination. Clin-Infect-Dis,2003,36:954-962
(4) Covert, T.C., M.R.Rodgers, A.L.Reyes, et al. Occurrence of nontuberculous mycobateria in environmental samples. Environ-Microbiol,1999; Jun;65(6) 2492-2496
(5) Patrick C. Y. Woo, Kit-Wah Leung, Samson S. Y. Wong, et al. Relatively Alcohol-Resistant Mycobacteria Are Emerging Pathogens in Patients ReceivingAcupuncture Treatment. J-Clin-Microbiol, 2002,40(4):1219-1224
(6) J. Daisy Vanitha, R. Venkatasubramani, K. dharmalingam, et al. Large-Restriction-fragment Polymorphism Analysis of Mycobacterium chelonae and Mycobacterium terrae Isolates.Applied-And-Enviroment-Microbiol, 2003,69(7):4337-4341
(7) Kwan Kew Lai, Barbara A. Brown, Judy A.Westerling, et al. Long-Term Laboratory Contamination by Mycobacterium abscessus Resulting in Two Pseudo-Outbreaks: Recognition with Use of Random Amplified Polymorphic DNA(RAPD) Plymerase Chain Reaction. Clin-Infect-Dis,1998,27:169-175
(8) Zhang Y, Ragagopalan M, Brown BA, et al. Random amplified polymorphic DNA-PCR for comparison of Mycobacterium abscessus strains from nonsocomial outbreaaks. J-Clin-Microbiol, 1997,37:3132-3139
(9) Leslie Hall, Kelly A. Doerr, Sherri L. Wohlfiel, et al.Evalution of the MicroSeq System for Identification of Mtcobacteria by 16S Ribosomal DNA Sequencing and Its Integration into a Routine Clinical Mycobacteriology Laboratory. J-Clin-Microbiol,2003,41(4):1447-1453
(10) Victoria J. Cook, Christine Y. Turenne,Joyce Wolfe, et al. Conventional Methods versus 16S Ribosomal DNA Sequencing for Identification of Nontuberculous Mycobacteria: Cost Analysis.J-Clin-Microbiol,2003,41(3):1410-1415
(11) Andreas Roth, Udo Reischl, Anna Streubel, et al. novel Diagnostic Algorithm for Identification of Mycobacteria Using Genus-Specific Amplification of the 16S-23S rRNA Gene spacer and Restriction Endonucleases. J-Clin-Microbiol,2000,38(3): 1094-1104
(12) Tomotada Iwamoto, Toshiaki Sonobe, Kozaburo Hayashi, et al. Novel Algorithm identifies Species in a Polymycobacterial Sample by Fluorescence Capillary Electrophoresis-Based Single-Strand Conformation Polymorphism Analysis. J-Clin-Microbiol,2002, 40 (12):4705-4712
(13) Francesca Brunello, Marco Ligozzi, Emanuela Cristelli, et al. Identification of 54 Mycobacterial Sepecies by PCR-Restriction Fragment Length Polymorphism Analysis of the hsp65 Gene. J-Clin-Microbiol,2001,39(8):2799-2806
(14) Mitchell A. Yakrus, S. Moises Hernandez, Margaret M. Floyd, et al. Comparison of Methods for Identification of Mycobacterium abscessus and M.chelonae Isolate. J-Clin-Microbiol,2001,39(11):4103-4110
(15)Hyeyoung Lee, Hye-Eun Bang, Gill-Han Bai, et al. Novel Polymorphic Region of the rpoB Gene Containing Mycobacterium Species-Sequence and Its Use in Identification of Mycobacteria. J-Clin-Microbiol,2003,41(5):2213-2218
(16) Bum-Joon Kim, Seong-Karp Hong, Keun-Hwa Lee, et al.Differential Identification of Mycobacterium tuberculosis Complex and nontuberculous Mycobateria by Duplex PCR Assay Using the Rna Polymerase Gene(rpoB). J-Clin-Microbiol, 2004,42(3):1308-1312
(17) Jean-Noel Dauendorffer, Isabelle Guillemin, Alexandra Aubry, et al. Identification of Mycobacterial Species by PCR Sequencing of Quinolone Resistance-Determing Regions of DNA Gyrase Gene.J-Clin-Microbiol,2003,41(3):1311-1315
(18) Orita, M.,Y.Suzuki, T. Sekiya, et al. A rapid and sensitive detection of point mutations and genetic polymorphisms using polymorphase chain reaction.Genomics,1989,5:874-879
(19) Lawrence M. Gillman, James Gunton, Christine Y. Turenne, et al. Identification of mycobacterium Species by Multiple-Fluorescence PCR- Single-Strand Conformation Polymorphism Analysis of the 16S rRNA Gene. J-Clin-Microbiol, 2001, 39(9): 3085-3091
(20) Nancimae Miller, Susanna Infante, Tim Cleary, et al. Evaluation of the LiPA MYCOBACTERIA Assay gor Identification of Mycobacterial Species from BACTEC 12B Bottles. J-Clin-Microbiol, 2000,38(5):1915-1918
(21)Enrico Tortoli, Alessandro Mariottini, Gianna Mazzarelli, et al. Evaluation of INNO-LiPA MYCOBACTERIA V2: Improved Reverse Hybridization Multiple DNA Probe Assay for Mycobacterial Identification. J-Clin-Microbiol, 2003,41(9):4418-4420
(22) Enrico Tortoli, Anna Nanetti, Claudio Piersimoni, et al. Performance Assessment of NewMultiplex Probe Assay forIdentification of Mycobacteria.J-Clin-Microbiol, 2001, 39(3):10798-1084
[2] Yang Y, Deng CS, Yao XJ,et al. Effect of Helicobacter pylori on morphology and growth of gastric epithelial cells[J].Shijie Huaren Xiaohua Zazhi. 2000,8(5):500- 504
[3] Liu HF,Liu WW,Fang DC,et al. Gastric epithelial apoptosis induced by Helicobacter pylori and its relationship with Bax protein expression[J].Shijie Huaren Xiaohua Zazhi. 2000,8(8):860-862
[4] Chen SY,Wang JY,Ji Y,et al. Effects of Helicobacter pylori and protein kinase C on gene mutation in gastric cancer and precancerous lesions[J].Shijie Huaren Xiaohua Zazhi. 2001,9(3):302-307
[5] International agency for reseach on cancer. Schistosomes, liver fluker and Helicobacter Pylori.IARC monographs on the evalition on carcinogenic risks to human[J]. Lyon: IASC. Vo161,1994
[6] Pounder RE, Ng D. The prevalence of Helicobacter Pylori infection in different countries[J].Science. 1995, 267:17
[7] Evans DG, Evans DJ Jr, Lampert HC,et al. Restriction fragment length polymorphism in the adhesin gene HpaA of Helicobacter pylori[J]. Am J Gastroenterol. 1995,90(8):1282-1288.
[8] Mao YF,Yan J,Li LW,et al.Construction of HpaA gene from a clinical isolate of Helicobacter pylori and identification of fusion protein[J]. World J Gastroenterol 2003,9(7):1529-1536
[9] Mao YF, Yan J, Li LW.Cloning, expression and identification of HpaA gene from a clinical isolate of Helicobacter pylori[J].Zhejiang Da Xue Xue Bao Yi Xue Ban. 2003, 32(1):9-12.
[10]Voland P, Hafsi N, Zeitner M,et al.Antigenic properties of HpaA and Omp18, two outer membrane proteins of Helicobacter pylori[J]. Infect Immun. 2003, 71(7): 3837-43
[11]Lundstrom AM, Bolin I, Bystrom M,et al.Recombinant HpaA purified from Escherichia coli has biological properties similar to those of native Helicobacterpylori HpaA[J].APMIS. 2003 ,111(3):389-97.
[12]Zhu S, Chen M, Chen J,et al. Construction of HpaA gene-engineered attenuated Salmonella typhimurium vaccine strain[J]. Wei Sheng Wu Xue Bao. 2002, 42(1): 27-32
[13]Mine Y,Kovacs-Nolan J.Chicken egg yolk antibodies as therapeutics in enteric infectious disease:a review[J].Med Food. 2002 Fall;5(3):159-69
[14]Shin Ji-Hyun,Mierha Yand,Seung Woo Nam,et al.Use of egg yolk-derived immunoglobulin as an alternative to antibiotic treatment for control of Helicobacter pylori infection[J].Clin Diagn Lab Immunol. 2002 Sep;9(5):1061-6
[15]J.萨姆布鲁克,E.F.弗里奇,T.曼尼阿蒂斯著,金冬雁,黎孟枫等译.分子克隆实验指南[M]. 第 2 版,北京:科学出版社,1999 年,p22-23.
[16]杨廷彬,尹学念.《实用免疫学》[M].长春出版社,1992 年, p443-453, p365-378.
[17]Mine Y,Kovacs N J. Chiken egg yolk antibodies as therapeutics in enteric infections disease[J].J Med Food. 2002;5(3):159-169.
[18]郭立君,李春晖,赵锋等.抗轮状病毒 IgY 的研制[J].中国生物制品学杂志. 2001,14(1):24-26.
[19]Sarker SA,Casswall TH,Juneja LR, et al.Randomized,placebo-contrlled,clinical trial of hyperimmunized chicken egg yolk immunoglobulin in children with rotavirus diarrhea[J].J Pediatr Gastroenterol Nutr. 2001Jan:32(1):19-25.
[20]王忠泽,侯小军,荫俊等.抗大肠杆菌 O157:H7 鸡蛋黄抗体的制备及其被动保护作用的研究[J].中国人兽共患病杂志. 2002,18(2):17-19.
[21]许扬,熊勇华,魏华等. 幽门螺杆菌全菌抗原对母鸡的免疫原性[J].中国生物制品学杂志. 2000;13(3):147-150.
[22]刘立华,杨贵珍,齐名等. IgY 抗体对幽门螺杆菌菌体抗原细胞毒抗原的中和作用[J]. 中国生物制品学杂志. 1999;12(3):139-141.
[23]陈翠萍,杨朝晖,王永谦等. IgY 抗体在体外和体内对幽门螺杆菌作用的研究. 中华微生物学和免疫学杂志. 2002;22(1):37-40.
[24] Ji-Hyun Shin,Mierha Yang,Seung Woo Nam, et al.Use of Egg Yolk-Derived Immunoglobulin as an Alternative to Antibiotic Treatment for Control of Helicobacter pylori infection.2002;9(5):1061-1066.
[25]Sturmer AM,Driscoll DP,Jackson-Matthews DE. A quantitative immunoassay usingchicken antibodies for detection of native and recombinant alpha-amidating enzyme[J].Immunol Methods. 1992,146(1):105-110
[26]Sasse M,Kruger M,Schade R, et al.Herstellung und Charakterisierung aviarer vitelliner Antikorper gegen Lipopolysaccharide(IgY) gegen Endotoxine[J].Berl Munch Tierarztl Wochenschr. 1998,111(4):121-126
[27]Svendsen L,Crowley A,Ostergaard LH, et al.Development and comparison of purification strategies for chicken antibodies from egg yolk[J].Lab Anim Sci. 1995,45(1):89-93
[1] Mine Y,Kovacs-Nolan J,et al.chicken egg yolk antibodies as therapeutics in enteric infectious disease:a review[J].J Med Food. 2002 Fall,5(3):159-66
[2] de Alemeida CM,Quintana-Flores VM,Medina-Acosta E, et al.Egg yolk anti-BfpA antibodies as a tool for recognizing and identifying enteropathogenic Escherichia coli[J].Scand J Immunol. 2003 Jun,57(6):573-82
[3] DI Lonardo AD,Marcante ML,Poggiali F et al.Egg yolk antibodies against the E7 oncogenic protein of human papillomavirus type 16[J].Arch Virol. 2001, 146(1): 117-25
[4] Camenisch G,Tini M,Chilov D,et al.General applicability of chicken egg yolk antibodies:the performance of IgY immunoglobulins raised against the hypoxia-inducible factor lalpha[J].FASEB J. 1999 Jan,13(1):81-8
[5] Ohnishi T,Kakimoto K,Hashida S et al. Development of highly sensitive enzyme-linked immunosorbent assays for hepatocyte growth factor/scatter factor (HGF/SF): determination of HGF/SF in serum and urine from normal human subjects[J].J Immunol Methods. 2000 Oct 20,244(1-2):163-73
[6] Lemamy GJ,Roger P,Mani JC, et al.High-affinity antibodies from hen’s-egg yolks against human mannose-6-phosphate/insulin-like growth factor-Ⅱ recepter (M6p/EGFⅡ-R):characterization and potential use in clinical cancer studies[J].Int J Cancer. 1999 Mar 15,80(6):896-902
[7] Sturegard E,Sjunnesson H,Ho B,et al.Severe gastritis in guinea-pigs infacted with Helicobacter pylori[J].J Med Microbiol. 1998,47(12):1123-1129.
[8] Rokitan K.Role of heat ahock proteins in gastric mucosal protection[J].J Castroenterol Hepatol. 2000,15:12-19.
[9] Sutton P,Wilson J,Lee A.Further development of the Helicobacter pylori mouse vaccination model[J].Vaccne. 2000,18(24):2677-2685
[10]Ruiz-Bustos-E,Sierra-Beltran-A,Romero MJ,et al.Protection of BALB/c mice against experimental Helicobacter pylori infection by oral immumization with H.pylori heparan sulphate-binding proteins coupled to cholera toxin beta-subunit[J].J Med Microbiol. 2000,49(6):535-541.
[11] Kyong Ae Lee, Sung Keun Chang, Yoon Jin Lee,et al. Acid Stability of Anti –Helic.obacter pyroli IgY in Aqueous Polyol Solution[J]. Journal of Biochemistry and Molecular Biology. 2002,35(5): 488-493.
[12] Cho, Y. and Song, K. B. Effect of g-irradiation on the molecular properties of bovine serum albumin and b-lactoglobulin[J].J. Biochem. Mol. Biol.2000,33:133-137.
(1) Yang Zhibang, Zhang BIXia,Lu Qishan et al. Large-Scale Outbreak of Infection with Mycobacterium chelonae subsp.abscessus after Penicillin Infection. J-Clin-Microbiol,2002,40(7):2626-2628
(2) 李良成,扈庆华,刘军等。一起由龟分支杆菌脓肿亚种引起院内术后暴发流行感染的病原学研究。中国防痨杂志,1998,20(4):198
(3) Tejpratap S.P.Tiwari, Beverly Ray, Kenneth C. Jost et al. Forty Years of Disinfectant Failure: Outbreak of Postinjection Mycobacterium abscessus Infection Caused by Contamination. Clin-Infect-Dis,2003,36:954-962
(4) Covert, T.C., M.R.Rodgers, A.L.Reyes, et al. Occurrence of nontuberculous mycobateria in environmental samples. Environ-Microbiol,1999; Jun;65(6) 2492-2496
(5) Patrick C. Y. Woo, Kit-Wah Leung, Samson S. Y. Wong, et al. Relatively Alcohol-Resistant Mycobacteria Are Emerging Pathogens in Patients ReceivingAcupuncture Treatment. J-Clin-Microbiol, 2002,40(4):1219-1224
(6) J. Daisy Vanitha, R. Venkatasubramani, K. dharmalingam, et al. Large-Restriction-fragment Polymorphism Analysis of Mycobacterium chelonae and Mycobacterium terrae Isolates.Applied-And-Enviroment-Microbiol, 2003,69(7):4337-4341
(7) Kwan Kew Lai, Barbara A. Brown, Judy A.Westerling, et al. Long-Term Laboratory Contamination by Mycobacterium abscessus Resulting in Two Pseudo-Outbreaks: Recognition with Use of Random Amplified Polymorphic DNA(RAPD) Plymerase Chain Reaction. Clin-Infect-Dis,1998,27:169-175
(8) Zhang Y, Ragagopalan M, Brown BA, et al. Random amplified polymorphic DNA-PCR for comparison of Mycobacterium abscessus strains from nonsocomial outbreaaks. J-Clin-Microbiol, 1997,37:3132-3139
(9) Leslie Hall, Kelly A. Doerr, Sherri L. Wohlfiel, et al.Evalution of the MicroSeq System for Identification of Mtcobacteria by 16S Ribosomal DNA Sequencing and Its Integration into a Routine Clinical Mycobacteriology Laboratory. J-Clin-Microbiol,2003,41(4):1447-1453
(10) Victoria J. Cook, Christine Y. Turenne,Joyce Wolfe, et al. Conventional Methods versus 16S Ribosomal DNA Sequencing for Identification of Nontuberculous Mycobacteria: Cost Analysis.J-Clin-Microbiol,2003,41(3):1410-1415
(11) Andreas Roth, Udo Reischl, Anna Streubel, et al. novel Diagnostic Algorithm for Identification of Mycobacteria Using Genus-Specific Amplification of the 16S-23S rRNA Gene spacer and Restriction Endonucleases. J-Clin-Microbiol,2000,38(3): 1094-1104
(12) Tomotada Iwamoto, Toshiaki Sonobe, Kozaburo Hayashi, et al. Novel Algorithm identifies Species in a Polymycobacterial Sample by Fluorescence Capillary Electrophoresis-Based Single-Strand Conformation Polymorphism Analysis. J-Clin-Microbiol,2002, 40 (12):4705-4712
(13) Francesca Brunello, Marco Ligozzi, Emanuela Cristelli, et al. Identification of 54 Mycobacterial Sepecies by PCR-Restriction Fragment Length Polymorphism Analysis of the hsp65 Gene. J-Clin-Microbiol,2001,39(8):2799-2806
(14) Mitchell A. Yakrus, S. Moises Hernandez, Margaret M. Floyd, et al. Comparison of Methods for Identification of Mycobacterium abscessus and M.chelonae Isolate. J-Clin-Microbiol,2001,39(11):4103-4110
(15)Hyeyoung Lee, Hye-Eun Bang, Gill-Han Bai, et al. Novel Polymorphic Region of the rpoB Gene Containing Mycobacterium Species-Sequence and Its Use in Identification of Mycobacteria. J-Clin-Microbiol,2003,41(5):2213-2218
(16) Bum-Joon Kim, Seong-Karp Hong, Keun-Hwa Lee, et al.Differential Identification of Mycobacterium tuberculosis Complex and nontuberculous Mycobateria by Duplex PCR Assay Using the Rna Polymerase Gene(rpoB). J-Clin-Microbiol, 2004,42(3):1308-1312
(17) Jean-Noel Dauendorffer, Isabelle Guillemin, Alexandra Aubry, et al. Identification of Mycobacterial Species by PCR Sequencing of Quinolone Resistance-Determing Regions of DNA Gyrase Gene.J-Clin-Microbiol,2003,41(3):1311-1315
(18) Orita, M.,Y.Suzuki, T. Sekiya, et al. A rapid and sensitive detection of point mutations and genetic polymorphisms using polymorphase chain reaction.Genomics,1989,5:874-879
(19) Lawrence M. Gillman, James Gunton, Christine Y. Turenne, et al. Identification of mycobacterium Species by Multiple-Fluorescence PCR- Single-Strand Conformation Polymorphism Analysis of the 16S rRNA Gene. J-Clin-Microbiol, 2001, 39(9): 3085-3091
(20) Nancimae Miller, Susanna Infante, Tim Cleary, et al. Evaluation of the LiPA MYCOBACTERIA Assay gor Identification of Mycobacterial Species from BACTEC 12B Bottles. J-Clin-Microbiol, 2000,38(5):1915-1918
(21)Enrico Tortoli, Alessandro Mariottini, Gianna Mazzarelli, et al. Evaluation of INNO-LiPA MYCOBACTERIA V2: Improved Reverse Hybridization Multiple DNA Probe Assay for Mycobacterial Identification. J-Clin-Microbiol, 2003,41(9):4418-4420
(22) Enrico Tortoli, Anna Nanetti, Claudio Piersimoni, et al. Performance Assessment of NewMultiplex Probe Assay forIdentification of Mycobacteria.J-Clin-Microbiol, 2001, 39(3):10798-1084