幽门螺杆菌卵黄抗体的研制
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摘要
试验以幽门螺杆菌(Helicobacter pylori，Hp)为材料，采用不同培养基进行培养和保存，并制备成完全抗原对刚开产或开产不久的母鸡进行免疫，用凝集试验法测定其抗体效价，并对免疫Hp母鸡免疫应答规律和卵黄抗体的抑菌活性及理化特性做了初步探讨。为制备高含量的抗Up的免疫卵黄及相关生物的制剂提供技术依据，也为鸡蛋及免疫蛋的开发利用和深入研究积累经验并提供借鉴。
1 幽门螺杆菌的培养与保存
本试验采用不同培养基对幽门螺杆菌(Helicobacter pylori，Hp)进行培养和保存，结果表明：固体培养Hp，培养基为哥伦比亚琼脂基础加混合抗生素(10mg·L~(-1)盐酸万古霉素+10mg·L~(-1)两性霉素B+2500iu·L~(-1)多粘菌素B+5mg·L~(-1)甲氧苄胺嘧啶)，并分别添加6％脱纤维绵羊血、6％马血清，在5％O_2，10％CO_2，85％N_2的微需氧条件下，37℃培养3d，均可培养出Hp；严格无菌的条件下，不添加混合抗生素，对细菌的生长没有影响，且不会造成杂菌污染。液体培养Hp，布氏肉汤基础中加入混合抗生素和6％马血清，Hp增菌迅速，但如不及时更换新鲜的微需氧环境，极易形成球形体。菌株采取冷冻保存液法保存于-70℃～-80℃低温条件下6个月，可以成功复苏，且复苏率可达100％。
2 抗幽门螺杆菌卵黄抗体的制备及其体外抑菌试验
试验以幽门螺杆菌(helicobacter pylori，Hp)全菌灭活抗原对150日龄伊沙褐壳母鸡进行首免，165日龄二免，180日龄三免，于首免后即开始收集鸡蛋，采用凝集试验法测定卵黄中的抗体效价。结果表明：每间隔15天，采取两次加强免疫方法，可有效的引起免疫应答。母鸡在初免45d后，抗体效价可达到1:1024，同批样品在此水平可维持达60d以上(效价大于1:128)，然后抗体效价呈缓慢下降趋势，经130d后，抗体效价下降至1:8。体外抑菌试验表明，高免卵黄具有明显的抑制HP生长的活性。
3 鸡抗幽门螺杆菌卵黄抗体的理化特性
试验对自制鸡抗幽门螺杆菌高免卵黄的理化特性进行了研究。结果发现：pH值、温度对其活性的影响较明显，胃蛋白酶则加强了这一作用。在pH值单独影响下，pH≤3为其敏感区，如果同时存在胃蛋白酶，则其敏感区为pH≤4，在巴氏消毒温度条件下其具有较好的热稳定性，这对于工业化生产卵黄抗体具有非常重要的意义。
Different culture media were tested to culture and preserve Helicobacter Pylori (Hp). The just mature chickens were procedurally immunized with complete Hp which cultured by the team, agglutination test were used to determine the egg yolk antibody level, and the pattern of immuno-responsion, bacteriostasis in Vitro, physical and chemical characteristic of immune yolk were searched. All these studies were of great significance to the industrial production of high immune yolk antibody against Hp and correlatively biologic medication.
1 Effect of different methods on Hp culture and preservation
The strain was cultured on Columbia Agar Base (CAB) supplemented with mixed antibiotics containing 2500IU/l Polymyxin B Sulfate, 5mg/l Trimethoprim, 10mg/l Vancomycin HC1 and 10mg/l Amphotericin B and 6% defibrinated sheep blood or 6% horse serum respectively in a microaerophilic atmosphere of 5% O_2, 10% CO_2 and 85% N_2 at 37℃ for 72h. With the two methods, good effect was observed. In condition of sterilization with no supplementation with mixed antibiotics, the strain can't be contaminated and grow well as well. When Brucella Broth was used an culture medium with combined antibiotics and horse serum in microaerophilic bag, Hp could grow rapidly but was easily to be spheroid without replacing the bag on time. And the liquid culture method was contaminated compared with solid method. The strain can be revived successively with cryopreservation at -70℃ to -80℃, 100 percents,
2 Preparation and Bacteriostasis in Vitro of Egg Yolk Antibody Against Helicobacter Pylori
Preparation and bacteriostasis in vitro of egg yolk antibody against Helicobacter pylori (Hp) have been studied. Chickens were procedurally immunized with complete bacterin at 150, 165 and 180 days old, successively. Agglutination test has shown that the egg yolk antibody against HP was produced from immunized hens, the antibody titer reached to 1:1024 at the 45th day after the first inoculability, the high level can last for 2 months, then go down gradually to 1:8 after about 130 days. Bacteriostasis in vitro shown that HP is hypersensitive to egg yolk antibody.
1 幽门螺杆菌的培养与保存
本试验采用不同培养基对幽门螺杆菌(Helicobacter pylori，Hp)进行培养和保存，结果表明：固体培养Hp，培养基为哥伦比亚琼脂基础加混合抗生素(10mg·L~(-1)盐酸万古霉素+10mg·L~(-1)两性霉素B+2500iu·L~(-1)多粘菌素B+5mg·L~(-1)甲氧苄胺嘧啶)，并分别添加6％脱纤维绵羊血、6％马血清，在5％O_2，10％CO_2，85％N_2的微需氧条件下，37℃培养3d，均可培养出Hp；严格无菌的条件下，不添加混合抗生素，对细菌的生长没有影响，且不会造成杂菌污染。液体培养Hp，布氏肉汤基础中加入混合抗生素和6％马血清，Hp增菌迅速，但如不及时更换新鲜的微需氧环境，极易形成球形体。菌株采取冷冻保存液法保存于-70℃～-80℃低温条件下6个月，可以成功复苏，且复苏率可达100％。
2 抗幽门螺杆菌卵黄抗体的制备及其体外抑菌试验
试验以幽门螺杆菌(helicobacter pylori，Hp)全菌灭活抗原对150日龄伊沙褐壳母鸡进行首免，165日龄二免，180日龄三免，于首免后即开始收集鸡蛋，采用凝集试验法测定卵黄中的抗体效价。结果表明：每间隔15天，采取两次加强免疫方法，可有效的引起免疫应答。母鸡在初免45d后，抗体效价可达到1:1024，同批样品在此水平可维持达60d以上(效价大于1:128)，然后抗体效价呈缓慢下降趋势，经130d后，抗体效价下降至1:8。体外抑菌试验表明，高免卵黄具有明显的抑制HP生长的活性。
3 鸡抗幽门螺杆菌卵黄抗体的理化特性
试验对自制鸡抗幽门螺杆菌高免卵黄的理化特性进行了研究。结果发现：pH值、温度对其活性的影响较明显，胃蛋白酶则加强了这一作用。在pH值单独影响下，pH≤3为其敏感区，如果同时存在胃蛋白酶，则其敏感区为pH≤4，在巴氏消毒温度条件下其具有较好的热稳定性，这对于工业化生产卵黄抗体具有非常重要的意义。
Different culture media were tested to culture and preserve Helicobacter Pylori (Hp). The just mature chickens were procedurally immunized with complete Hp which cultured by the team, agglutination test were used to determine the egg yolk antibody level, and the pattern of immuno-responsion, bacteriostasis in Vitro, physical and chemical characteristic of immune yolk were searched. All these studies were of great significance to the industrial production of high immune yolk antibody against Hp and correlatively biologic medication.
1 Effect of different methods on Hp culture and preservation
The strain was cultured on Columbia Agar Base (CAB) supplemented with mixed antibiotics containing 2500IU/l Polymyxin B Sulfate, 5mg/l Trimethoprim, 10mg/l Vancomycin HC1 and 10mg/l Amphotericin B and 6% defibrinated sheep blood or 6% horse serum respectively in a microaerophilic atmosphere of 5% O_2, 10% CO_2 and 85% N_2 at 37℃ for 72h. With the two methods, good effect was observed. In condition of sterilization with no supplementation with mixed antibiotics, the strain can't be contaminated and grow well as well. When Brucella Broth was used an culture medium with combined antibiotics and horse serum in microaerophilic bag, Hp could grow rapidly but was easily to be spheroid without replacing the bag on time. And the liquid culture method was contaminated compared with solid method. The strain can be revived successively with cryopreservation at -70℃ to -80℃, 100 percents,
2 Preparation and Bacteriostasis in Vitro of Egg Yolk Antibody Against Helicobacter Pylori
Preparation and bacteriostasis in vitro of egg yolk antibody against Helicobacter pylori (Hp) have been studied. Chickens were procedurally immunized with complete bacterin at 150, 165 and 180 days old, successively. Agglutination test has shown that the egg yolk antibody against HP was produced from immunized hens, the antibody titer reached to 1:1024 at the 45th day after the first inoculability, the high level can last for 2 months, then go down gradually to 1:8 after about 130 days. Bacteriostasis in vitro shown that HP is hypersensitive to egg yolk antibody.
引文
[1] Warren JR, Marshall BJ. Unidentified curved bacilli on gastric epithelium in active chronic gastritis[J]. Lancet 1953(4):1273～1275.
[2] Biaser MJ. Helicobacter pylori and the pathogenesis of gastroduodenal inflammation[J]. J Infect Dis 1990 161:626～633.
[3] Lee A, Fos J, Hazell SL. Pathogenicity of Helicobacterpylori: a perspective[J]. Infect Immun. 1993,61:1601～1610.
[4] Seolnick JV, Tompkins LS. Helieobacter pylori and gastroduodenal disease: pathogenesis and host-parasite interaction[J]. Infect Agents Dis. 1993,1:294～309.
[5] Cover TL, Blaser MJ. Helicobacter pylori and gastroduodenal disease[J]. Annu Rev Med. 1992,43:135～145.
[6] Wotherspoon AC, Doglioni C, Diss TC, et al. Regression of primary low-grade B-cell gastric lymphoma of mucosa-associated lymphoid tissue type after eradication of Helicobacter pylori[J]. Lancet. 1993,2:575～577.
[7] Akira G, Yoshiyuki K, Takaynki A, et al. Susceptibility of Helicobacter pylori isolates against agents commonly administered for eradication therapy and the efficacy of chemotherapy[J]. Microbiol Immunol. 1997,41:7-12.
[8] Queiroz DMM,, Coimbra RS, Mendes EN, et al. Metronidazole-resistant Helicobacter pylori in a developing country[J]. Am J Gastroenterol. 1993,88:322～323.
[9] Graham DY MACG, Wink A, DE Boer, et al. Choosing the best anti-Helieobacter pylori therapy: effect of antimierobiol resistance[J]. Am J Gastroenterol, 1996,91: 1072-1076.
[10] 范学工，夏华向．幽门螺杆菌感染—基础与临床[M]．湖南科学技术出版社，1998：156～225．
[11] Pina M, Occhialini A, Monteiro L, et al. Detection of point mutations association with resistance, of Helicobacter pylori to elarithromyein by hybridization in liquid phase[J]. J Clin Microbiol, 1998;36:3285-3290.
[12] Di Mario F, et al. Azithromyein for the cure of Helicobaeter pylori infection[J]. Am J Gastroenterol, 1996;91:264.]
[13] 杨惠芳，乔家国．幽门螺杆菌的中西药敏感试验对照研究[J]．江苏临床医学杂志，1997；1(2)：103．
[14] Vakli N, Fermerty MB, et al. Cost effectiveless of treatment regimems for the eradication of Helieobacter pylori in duodenal ulcer[J]. Am J Gastroenterol, 1996.91 (2):239.
[15] Jenks PJ, Labigne A, Ferrero RL. Exposure to metronidazole in vivo readily induces resistance in Helicobacter pylori and reduces the efficacy of eradication therapy in mice[J]. Antimicrob Agents Chemother, 1999;43:777-781.
[16] 姚希贤．治疗幽门螺杆菌感染的研究进展[J]．新消化病学杂志，1997；5(9)；545-546．
[17] 杨秀珍，鲍蕾．氧氟沙星抗生素对幽门螺杆菌的敏感性试验[J]．中国微生态学杂志，1999；11(3)：152．[18]陈洁平，徐采朴．幽门螺杆菌感染的治疗[J]．新消化病学杂志，1997；5(4)258～59．
[19]陈洁平，徐采朴，王振华等．抗幽门螺杆菌感染中、西药物的体内外药敏试验研究[J]．新消化病学杂志，1997；5(10)：629-631．
[20]范学工，夏华向．幽门螺杆菌感染—基础与临床[M]．湖南科学技术出版社，1998：156～225．
[21]Cheng CY, Lin CK, Wu SH, et al. Two days, high-dosage, triple therapy with omeperazol for eradication of Helicobacter pylori[J]. Gastroenterology, 1996; 110(suppl 1):43-47.
[22]袁佩英．鸦胆子乳剂抑制螺菌的临床研究[J]．山西中医，1992，(6)：20．
[23]张琳．幽门螺杆菌与慢性萎缩性胃炎发病关系及防治研究[J]．中国中西医结合杂志，1992，12(9)：521．
[24]徐州．中药对幽门螺杆菌抑杀作用的实验研究[J]．中国医学报，1993，8(5)：25．
[25]邓世荣．槟榔治疗幽门螺杆菌感染的临床观察[J]．中医杂志，1993，(10)：605．
[26]周增芬，张永生，崔荣等．大蒜对幽门螺杆菌抑制作用的实验研究[J]．中国新药杂志，1999，8(4)：243～245．
[27]张安田．锡类散治疗消化性溃疡作用机理的研究[J]．中国中西医结合杂志，1995，15(4)：216．
[28]董国华．龙胆泻肝丸治疗幽门螺杆菌相关性胃炎47例[J]．上海中医药杂志，1996，(6)：38．
[29]武和平．珠黄散加德诺治疗幽门螺杆菌相关性溃疡的临床观察[J]．上海中医药杂志，1996，(8)：28．
[30]姚希贤．治疗幽门螺杆菌感染的研究进展[J]．新消化病学杂志，1997；5(9)；545-546．
[31]周增芬，张永生，崔蓉等．大蒜对幽门螺杆菌抑菌作用的实验研究[J]．中国新药杂志，1999；8(4)；243-245．
[32]Thomas JE Austin S Dale A et al. Protection by human milk IgA against Helicobacter pylori infection in infancy(letter) [J]. Lancet 1993 342:121.
[33]Czinn SJ, Cai A and Nedrud JG. Protection of germ free mice from infection by Helicobacter felis after active oral or passive IgA immunization[J]. Vaccine, 1993,11:637～642.
[34]Lee CK, Weltzin R, Thomas WD, et al. Oral immunization with recombinant Helicobacter pylori urease induces secretory IgA antibodies and protects mice from challenge with Helicobacter felis[J]. J Infect Dis, 1995,172:161～172.
[35]Blanchard TG, Czinn S J, Maurer R, et al. Urease-specific monoclonal antibodies prevent Helicobacter pelis infection in mice[J]. Infect Immun, 1995,63: 1394-1399.
[36]张和平，郭军．免疫乳科学与技术tM]．北京：中国轻工业出版社，2002．[1] Locken MR. Roth TF. Analysis of maternal IgG subpopulations wbich are transfer to egg yolk in chickens[J]. J Immunol. 1962.89:272～278.
[2] Morrison SL. Mohammed MS. Wims LA, et al. Sequences in antibody molecules important for receptant for receptor-mediated transport into the chicken egg yolk[J]. Molecular Immunoloy. 2002,38(8):619～625.
[3] 王炯，龚春梅，赵树栋等．鸡卵抗体(IgY)理化特性的研究[J]．中国生物制品学杂志，1997，3：140～143．
[4]Song CS etal[J]. J Immunol,1985;135(5):3354～3359.
[5]龙中儿，钟青萍，刘活林等．卵黄免疫球蛋白的稳定性研究[J]．中国生物制品学杂志，1997；4：218～221．
[6]易全茂，谢艳霞．鸡卵黄抗体研究进展[J]．动物科学与动物医学，2004；2：44～46．
[7]Otani H, Matsumoto K, Saeki A, et al. Comparative studies on porperties on hen egg yolk IgY and rabbit serum IgG antibodies[J]. Lebensm--Wiss u Technol,1991,24:152.
[8]陈斌，杨志华．鸡卵黄免疫球蛋白(IgY)的作用机理及应用前景[J]．浙江畜牧兽医，2005．1：9～10．
[9]Woolly JA et al[J]. J Immunol Methods, 1995;178:253--263.
[10]Hassl A et al. Zbl Bake Hyg A, 1987;267:247-253.1111.Mclaren R D et al[J]. J Immunol??Methods, 1994;177:175-184.
[11] Hatta H et al[J]. Agric Biol Chem, 1990;54:2531.
[12]康亦兼，咸漠，李文兴．水稀释法分离卵黄IgY[J]．吉林大学自然科学学报，2001，3：84-86．
[13]肖驰，周淑兰，涂志等．鸡抗猪大肠埃希氏菌卵黄抗体的研制与应用[J]．中国兽医科技，1998,4：21～23．
[14]俞海峰．饲料营养元素与动物免疫研究进展[J]．饲料广角，2003，12：34-36．
[15]杨晓梅，刘万钧，李力等．鸡抗鸭病毒性肝炎高免卵黄抗体的研制与应用[J]．四川畜牧兽医，1994,2：9～10．
[16]汪铭书，程安春，陈孝跃．应用高免卵黄抗体防治鸭病毒性肝炎的研究[J]．中国预防兽医学报，1997,5：14～17．
[17]王玉兰，张英．小鹅瘟与鹅禽霍乱防制措施的探讨[J]．辽宁畜牧兽医，1994,6：28～29．
[18]边传周，袁道强，任敏等．禽流感高免卵黄抗体的研制与应用[J]．郑州牧业工程高等专科学校学报，2000,2：84～85．
[19]詹丽娥，乔忠，孙世兴．鸡三联蛋黄抗体的研制及应用[J]．中国兽医科技，1994,11：30～31．
[20]郑厚旌，巩霞，王同亮等．应用改良琼指扩散试验检测鸡传染性法氏囊病病毒的研究[J]．中国预防兽医学报，1996,2：11-14．
[21]李槿年，余为一．利用卵黄监测鳖群中病原菌感染[J]．淡水渔业，1999,12：7～9．[1] Meguaud F.Epidemiology of Helicobacter pylori Infection [J]. Gas Clin Nor Am, 1993, 22:73-88.
[2] Parsonnet J. The incidence of Helicobacter pylori Infection [J]. Ali Pha Ther, 1995,9:45-51.
[3] Li YY, Hu P J, Du GG, et al. The prevalence Helicobacter pylori Infection in P.R. China [J]. Am J Gastroenterol, 1991,86:446～449.
[4] Suerbaum S, Hur C, Josenhans C, et al. Pathogenesis and virulence factors of Helicobacter pylori [J]. Curr Opin Gastroenterol, 1999,15:s11～16.
[5] Rudnicks W, Andersen LP. Inflammation and host response[J]. Curr Opin Gastroenterol, 1999, 15:s17～22.
[6] Rokkas T, Wotherspoon A. Carcinogenesis [J]. Curr Opin Gastroenterol, 1999, 15:s23-28.
[7] Marshall BJ. Helicobacter pylori [J]. Am J Gastroenterol, 1994,89:s116.[8] Edit S, Stolte M. The significance of Helicobacter pylori in relation to gastric cancer and lymphoma [J]. Eur .1 Gastroenterol Hepatol, 1995,7:318.
[9] Mohamed MA, Goggin PM, Molineaux N, et al. Relation ofHelicobacter pylori infection and coronary heart disease[J]. Br Heart J, 1994,71:437-439.
[10] Markus HS, Brown M, Levy J, et al. H. pylori: a new risk factor cerebrovascular disease and carotid atheroma [J]. Cerebrovasc Dis, 1995,5:251.
[11] Gasbarrini A, De Luca A, Fiore G, et al. Beneficial effects of Helicobacter pylori eradicatin on migraine [J]. Hepatogas torenterology, 1998,45:765-770.
[12] Gasbrrini A, Serricchio M, Tindi P, et al. Association of Helicobacter pylori infection with primary Raynaud phenomenon [J]. Lancet, 1996,348:966-967.
[13] Hunt RH, Malfertheiner P, Yeomans ND, et al. Critical issues in the pathophysiology and management of peptic ulcer aisease [J]. Eur J Gastroenterol Hepatol, 1995, 7:685.
[14] Harry K, Gwendolyn AM, Kathleen M, et al. Rectal and Intranasal Immunizations with Recombinant Urease Induce Distinct Local and Serum Immune Response in Mice and Protect against Helicobacter pylori Infection [J]. Infection and Immunity, 1998, 66: 2879-2886.
[15] Wouw, BAM, Boer WA, Jansz AR, et al. Serodiagnosis of Helicobacterpylori Infection: an evaluation of a commercially available ELISA-IgG [J]. Net J Med, 1995, 47:272-277.
[16] 徐顺福，袁建平，张红杰等．幽门螺杆菌的培养研究[J]．南京医科大学学报(中文版)，1997，17：281-283．
[17] 胡伏莲，周殿元．幽门螺杆菌感染的基础与临床[M]．北京：中国科学技术出版社，2002,245．
[18] West AP, Millar MR, Tompkins DS. Survival Of Helicobacterpylori in water and saline [J]. J Clin Pathol, 1990, 43:609.
[19] 胡伏莲，周殿元．幽门螺杆菌感染的基础与临床[M]．北京：中国科学技术出版社，2002．243．
[20] 黄文．幽门螺杆菌免疫防治研究[J]．国际医药卫生导报，2002,1；50～52．[1] Warren JR and Marshall BJ. Unidentified curved bacilli on gastic epitheliun in active chronic gastritis[J]. Lancet 1983(1): 1273-1275.
[2] Peterson WL. Helicobacter pylori and peptic ulcer disease[J]. N Engl J Med 1991 324:1043-1048.
[3] Hussell T Isaacson PG Crabtree JE and Spencer J. The response of cells from low-grade B-cell gastric lymphomas of mucosa-associated lymphoid tissue to Helicobactor pylori[J]. Lancet 1993 342: 571-574.
[4] Eurogast Study Group. A international association between Helicobacter pylori infection and gastric cancer[J]. Lancet 1993 341: 1359-1362.
[5] 毛小琴，杨致邦．IgY在人类疾病诊断与防治中的作用[J]．国外医学临床生物化学与检验学分册，2004，5：423～425．
[6] 穆海波，王洪新．卵黄球蛋白IgY的提纯及应用研究进展[J]．中国家禽，2002，24(20)35～37．
[7] 王健，沈建根，朱永良．抗oh~8dG单克隆抗体的制备及生物学特性研究[J]．浙江大学学报(医学版)，2003，1(32)：33～36．
[8] 吴超，冯强，曾韦锟等．重组大肠杆菌不耐热肠毒素B亚单位的表达、纯化及粘膜佐剂活性研究[J]．中华微生物学和免疫学杂志，2005，4：307～311．
[9] Shin JH, Nam SW, Kim JT, et al. Identification of immunodominant Helicobacter pylori proteins with reactivity to H pylori-specific egg-yolk immunoglobulin [J]. J Med Microbiol, 2003,52(Pt3):217～22.
[10] 黄文．幽门螺杆菌免疫防治研究[J]．国际医药卫生导报，2002，1：50～52．
[11] 史彤，刘文忠．幽门螺杆菌疫苗免疫保护机制及其研究进展[J]．胃肠病学，2002，1(7)：45～49．
[12] Shigeo O, Masatomo H, Ken T. Application of lgY for protein against oral disease and gastrointestinal infection [J]. Nippon Shokuhin Kaishi, 1993,67(10): 1437～1439.
[13] Hansen Per, Australia S. Isolation and purification of immunogiobulins from chicken eggs using thiophilin interaction chromatography [J]. Immunological Meths, 1998,215:1～7.[1]穆海波，王洪新．卵黄球蛋白IgY的提纯及应用研究进展[J]．中国家禽,2002,24(20)35～37．
[2]谢献胜，王锋，周新民等．抗幽门螺杆菌卵黄抗体的制备及其体外抑菌试验[J]．中国家禽,2005,7(13)12～14．
[3]陈剑秋，许兰薇，张颖等．健康成人胃内24小时pH节律的观察[J]．天津医药，1997，11：667～671．
[4]Burget DW, Chiverton SG, Hunt RH, et al. Is there an optimal degree of acid suppression for healing of duodenal ulcer[J]? Gastroenterology, 1999; 99: 345.
[5]李春晖，郭立君，于洪涛等．胃蛋白酶对抗狂犬病毒IgY活性的影响[J]．中国生物制品学杂志，2002,1：45～47．
[6]熊勇华，许杨，魏华等．抗幽门螺杆菌牛初乳IgG酶解特性的研究(in Vitro)——抗幽门螺杆菌牛初乳IgG抗胃蛋白酶及胃酸屏障的研究[J]．食品科学，2003,7：48～51．
[7]Berstad A,Olafsson S, Tfera S,et al. Bismuth therapy for Helicobacter pylori infection. A review of five years experience at university hospital in NorWay[J]. J Physiol Pharmacol, 1996;47:31～49.
[8]张东海，王永琴．胶态铋剂在消化性溃疡中的应用[J]．齐齐哈尔医学院学报，2002,01：12．
[2] Biaser MJ. Helicobacter pylori and the pathogenesis of gastroduodenal inflammation[J]. J Infect Dis 1990 161:626～633.
[3] Lee A, Fos J, Hazell SL. Pathogenicity of Helicobacterpylori: a perspective[J]. Infect Immun. 1993,61:1601～1610.
[4] Seolnick JV, Tompkins LS. Helieobacter pylori and gastroduodenal disease: pathogenesis and host-parasite interaction[J]. Infect Agents Dis. 1993,1:294～309.
[5] Cover TL, Blaser MJ. Helicobacter pylori and gastroduodenal disease[J]. Annu Rev Med. 1992,43:135～145.
[6] Wotherspoon AC, Doglioni C, Diss TC, et al. Regression of primary low-grade B-cell gastric lymphoma of mucosa-associated lymphoid tissue type after eradication of Helicobacter pylori[J]. Lancet. 1993,2:575～577.
[7] Akira G, Yoshiyuki K, Takaynki A, et al. Susceptibility of Helicobacter pylori isolates against agents commonly administered for eradication therapy and the efficacy of chemotherapy[J]. Microbiol Immunol. 1997,41:7-12.
[8] Queiroz DMM,, Coimbra RS, Mendes EN, et al. Metronidazole-resistant Helicobacter pylori in a developing country[J]. Am J Gastroenterol. 1993,88:322～323.
[9] Graham DY MACG, Wink A, DE Boer, et al. Choosing the best anti-Helieobacter pylori therapy: effect of antimierobiol resistance[J]. Am J Gastroenterol, 1996,91: 1072-1076.
[10] 范学工，夏华向．幽门螺杆菌感染—基础与临床[M]．湖南科学技术出版社，1998：156～225．
[11] Pina M, Occhialini A, Monteiro L, et al. Detection of point mutations association with resistance, of Helicobacter pylori to elarithromyein by hybridization in liquid phase[J]. J Clin Microbiol, 1998;36:3285-3290.
[12] Di Mario F, et al. Azithromyein for the cure of Helicobaeter pylori infection[J]. Am J Gastroenterol, 1996;91:264.]
[13] 杨惠芳，乔家国．幽门螺杆菌的中西药敏感试验对照研究[J]．江苏临床医学杂志，1997；1(2)：103．
[14] Vakli N, Fermerty MB, et al. Cost effectiveless of treatment regimems for the eradication of Helieobacter pylori in duodenal ulcer[J]. Am J Gastroenterol, 1996.91 (2):239.
[15] Jenks PJ, Labigne A, Ferrero RL. Exposure to metronidazole in vivo readily induces resistance in Helicobacter pylori and reduces the efficacy of eradication therapy in mice[J]. Antimicrob Agents Chemother, 1999;43:777-781.
[16] 姚希贤．治疗幽门螺杆菌感染的研究进展[J]．新消化病学杂志，1997；5(9)；545-546．
[17] 杨秀珍，鲍蕾．氧氟沙星抗生素对幽门螺杆菌的敏感性试验[J]．中国微生态学杂志，1999；11(3)：152．[18]陈洁平，徐采朴．幽门螺杆菌感染的治疗[J]．新消化病学杂志，1997；5(4)258～59．
[19]陈洁平，徐采朴，王振华等．抗幽门螺杆菌感染中、西药物的体内外药敏试验研究[J]．新消化病学杂志，1997；5(10)：629-631．
[20]范学工，夏华向．幽门螺杆菌感染—基础与临床[M]．湖南科学技术出版社，1998：156～225．
[21]Cheng CY, Lin CK, Wu SH, et al. Two days, high-dosage, triple therapy with omeperazol for eradication of Helicobacter pylori[J]. Gastroenterology, 1996; 110(suppl 1):43-47.
[22]袁佩英．鸦胆子乳剂抑制螺菌的临床研究[J]．山西中医，1992，(6)：20．
[23]张琳．幽门螺杆菌与慢性萎缩性胃炎发病关系及防治研究[J]．中国中西医结合杂志，1992，12(9)：521．
[24]徐州．中药对幽门螺杆菌抑杀作用的实验研究[J]．中国医学报，1993，8(5)：25．
[25]邓世荣．槟榔治疗幽门螺杆菌感染的临床观察[J]．中医杂志，1993，(10)：605．
[26]周增芬，张永生，崔荣等．大蒜对幽门螺杆菌抑制作用的实验研究[J]．中国新药杂志，1999，8(4)：243～245．
[27]张安田．锡类散治疗消化性溃疡作用机理的研究[J]．中国中西医结合杂志，1995，15(4)：216．
[28]董国华．龙胆泻肝丸治疗幽门螺杆菌相关性胃炎47例[J]．上海中医药杂志，1996，(6)：38．
[29]武和平．珠黄散加德诺治疗幽门螺杆菌相关性溃疡的临床观察[J]．上海中医药杂志，1996，(8)：28．
[30]姚希贤．治疗幽门螺杆菌感染的研究进展[J]．新消化病学杂志，1997；5(9)；545-546．
[31]周增芬，张永生，崔蓉等．大蒜对幽门螺杆菌抑菌作用的实验研究[J]．中国新药杂志，1999；8(4)；243-245．
[32]Thomas JE Austin S Dale A et al. Protection by human milk IgA against Helicobacter pylori infection in infancy(letter) [J]. Lancet 1993 342:121.
[33]Czinn SJ, Cai A and Nedrud JG. Protection of germ free mice from infection by Helicobacter felis after active oral or passive IgA immunization[J]. Vaccine, 1993,11:637～642.
[34]Lee CK, Weltzin R, Thomas WD, et al. Oral immunization with recombinant Helicobacter pylori urease induces secretory IgA antibodies and protects mice from challenge with Helicobacter felis[J]. J Infect Dis, 1995,172:161～172.
[35]Blanchard TG, Czinn S J, Maurer R, et al. Urease-specific monoclonal antibodies prevent Helicobacter pelis infection in mice[J]. Infect Immun, 1995,63: 1394-1399.
[36]张和平，郭军．免疫乳科学与技术tM]．北京：中国轻工业出版社，2002．[1] Locken MR. Roth TF. Analysis of maternal IgG subpopulations wbich are transfer to egg yolk in chickens[J]. J Immunol. 1962.89:272～278.
[2] Morrison SL. Mohammed MS. Wims LA, et al. Sequences in antibody molecules important for receptant for receptor-mediated transport into the chicken egg yolk[J]. Molecular Immunoloy. 2002,38(8):619～625.
[3] 王炯，龚春梅，赵树栋等．鸡卵抗体(IgY)理化特性的研究[J]．中国生物制品学杂志，1997，3：140～143．
[4]Song CS etal[J]. J Immunol,1985;135(5):3354～3359.
[5]龙中儿，钟青萍，刘活林等．卵黄免疫球蛋白的稳定性研究[J]．中国生物制品学杂志，1997；4：218～221．
[6]易全茂，谢艳霞．鸡卵黄抗体研究进展[J]．动物科学与动物医学，2004；2：44～46．
[7]Otani H, Matsumoto K, Saeki A, et al. Comparative studies on porperties on hen egg yolk IgY and rabbit serum IgG antibodies[J]. Lebensm--Wiss u Technol,1991,24:152.
[8]陈斌，杨志华．鸡卵黄免疫球蛋白(IgY)的作用机理及应用前景[J]．浙江畜牧兽医，2005．1：9～10．
[9]Woolly JA et al[J]. J Immunol Methods, 1995;178:253--263.
[10]Hassl A et al. Zbl Bake Hyg A, 1987;267:247-253.1111.Mclaren R D et al[J]. J Immunol??Methods, 1994;177:175-184.
[11] Hatta H et al[J]. Agric Biol Chem, 1990;54:2531.
[12]康亦兼，咸漠，李文兴．水稀释法分离卵黄IgY[J]．吉林大学自然科学学报，2001，3：84-86．
[13]肖驰，周淑兰，涂志等．鸡抗猪大肠埃希氏菌卵黄抗体的研制与应用[J]．中国兽医科技，1998,4：21～23．
[14]俞海峰．饲料营养元素与动物免疫研究进展[J]．饲料广角，2003，12：34-36．
[15]杨晓梅，刘万钧，李力等．鸡抗鸭病毒性肝炎高免卵黄抗体的研制与应用[J]．四川畜牧兽医，1994,2：9～10．
[16]汪铭书，程安春，陈孝跃．应用高免卵黄抗体防治鸭病毒性肝炎的研究[J]．中国预防兽医学报，1997,5：14～17．
[17]王玉兰，张英．小鹅瘟与鹅禽霍乱防制措施的探讨[J]．辽宁畜牧兽医，1994,6：28～29．
[18]边传周，袁道强，任敏等．禽流感高免卵黄抗体的研制与应用[J]．郑州牧业工程高等专科学校学报，2000,2：84～85．
[19]詹丽娥，乔忠，孙世兴．鸡三联蛋黄抗体的研制及应用[J]．中国兽医科技，1994,11：30～31．
[20]郑厚旌，巩霞，王同亮等．应用改良琼指扩散试验检测鸡传染性法氏囊病病毒的研究[J]．中国预防兽医学报，1996,2：11-14．
[21]李槿年，余为一．利用卵黄监测鳖群中病原菌感染[J]．淡水渔业，1999,12：7～9．[1] Meguaud F.Epidemiology of Helicobacter pylori Infection [J]. Gas Clin Nor Am, 1993, 22:73-88.
[2] Parsonnet J. The incidence of Helicobacter pylori Infection [J]. Ali Pha Ther, 1995,9:45-51.
[3] Li YY, Hu P J, Du GG, et al. The prevalence Helicobacter pylori Infection in P.R. China [J]. Am J Gastroenterol, 1991,86:446～449.
[4] Suerbaum S, Hur C, Josenhans C, et al. Pathogenesis and virulence factors of Helicobacter pylori [J]. Curr Opin Gastroenterol, 1999,15:s11～16.
[5] Rudnicks W, Andersen LP. Inflammation and host response[J]. Curr Opin Gastroenterol, 1999, 15:s17～22.
[6] Rokkas T, Wotherspoon A. Carcinogenesis [J]. Curr Opin Gastroenterol, 1999, 15:s23-28.
[7] Marshall BJ. Helicobacter pylori [J]. Am J Gastroenterol, 1994,89:s116.[8] Edit S, Stolte M. The significance of Helicobacter pylori in relation to gastric cancer and lymphoma [J]. Eur .1 Gastroenterol Hepatol, 1995,7:318.
[9] Mohamed MA, Goggin PM, Molineaux N, et al. Relation ofHelicobacter pylori infection and coronary heart disease[J]. Br Heart J, 1994,71:437-439.
[10] Markus HS, Brown M, Levy J, et al. H. pylori: a new risk factor cerebrovascular disease and carotid atheroma [J]. Cerebrovasc Dis, 1995,5:251.
[11] Gasbarrini A, De Luca A, Fiore G, et al. Beneficial effects of Helicobacter pylori eradicatin on migraine [J]. Hepatogas torenterology, 1998,45:765-770.
[12] Gasbrrini A, Serricchio M, Tindi P, et al. Association of Helicobacter pylori infection with primary Raynaud phenomenon [J]. Lancet, 1996,348:966-967.
[13] Hunt RH, Malfertheiner P, Yeomans ND, et al. Critical issues in the pathophysiology and management of peptic ulcer aisease [J]. Eur J Gastroenterol Hepatol, 1995, 7:685.
[14] Harry K, Gwendolyn AM, Kathleen M, et al. Rectal and Intranasal Immunizations with Recombinant Urease Induce Distinct Local and Serum Immune Response in Mice and Protect against Helicobacter pylori Infection [J]. Infection and Immunity, 1998, 66: 2879-2886.
[15] Wouw, BAM, Boer WA, Jansz AR, et al. Serodiagnosis of Helicobacterpylori Infection: an evaluation of a commercially available ELISA-IgG [J]. Net J Med, 1995, 47:272-277.
[16] 徐顺福，袁建平，张红杰等．幽门螺杆菌的培养研究[J]．南京医科大学学报(中文版)，1997，17：281-283．
[17] 胡伏莲，周殿元．幽门螺杆菌感染的基础与临床[M]．北京：中国科学技术出版社，2002,245．
[18] West AP, Millar MR, Tompkins DS. Survival Of Helicobacterpylori in water and saline [J]. J Clin Pathol, 1990, 43:609.
[19] 胡伏莲，周殿元．幽门螺杆菌感染的基础与临床[M]．北京：中国科学技术出版社，2002．243．
[20] 黄文．幽门螺杆菌免疫防治研究[J]．国际医药卫生导报，2002,1；50～52．[1] Warren JR and Marshall BJ. Unidentified curved bacilli on gastic epitheliun in active chronic gastritis[J]. Lancet 1983(1): 1273-1275.
[2] Peterson WL. Helicobacter pylori and peptic ulcer disease[J]. N Engl J Med 1991 324:1043-1048.
[3] Hussell T Isaacson PG Crabtree JE and Spencer J. The response of cells from low-grade B-cell gastric lymphomas of mucosa-associated lymphoid tissue to Helicobactor pylori[J]. Lancet 1993 342: 571-574.
[4] Eurogast Study Group. A international association between Helicobacter pylori infection and gastric cancer[J]. Lancet 1993 341: 1359-1362.
[5] 毛小琴，杨致邦．IgY在人类疾病诊断与防治中的作用[J]．国外医学临床生物化学与检验学分册，2004，5：423～425．
[6] 穆海波，王洪新．卵黄球蛋白IgY的提纯及应用研究进展[J]．中国家禽，2002，24(20)35～37．
[7] 王健，沈建根，朱永良．抗oh~8dG单克隆抗体的制备及生物学特性研究[J]．浙江大学学报(医学版)，2003，1(32)：33～36．
[8] 吴超，冯强，曾韦锟等．重组大肠杆菌不耐热肠毒素B亚单位的表达、纯化及粘膜佐剂活性研究[J]．中华微生物学和免疫学杂志，2005，4：307～311．
[9] Shin JH, Nam SW, Kim JT, et al. Identification of immunodominant Helicobacter pylori proteins with reactivity to H pylori-specific egg-yolk immunoglobulin [J]. J Med Microbiol, 2003,52(Pt3):217～22.
[10] 黄文．幽门螺杆菌免疫防治研究[J]．国际医药卫生导报，2002，1：50～52．
[11] 史彤，刘文忠．幽门螺杆菌疫苗免疫保护机制及其研究进展[J]．胃肠病学，2002，1(7)：45～49．
[12] Shigeo O, Masatomo H, Ken T. Application of lgY for protein against oral disease and gastrointestinal infection [J]. Nippon Shokuhin Kaishi, 1993,67(10): 1437～1439.
[13] Hansen Per, Australia S. Isolation and purification of immunogiobulins from chicken eggs using thiophilin interaction chromatography [J]. Immunological Meths, 1998,215:1～7.[1]穆海波，王洪新．卵黄球蛋白IgY的提纯及应用研究进展[J]．中国家禽,2002,24(20)35～37．
[2]谢献胜，王锋，周新民等．抗幽门螺杆菌卵黄抗体的制备及其体外抑菌试验[J]．中国家禽,2005,7(13)12～14．
[3]陈剑秋，许兰薇，张颖等．健康成人胃内24小时pH节律的观察[J]．天津医药，1997，11：667～671．
[4]Burget DW, Chiverton SG, Hunt RH, et al. Is there an optimal degree of acid suppression for healing of duodenal ulcer[J]? Gastroenterology, 1999; 99: 345.
[5]李春晖，郭立君，于洪涛等．胃蛋白酶对抗狂犬病毒IgY活性的影响[J]．中国生物制品学杂志，2002,1：45～47．
[6]熊勇华，许杨，魏华等．抗幽门螺杆菌牛初乳IgG酶解特性的研究(in Vitro)——抗幽门螺杆菌牛初乳IgG抗胃蛋白酶及胃酸屏障的研究[J]．食品科学，2003,7：48～51．
[7]Berstad A,Olafsson S, Tfera S,et al. Bismuth therapy for Helicobacter pylori infection. A review of five years experience at university hospital in NorWay[J]. J Physiol Pharmacol, 1996;47:31～49.
[8]张东海，王永琴．胶态铋剂在消化性溃疡中的应用[J]．齐齐哈尔医学院学报，2002,01：12．