幽门螺杆菌卵黄抗体的研制及其功能的初步评价
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摘要
幽门螺杆菌(Helicobacter pylori, Hp)是引起慢性活动性胃炎、消化性溃疡的最常见的病原,也是引起胃癌发生的关键因素。抗生素的多联治疗对消除Hp的感染有较好的疗效,然而抗生素治疗在体内仍不能完全有效,而且极易产生抗药性。因此,探讨其它的治疗途径是一种必然的研究趋势。本研究以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,并对免疫接种Hp母鸡免疫应答规律和卵黄抗体的抑菌活性及理化特性等做了初步探讨,这为制备高含量的抗Hp免疫卵黄及相关的生物制剂提供技术依据,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%02,10%C02,85%N2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
以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15d,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,抗体效价最高可达到1：1024,抗体效价大于1：128可维持60d以上,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,高免卵黄抗体具有明显的抑制Hp生长的活性。
3鸡抗幽门螺杆菌卵黄抗体的理化特性
对鸡抗幽门螺杆菌高免卵黄抗体的理化特性进行了研究。结果表明：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。
4幽门螺杆菌感染蒙古沙土鼠疾病模型的建立
利用蒙古沙土鼠建立Hp NCTC11637株胃内感染模型。感染方法有两种,方法Ⅰ是沙土鼠禁食18h后用50%酒精0.5mL预处理,6h后接种Hp,间隔12h、24h分别再次接种。方法Ⅱ的接种方法同方法Ⅰ,但接种后每日口服雷尼替丁5mg/kg体重。胃内接种Hp后,于第7d、15d、35d、45d分别用Hp抗原酶联免疫诊断试剂盒、细菌培养和胃组织切片的方法观察沙土鼠胃内细菌感染情况和胃病理组织学变化。结果表明,给沙土鼠胃内灌服酒精以损伤胃黏膜并每日口服雷尼替丁,在人工接种35d后,胃内见有多量Hp生长,胃组织病变与Hp自然感染的病例相似。
5卵黄抗体对幽门螺杆菌感染沙土鼠的预防试验
在成功研制出抗Hp卵黄抗体的基础上,利用蒙古沙土鼠作为实验动物,观察抗Hp卵黄抗体对Hp感染的防制作用。实验鼠随机分为四组(36只／组),Ⅰ、Ⅱ、Ⅲ组分别灌服生理盐水、复合抗生素和抗Hp卵黄抗体,1次/d,连续13d。Ⅳ组皮下注射抗Hp卵黄抗体,1次/d,48h后再注射1次。在首次用药后第48h口服接种幽门螺杆菌(ATCC43504)2.75×108CFU(布氏培养液)。在接种后第7、15、30、45d,Ⅰ组鼠胃内均有大量Hp定植,感染率100%；Ⅱ、Ⅲ、Ⅳ组的感染率均低于23%。结果表明灌服和注射抗Hp卵黄抗体,可以抑制沙土鼠感染Hp,其抑制效果与抗生素组无明显差异(p<0.05)。
6卵黄抗体对沙土鼠胃内感染幽门螺杆菌的治疗试验
利用蒙古沙土鼠作为实验动物,观察Hp特异卵黄抗体对胃内Hp感染的治疗效果。实验鼠间隔48h两次口服接种Hp (ATCC43504)布氏培养液1ml(1.15×108CFU),首次接种7d后将实验鼠随机分为四组(16只/组),Ⅰ、Ⅱ、Ⅲ组分别灌服生理盐水、复合抗生素和抗Hp卵黄抗体,1次/d,连续12d;Ⅳ组间隔48h皮下注射抗Hp卵黄抗体两次。在用药前实验鼠胃内均有大量Hp定植,感染率100%；用药后第7d,Ⅱ组鼠的Hp清除率为60%；Ⅲ组、Ⅳ组鼠胃内均有少量Hp存在。用药12d后,Ⅱ、Ⅲ、Ⅳ组的胃内Hp清除率分别为60%、60%、40%。灌服和注射抗Hp卵黄抗体,可以抑制沙土鼠胃内Hp感染,其抑制效果与抗生素相似。
7幽门螺杆菌UreB基因的克隆及原核表达
为研制幽门螺杆菌基因工程疫苗或为制备卵黄抗体提供免疫原,以原核载体表达幽门螺杆菌的保护性抗原成分UreB蛋白。PCR方法扩增UreB基因片段,将其克隆至pGEM-T Easy,测序证明UreB基因序列的正确性。酶切后连接至IJpET32a(+)质粒上,转化E.coli BL21(DE3), IPTG诱导后表达UreB融合蛋白,经SDS-PAGE检测表明,融合表达的UreB蛋白分子质量约为80kDa。30℃诱导可使融合蛋白呈可溶性表达,经Ni2+柱亲和层析纯化可得到纯化蛋白。Western blot检测表明融合蛋白可以被Hp免疫小鼠血清的相应抗体识别,具有良好的免疫学活性。
Helicobacter pylori (H. pylori, Hp) is the most common cause of gastritis and gastric ulcers and plays a pivotal role in the development of gastric carcinomas. Successful treatment of Hp infections most often employs antibiotic therapy, consisting of some combination of antibiotic, and either bismuth or a proton pump inhibitor. However, antibiotic therapy fails in10%to15%of cases due to the development of antibiotic resistance. It is important to seek new therapies for a wider means of treating, suppressing, or preventing H. pylori infection without drug resistance problems. In this study, different culture media were tested to culture and preserve Hp. The mature chickens were procedurally immunized with inactivated Hp cells, agglutination test were used to determine the titer of egg yolk antibody, and the pattern of immune response, bacteriostasis in vitro, physical and chemical characteristic of immune yolk were investigated. All these studies were of great significance to the industrial production of high immune yolk antibody against Hp and correlatively biologic medication.
1Effect of different methods on Hp culture and preservation
The strain was cultured on Columbia Agar Base (CAB) supplemented with mixed antibiotics containing2500IU/l Polymyxin B Sulfate,5mg/l Trimethoprim,10mg/l Vancomycin HCl and10mg/l Amphotericin B and6%defibrinated sheep blood or6%horse serum respectively in a microaerophilic atmosphere of5%O2,10%CO2and85%N2at37℃for72h. With the two methods, good effect was observed. In condition of sterilization with no supplementation with mixed antibiotics, the strain grew well without contamination. When Brucella Broth was used an culture medium with combined antibiotics and horse serum in microaerophilic bag, Hp grew rapidly but was easily to form spheroid without replacing the bag on time. The strain was revived successively with cryopreservation at-70℃to-80℃.
2Preparation and bacteriostasis in vitro of egg yolk antibody against Helicobacter pylori
Preparation and bacteriostasis in vitro of egg yolk antibody against Hp were studied. Chickens were procedurally immunized with inactivated bacterial cells at150,165and180days old, successively. Agglutination test showed that the egg yolk antibody against HP was produced from immunized hens, the antibody titer reached to1:1024at the45th day after the first immunization, the high level of antibody (>1:128) lasted for2months, then went down gradually to1:8after130days of age. Bacteriostasis in vitro showed that Hp was hypersensitive to egg yolk antibody.
3Physical and chemical characteristic of high immune yolk against Helicobacter pylori
The physical and chemical characteristic of high immune yolk (HIY) against Hp was studied. The results showed that the pH value and temperature of environment had obvious influence on anti-Hp HIY, while pepsin enhaced the influence. When only with pH value, the sensitive range was pH<3, when with pH value and pepsin, the sensitive range was pH≤4. HIY had better thermal stability at the temperature of pasteurize. All these characteristics were of great significance to the industrial production of high immune yolk antibody against Hp.
4Establishment of Helicobacter pylori infection model in Mongolian gerbils
Two methods were used to establish models of Hp (NCTC11637) infection in the Mongolian gerbil stomach. In the first method, Mongolian gerbils were forbidden to eat for18hours, then feeded0.5mL of50%alcohol and inoculated Hp three times at a6h-,12h-, and24h-interval. In the second method, Mongolian gerbils was treated as same as the above method, but taken Ranitidine orally everyday, with5mg/kg body weight. On the7th,15th,35th, and the45th day after inoculation, the infection and pathological changes in gastric mucosa were detected by Hp antigen diagnostic ELISA kit, bacterial culture, and histological section. The results showed that there were much Helicobacter pylori in the stomach in35days postinfection, combining the gastric mucous membrane damaged by alcohol with Ranitidine by oral administration per day, and the pathological changes were the same with that in natural infected cases.
5Inhibitory effect of egg-yolk antibody on experimental Helicobacter pylori infection in Mongolian gerbils
Inhibitory role of egg-yolk antibody on experimental Hp infection in Mongolian gerbil were observed as soon as egg-yolk antibody against Hp was prepared. The experiment gerbils were divided into4groups(36mice/group) and then taken orally normal saline in group Ⅰ, combination of antibiotics in Group Ⅱ, egg-yolk antibody against Hp in GroupⅢ for13days(one time/d). The egg-yolk antibody was injected hypodermically twice with a48h interval in groupIV.48h after first time administration the mice were inoculated with2.75×108colony forming units(CFUs) of Hp (ATCC43504) bacteria grown in Brucella broth. There were much Hp in the stomach on the7th,15th,30th,45th day after inoculation. The infection rate was100%in group Ⅰ, but low than23%in Group Ⅱ, GroupⅢ or groupⅣ. The results showed that the egg-yolk antibody against Hp by oral or injected route might decrease Hp infection in the stomach of Mongolian gerbils, and there is no significantly difference between group with egg-yolk antibody and group with antibiotic (p<0.05)
6Effects of egg-yolk antibody on the treatment of Helicobacter pylori infection in the stomach of Mongolian gerbils
To observe the effects of treatment by egg-yolk antibody aganist H. pylori infection, the experiment Mongolian gerbils were orally inoculated with H. pylori grown in Brucella broth (ATCC43504,1.15×108CFU) twice at48-hour-interval, and divided into4groups (16mice/group) on the seventh day after first inoculation time. Then the gerbils took orally normal saline in group Ⅰ, association of antibiotics in Group Ⅱ, egg-yolk antibody against H. pylori in GroupⅢ for12days (one time/d). The egg-yolk antibody was injected hypodermically twice with a48-hour-interval in groupⅣ. There were much H. pylori in the stomach before medication, with infection rate of100%. On the seventh day after medication the clearance rate of H. pylori was60%in group Ⅱ, and there were few H. pylori in group Ⅲ and group Ⅳ. On the twelfth day after taking orally drugs, the clearance rate was60%in both of group Ⅱ and group Ⅲ, but40%in group Ⅳ. Taking orally or injecting the egg-yolk antibody against H. pylori might decrease H. pylori infection in the stomach of Mongolian gerbil, and there is significantly difference between group treated with egg-yolk antibody and group treated with antibiotic.
7Clone and expression of UreB gene from Helicobacter pylori in prokaryotic expression vector
In order to develop genetic engineering vaccine against Helicobacter pylori or provide immunogen for preparation of egg-yolk antibody, a prokaryotic expression vector expressed UreB gene encoded protective antigen from Hp was constructed. UreB gene was PCR amplified and cloned in pGEM-T Easy vector. After sequence analysis of verification, the UreB gene was digested with restriction endoenzyme and ligated to pET32a (+), transformed to E.coli BL21(DE3). The molecular weight of fusion protein was about80KDa when the recombinant bacterium was induced by IPTG and detected by SDS-PAGE. The fusion protein was obtained in the supernatant of recombinant bacterium and purified by Ni+affinity chromatography when it was induced at30℃. The fusion protein could be recognized by corresponding antibody of mice sera immunized by inactivated Hp in western bolt, indicating this fusion protein had good immunocompetence.
1幽门螺杆菌的培养与保存
本试验采用不同培养基对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%02,10%C02,85%N2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
以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15d,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,抗体效价最高可达到1：1024,抗体效价大于1：128可维持60d以上,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,高免卵黄抗体具有明显的抑制Hp生长的活性。
3鸡抗幽门螺杆菌卵黄抗体的理化特性
对鸡抗幽门螺杆菌高免卵黄抗体的理化特性进行了研究。结果表明：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。
4幽门螺杆菌感染蒙古沙土鼠疾病模型的建立
利用蒙古沙土鼠建立Hp NCTC11637株胃内感染模型。感染方法有两种,方法Ⅰ是沙土鼠禁食18h后用50%酒精0.5mL预处理,6h后接种Hp,间隔12h、24h分别再次接种。方法Ⅱ的接种方法同方法Ⅰ,但接种后每日口服雷尼替丁5mg/kg体重。胃内接种Hp后,于第7d、15d、35d、45d分别用Hp抗原酶联免疫诊断试剂盒、细菌培养和胃组织切片的方法观察沙土鼠胃内细菌感染情况和胃病理组织学变化。结果表明,给沙土鼠胃内灌服酒精以损伤胃黏膜并每日口服雷尼替丁,在人工接种35d后,胃内见有多量Hp生长,胃组织病变与Hp自然感染的病例相似。
5卵黄抗体对幽门螺杆菌感染沙土鼠的预防试验
在成功研制出抗Hp卵黄抗体的基础上,利用蒙古沙土鼠作为实验动物,观察抗Hp卵黄抗体对Hp感染的防制作用。实验鼠随机分为四组(36只／组),Ⅰ、Ⅱ、Ⅲ组分别灌服生理盐水、复合抗生素和抗Hp卵黄抗体,1次/d,连续13d。Ⅳ组皮下注射抗Hp卵黄抗体,1次/d,48h后再注射1次。在首次用药后第48h口服接种幽门螺杆菌(ATCC43504)2.75×108CFU(布氏培养液)。在接种后第7、15、30、45d,Ⅰ组鼠胃内均有大量Hp定植,感染率100%；Ⅱ、Ⅲ、Ⅳ组的感染率均低于23%。结果表明灌服和注射抗Hp卵黄抗体,可以抑制沙土鼠感染Hp,其抑制效果与抗生素组无明显差异(p<0.05)。
6卵黄抗体对沙土鼠胃内感染幽门螺杆菌的治疗试验
利用蒙古沙土鼠作为实验动物,观察Hp特异卵黄抗体对胃内Hp感染的治疗效果。实验鼠间隔48h两次口服接种Hp (ATCC43504)布氏培养液1ml(1.15×108CFU),首次接种7d后将实验鼠随机分为四组(16只/组),Ⅰ、Ⅱ、Ⅲ组分别灌服生理盐水、复合抗生素和抗Hp卵黄抗体,1次/d,连续12d;Ⅳ组间隔48h皮下注射抗Hp卵黄抗体两次。在用药前实验鼠胃内均有大量Hp定植,感染率100%；用药后第7d,Ⅱ组鼠的Hp清除率为60%；Ⅲ组、Ⅳ组鼠胃内均有少量Hp存在。用药12d后,Ⅱ、Ⅲ、Ⅳ组的胃内Hp清除率分别为60%、60%、40%。灌服和注射抗Hp卵黄抗体,可以抑制沙土鼠胃内Hp感染,其抑制效果与抗生素相似。
7幽门螺杆菌UreB基因的克隆及原核表达
为研制幽门螺杆菌基因工程疫苗或为制备卵黄抗体提供免疫原,以原核载体表达幽门螺杆菌的保护性抗原成分UreB蛋白。PCR方法扩增UreB基因片段,将其克隆至pGEM-T Easy,测序证明UreB基因序列的正确性。酶切后连接至IJpET32a(+)质粒上,转化E.coli BL21(DE3), IPTG诱导后表达UreB融合蛋白,经SDS-PAGE检测表明,融合表达的UreB蛋白分子质量约为80kDa。30℃诱导可使融合蛋白呈可溶性表达,经Ni2+柱亲和层析纯化可得到纯化蛋白。Western blot检测表明融合蛋白可以被Hp免疫小鼠血清的相应抗体识别,具有良好的免疫学活性。
Helicobacter pylori (H. pylori, Hp) is the most common cause of gastritis and gastric ulcers and plays a pivotal role in the development of gastric carcinomas. Successful treatment of Hp infections most often employs antibiotic therapy, consisting of some combination of antibiotic, and either bismuth or a proton pump inhibitor. However, antibiotic therapy fails in10%to15%of cases due to the development of antibiotic resistance. It is important to seek new therapies for a wider means of treating, suppressing, or preventing H. pylori infection without drug resistance problems. In this study, different culture media were tested to culture and preserve Hp. The mature chickens were procedurally immunized with inactivated Hp cells, agglutination test were used to determine the titer of egg yolk antibody, and the pattern of immune response, bacteriostasis in vitro, physical and chemical characteristic of immune yolk were investigated. All these studies were of great significance to the industrial production of high immune yolk antibody against Hp and correlatively biologic medication.
1Effect of different methods on Hp culture and preservation
The strain was cultured on Columbia Agar Base (CAB) supplemented with mixed antibiotics containing2500IU/l Polymyxin B Sulfate,5mg/l Trimethoprim,10mg/l Vancomycin HCl and10mg/l Amphotericin B and6%defibrinated sheep blood or6%horse serum respectively in a microaerophilic atmosphere of5%O2,10%CO2and85%N2at37℃for72h. With the two methods, good effect was observed. In condition of sterilization with no supplementation with mixed antibiotics, the strain grew well without contamination. When Brucella Broth was used an culture medium with combined antibiotics and horse serum in microaerophilic bag, Hp grew rapidly but was easily to form spheroid without replacing the bag on time. The strain was revived successively with cryopreservation at-70℃to-80℃.
2Preparation and bacteriostasis in vitro of egg yolk antibody against Helicobacter pylori
Preparation and bacteriostasis in vitro of egg yolk antibody against Hp were studied. Chickens were procedurally immunized with inactivated bacterial cells at150,165and180days old, successively. Agglutination test showed that the egg yolk antibody against HP was produced from immunized hens, the antibody titer reached to1:1024at the45th day after the first immunization, the high level of antibody (>1:128) lasted for2months, then went down gradually to1:8after130days of age. Bacteriostasis in vitro showed that Hp was hypersensitive to egg yolk antibody.
3Physical and chemical characteristic of high immune yolk against Helicobacter pylori
The physical and chemical characteristic of high immune yolk (HIY) against Hp was studied. The results showed that the pH value and temperature of environment had obvious influence on anti-Hp HIY, while pepsin enhaced the influence. When only with pH value, the sensitive range was pH<3, when with pH value and pepsin, the sensitive range was pH≤4. HIY had better thermal stability at the temperature of pasteurize. All these characteristics were of great significance to the industrial production of high immune yolk antibody against Hp.
4Establishment of Helicobacter pylori infection model in Mongolian gerbils
Two methods were used to establish models of Hp (NCTC11637) infection in the Mongolian gerbil stomach. In the first method, Mongolian gerbils were forbidden to eat for18hours, then feeded0.5mL of50%alcohol and inoculated Hp three times at a6h-,12h-, and24h-interval. In the second method, Mongolian gerbils was treated as same as the above method, but taken Ranitidine orally everyday, with5mg/kg body weight. On the7th,15th,35th, and the45th day after inoculation, the infection and pathological changes in gastric mucosa were detected by Hp antigen diagnostic ELISA kit, bacterial culture, and histological section. The results showed that there were much Helicobacter pylori in the stomach in35days postinfection, combining the gastric mucous membrane damaged by alcohol with Ranitidine by oral administration per day, and the pathological changes were the same with that in natural infected cases.
5Inhibitory effect of egg-yolk antibody on experimental Helicobacter pylori infection in Mongolian gerbils
Inhibitory role of egg-yolk antibody on experimental Hp infection in Mongolian gerbil were observed as soon as egg-yolk antibody against Hp was prepared. The experiment gerbils were divided into4groups(36mice/group) and then taken orally normal saline in group Ⅰ, combination of antibiotics in Group Ⅱ, egg-yolk antibody against Hp in GroupⅢ for13days(one time/d). The egg-yolk antibody was injected hypodermically twice with a48h interval in groupIV.48h after first time administration the mice were inoculated with2.75×108colony forming units(CFUs) of Hp (ATCC43504) bacteria grown in Brucella broth. There were much Hp in the stomach on the7th,15th,30th,45th day after inoculation. The infection rate was100%in group Ⅰ, but low than23%in Group Ⅱ, GroupⅢ or groupⅣ. The results showed that the egg-yolk antibody against Hp by oral or injected route might decrease Hp infection in the stomach of Mongolian gerbils, and there is no significantly difference between group with egg-yolk antibody and group with antibiotic (p<0.05)
6Effects of egg-yolk antibody on the treatment of Helicobacter pylori infection in the stomach of Mongolian gerbils
To observe the effects of treatment by egg-yolk antibody aganist H. pylori infection, the experiment Mongolian gerbils were orally inoculated with H. pylori grown in Brucella broth (ATCC43504,1.15×108CFU) twice at48-hour-interval, and divided into4groups (16mice/group) on the seventh day after first inoculation time. Then the gerbils took orally normal saline in group Ⅰ, association of antibiotics in Group Ⅱ, egg-yolk antibody against H. pylori in GroupⅢ for12days (one time/d). The egg-yolk antibody was injected hypodermically twice with a48-hour-interval in groupⅣ. There were much H. pylori in the stomach before medication, with infection rate of100%. On the seventh day after medication the clearance rate of H. pylori was60%in group Ⅱ, and there were few H. pylori in group Ⅲ and group Ⅳ. On the twelfth day after taking orally drugs, the clearance rate was60%in both of group Ⅱ and group Ⅲ, but40%in group Ⅳ. Taking orally or injecting the egg-yolk antibody against H. pylori might decrease H. pylori infection in the stomach of Mongolian gerbil, and there is significantly difference between group treated with egg-yolk antibody and group treated with antibiotic.
7Clone and expression of UreB gene from Helicobacter pylori in prokaryotic expression vector
In order to develop genetic engineering vaccine against Helicobacter pylori or provide immunogen for preparation of egg-yolk antibody, a prokaryotic expression vector expressed UreB gene encoded protective antigen from Hp was constructed. UreB gene was PCR amplified and cloned in pGEM-T Easy vector. After sequence analysis of verification, the UreB gene was digested with restriction endoenzyme and ligated to pET32a (+), transformed to E.coli BL21(DE3). The molecular weight of fusion protein was about80KDa when the recombinant bacterium was induced by IPTG and detected by SDS-PAGE. The fusion protein was obtained in the supernatant of recombinant bacterium and purified by Ni+affinity chromatography when it was induced at30℃. The fusion protein could be recognized by corresponding antibody of mice sera immunized by inactivated Hp in western bolt, indicating this fusion protein had good immunocompetence.
引文
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