【作者】 陈建国；

【导师】 杨利国；

【作者基本信息】 华中农业大学 ， 动物遗传育种与繁殖， 2013， 博士

【摘要】 幽门螺杆菌(Helicobacter pylori, H. pylori, Hp)广泛存在于人类的胃中,是导致慢性胃炎、胃肠溃疡、胃腺癌等胃肠疾病的主要病原菌,已严重影响着人们的身体健康。尽管国内外采用抗生素三联、四联疗法在治疗H. pylori引起相关的胃炎和胃溃疡等胃肠疾病方面取得了较大成果,但临床发现广谱抗生素的治疗导致幽门螺杆菌耐药菌株不断出现和治愈后复发率居高不下,临床上将很快进入“感染-治愈-复发-再治疗-耐药”的恶性循环,不能彻底解决问题。因此希望通过疫苗免疫奶牛使在牛奶中产生高水平的抗幽门螺杆菌抗体,通过口服该抗体,来预防和治疗H. pylori引起的慢性胃炎、胃肠溃疡等胃肠疾病。因此希望使用安全、高效、使用方便的疫苗来免疫动物产生高浓度抗体或用该疫苗免疫动物使之对幽门螺杆菌产生有效的抵抗力。本研究首先选取生产后10天健康、无乳腺疾病和生殖器官疾病、体温正常的奶牛14头,随机分为两组：PBS对照组和幽门螺杆菌全菌蛋白免疫注射组,每组7头。用浓度为2×1010cfu/mL的幽门螺杆菌(H. polyri)NCTC11637全菌经超声破碎后与等剂量的完全(不完全)弗氏佐剂充分乳化后分别于0、14、28天三次多点肌肉注射全菌蛋白疫苗,每次4mL,免疫产后泌乳的健康奶牛,对照组每次注射PBS与弗氏佐剂乳化物4mL。每周采血和收集牛奶各一次,离心收集血清,以酶联免疫吸附试验测定血清、乳汁的IgG抗体,同时测量体温,观察发情和采食泌乳等健康情况。研究结果显示经肌注幽门螺杆菌全菌蛋白免疫奶牛后可在血清和乳汁中产生特异性抗体,抗体浓度显著高于对照组(P<0.05),但免疫结束后不久逐渐下降。免疫奶牛的体温、发情和采食泌乳等情况正常。但全菌蛋白疫苗费用高,制作麻烦,注射时阻力大、奶牛反应强烈,且需要多次多点注射。结果表明肌肉注射幽门螺杆菌全菌蛋白疫苗是一个安全、有效的免疫途径。但全菌蛋白疫苗制作麻烦,费用高,注射时奶牛反应大。因此希望构建更加安全、高效和使用更为方便的疫苗代替幽门螺杆菌全菌蛋白疫苗。考虑到幽门螺杆菌是肠道菌群,通过口服引起胃肠道粘膜免疫是一种可行途径。但口服的蛋白疫苗的制作复杂,提纯麻烦,且时间较长,同时需加相应的免疫佐剂提高免疫原性,在口服过程中蛋白疫苗还容易被胃酸及胃蛋白酶破坏而丧失免疫功能。因此考虑用大肠杆菌或减毒沙门氏菌来作为载体。本实验室多次采用减毒猪霍乱沙门氏菌C500作为载体,安全且免疫效果好,希望构建幽门螺杆菌的减毒猪霍乱沙门菌C500口服活载体疫苗。本实验选取幽门螺杆菌中已证实具有免疫原性的两个基因：尿素酶B(urease B, UreB)和细胞毒素相关抗原(cytotoxin-associated antigen, CagA)。先采用PCR技术从幽门螺杆菌标准株SS1扩增Hp-UreB和Hp-CagA基因,将其连接至原核表达质粒pYA3493中,使之构建为pYA3493-UreB-CagA重组质粒,经PCR及酶切鉴定后测定其基因序列,并与GenBank中已发表的相关序列的进行同源性分析。重组质粒pYA3493-UreB-CagA电击转入减毒猪霍乱沙门菌C500,培养后筛选阳性菌落,抽提质粒进行PCR及酶切鉴定。表达的UreB-CagA蛋白用SDS-PAGE和Western Blotting进行鉴定。然后将无特定病原菌昆明小鼠分成5组,每组10只,分别为肌肉注射幽门螺杆菌全菌蛋白疫苗和灌胃分别给予含质粒pYA3493-UreB-CagA的减毒沙门氏菌C500(0.2mL,1×1010CFUmL-1)、含空质粒pYA3493的减毒沙门氏菌C500(0.2mL,1×1010CFUmL-1)、减毒沙门氏菌C500(0.2mL,1×1010CFUmL-1)以及PBS (0.2mL)。每周免疫一次,连续五次。第六周后再用活H. pylori SS1(0.2mL,1×1010CFUmL-1)灌胃攻击,每天一次,连续三天。第九周采血后处死实验小鼠,取一半胃粘膜和十二指肠研磨进行细菌培养检查H. pylori在胃肠定植情况,另一半用于组织切片和免疫组化观察胃肠损伤情况和免疫蛋白分泌的情况,同时对重要脏器肝、脾、肺进行组织切片观察损伤情况。实验前和实验后每隔一周或二周采血一次,连续8次,离心分离血清,采用ELISA检测小鼠血清中的抗体水平。检验pYA3493-UreB-CagA减毒猪霍乱沙门菌C500口服疫苗对小鼠的免疫效果和口服疫苗的安全性评价。结果：核苷酸序列测定及同源性分析证实,克隆的Hp-CagA和Hp-UreB基因与GenBank中相关序列的同源性分别为98%和100%(472／480和1134／1134)。重组质粒PYA3493-UreB-CagA转染减毒猪霍乱沙门菌C500,可表达约59kD的UreB-CagA蛋白。全菌蛋白注射组和含质粒pYA3493-UreB-CagA的减毒沙门氏菌C500组的血清中幽门螺杆菌特异抗体滴度显著高于含质粒pYA3493的减毒沙门氏菌C500组、减毒沙门氏菌C500组以及PBS组(P<0.01)。但均未完全保护月=pylori SS1对胃的攻击,五组中胃和十二指肠均有H. pylori SS1的定植。但全菌蛋白注射组和含质粒pYA3493-UreB-CagA的减毒沙门氏菌C500组小鼠胃肠内的菌落数量显著低于含质粒pYA3493的减毒沙门氏菌C500组、减毒沙门氏菌C500组以及PBS组(P<0.01)。免疫组化表明全菌注射组和口服疫苗中胃和十二指肠有明显的抗体存在,且所有组胃、十二指肠、肝、脾以及肺均无明显损伤,也无小鼠死亡。结果表明建立了同时表达Hp-CagA和Hp-UreB基因的减毒猪霍乱沙门菌疫苗株,该口服减毒沙门氏菌载体疫苗有明显的免疫预防效果,而且比较安全,但不能完全抑制幽门螺杆菌在胃和十二指肠中定植。以上是UreB-CagA双价口服活载体疫苗在小鼠体内的免疫效果,安全有效。体外实验希望通过构建H. pylori的CagA和UreB基因重组真核表达质粒用电击法转染到胃粘膜上皮细胞GES-1后,用幽门螺杆菌SSl攻击转染成功的胃上皮细胞((?)ES-1,观察对细胞凋亡及细胞形态的影响,探讨细胞凋亡机制。采用PCR技术从幽门螺杆菌标准株SS1中获取CagA部分基因和UreB全长基因与真核表达载体pEGFP-N1相连,构建幽门螺杆菌CagA/UreB真核荧光表达载体pEGFP-N1-CagA-UreB,并用脂质体的方法将质粒pEGFP-N1-UreB-CagA和空质粒pEGFP-N1分别转染到胃粘膜上皮细胞GES-1中,用(3418筛选阳性细胞,用幽门螺杆菌SSl分别处理转染pEGFP-N1-CagA-UreB的细胞、转染pEGFP-N1的细胞以及没转染质粒的细胞。用Annexin V-APC和7-AAD染色后,流式细胞术检测胃上皮细胞GES-1凋亡和死亡的情况,同时透射电镜观察胃上皮细胞GES-1的形态变化。结果发现用脂质体转染质粒到细胞GES-1,转染效率可高达60%以上,500μg G418可对转染细胞进行筛选,筛选后的阳性细胞用幽门螺杆菌处理后,凋亡率要小于空质粒转染的细胞和未转染质粒的细胞,但长时间处理后GES-1细胞均死亡,电镜结果显示细胞凋亡和坏死。结果表明质粒pEGFP-N1-UreB-CagA可转染到胃上皮细胞GES-1在培养细胞内的表达可抵抗幽门螺杆菌SSl对细胞GES-1的破坏,减少细胞凋亡。但用幽门螺杆菌长时间、大剂量处理也会导致细胞凋亡和坏死。这说明尿素酶B和CagA可导致细胞凋亡,但同时也有其他途径参与凋亡过程。更多还原

【Abstract】 Helicobacter pylori is a gram-negative bacterium, specialized in the colonization of the stomach, and well known as the major gastro-duodenal pathogen of peptic ulcer disease, mucosa-associated lymphoid tissue lymphoma, and gastric cancer. In90%of H. pylori-infected patients treated with antibiotics and strong acid suppressor drug such as proton pump inhibitors, its eradication is successful. However, failure of H. pylori eradication treatment has recently increased due to the proliferation of antibiotic-resistant strains. Artificial passive immunization is one way to against H. pylori, and vaccination against H. pylori is therefore one of the most effective ways to control H. pylori infection and, indeed, administration of oral bacterial antigens can protect mice against H. pylori infection.To observe the effect of intra-muscular injection with H. pylori NCTC11637on the induction of immunity,14lactating cows were randomly divided into2groups:whole bacterial antigen of H. pylori (2×1010cfu) with Freund’s adjuvant and PBS with adjuvant. The immunizations were performed on days0,14and28, and milk and serum samples were taken by at intervals of7days and then centrifugalizated and stored at-20℃until use. Serum and milk samples were collected and the levels of anti-Hp IgG assayed by indirect enzyme-linked immunosorbent assay. The results of ELISA proved that specific IgG in serum and milk increased significantly(P≤0.05). But the levels of anti-Hp IgG in serum increaser quickly than that the milk. The temperature, feed and oestrus have no changing. But the whole bacterial antigen of H.pylori made more trouble, the cows had pain when intra-muscular injected, and had several times vaccination. The intra-muscular injection of whole bacterial antigen with adjuvant can induce systemic immune response, and may be a safe and effective immunization route. But the whole bacterial antigen of H.pylori made more trouble, expensive and trouble in used.So one other vaccine which been safe, effective and convenient should be constructed. Recombinant H. pylori vaccine comprising a single subunit antigen can only induce immune response with limited protection efficiency. Development of oral vaccine would be a new effective strategy for prevention of H. pylori infection. In this study, the protective effect of H. pylori multicomponent vaccine consisting of UreB and CagA subunit antigens was constructed and investigated in mice. To develop an attenuated salmonella choleraesuis vaccine strain C500expressing Helicobacter pylori CagA and UreB, and to investigate the effects of the two-valence vaccine consisting of Helicobacter pylori CagA and UreB in preventing H. pylori infection in mice and the safety of vaccine. The Hp-UreB and Hp-CagA gene were amplified by PCR respectively and were cloned into prokaryotic expression plasmid pYA3493to construct recombinant plasmid pYA3493-UreB-CagA. The pYA3493-UreB-CagA was performed successively by PCR, enzyme digestion analysis and sequencing. The homology was compared between the cloned Hp-CagA and Hp-UreB gene and related genes in GenBank. The plasmid pYA3493-UreB-CagA was purified and transformed into Salmonella choleraesuis C500strain by electroporation. After be cultivated, positive colonies were picked out and the plasmid was amplified by PCR and enzyme digestion analysis again. The expression of pYA3493-UreB-CagA protein in the C500bacteria was proved by SDS-PAGE and Western Blotting.50Kunming mice were randomly divided into5groups:inoculated orally0.2mL (1×1010CFUmL-1) of C500expressing PYA3493-UreB-CagA, C500with pYA3493, C500, PBS and injection0.2mL (2×109) of H, pylori SS1with adjuvant. Oral doses of1×1010CFUmL-1were administered via an intragastric gavage. Immunizations were performed on days0,7,14,21and28, and serum samples were taken by at intervals of7days or14days. Vaccinated mice were challenged with0.2mL (2×109) H. pylori SSI instilled into stomach one time every day for three days after vaccination in6th week.The immune response was assessed by mice immunity IgG ELISA, Immunohistochemistry and Helicobacter pylori SSl attacking. The safety of vaccine was assessed by the hematoxylin-eosin staining.The results show the equencing and homologous analysis showed that the homology between the cloned Hp-CagA and Hp-UreB gene and related genes in GenBank reached98%(472/480) and100%(1134/1134) respectively for their nucleotide and deduced amino acid sequences. The expression of UreB-CagA protein could be detected in the culture fluid of C500bacteria. Noticeable IgG response was induced in the sera of mice orally immunized with the Salmonella choleraesuis C500strain consisting of UreB and CagA subunit antigens. Mice vaccinated orally were significantly protected against gastric helicobacter infection following a challenge with Helicobacter pylori strain SSI and the liver, lung, spleen, duodenum and pylorus had no lesions. The immunohistochemical analysis showed that the anti-UreB antibody was positively expressed in both the duodenum and pylorus in immunized orally with C500that expresses the UreB-CagA protein. On the contrary, there was no positive reaction in control groups.The results showed that the attenuated salmonella choleraesuis strain C500has been established successfully, which lays the foundation for further developing oral H. pylori vaccine. The orally vaccination with expressing UreB-CagA could prevent gastric infection with helicobacter pylori and safe. But didn’t against the helicobacter pylori infection completely.Experiment in vivo showed the orally vaccination with expressing UreB-CagA could prevent gastric infection with helicobacter pylori and safe. But effective in vitro has not been reported.To investigate the effect of helicobacter pylori on apoptosis of the GES-l transfected pEGFP-N1-UreB-CagA eukaryotic expression plasmid in vitro, the Hp-UreB and Hp-CagA gene were amplified by PCR respectively and were cloned into eukaryotic expression plasmid pEGFP-NI to construct recombinant plasmid pEGFP-NI-UreB-CagA and transfected into GES-1cells and choiced by G418. GES-1cells transfected into plasmid pEGFP-NI-UreB-CagA were cocultured with H. pylori strain SSI and its apoptosis was quantified by flow cytometry using Annexin V-APC and AAD. The recombinant PEGFP-N1-UreB-CagA was constructed and stable transfected into GES-1cells. The apoptotic rates of cells transfected into pEGFP-N1-UreB-CagA plasmids were significantly lower than those of control cells (P<0.01). The GES-1cells were died when H. pylori strain SS1cocultured with cells for a long time.The results showed that the GES-1cells which transfected into pEGFP-Nl-UreB-CagA plasmids can block apoptisis of GES-1cells. But helicobacter pylori also caused cell necrosis for a long time and high dose.更多还原