幽门螺杆菌基因hp0410的克隆表达及HP0410的免疫原性研究
发布时间：2018-09-04 17:21
【摘要】： 一、研究背景和目的 长期以来，医学界普遍认为，精神压力和生活方式等因素是慢性胃炎和消化性溃疡发病的主要原因。1983年，澳大利亚科学家J Robin Warren和Barry J Marshall首次分离培养出幽门螺杆菌(Helicobacter pylori，Hp)，并证实Hp感染可以引起胃炎和消化性溃疡，为此荣膺2005年诺贝尔生理学与医学奖。幽门螺杆菌是世界范围内感染率最高的致病菌，也是慢性胃炎和消化性溃疡主要病原菌，并与胃腺癌和胃黏膜相关B细胞淋巴瘤的发生密切相关。怎样有效控制Hp感染，一直是国内外学者所关注的焦点。 目前，国内外常用三联药物(如铋剂+甲硝唑+抗生素)治疗胃炎和消化性溃疡。但是，Hp容易产生耐药性，导致抗生素治疗失效。如果Hp未能根除，多达75％-80％的患者很快又旧病复发。鉴于抗生素疗法的弊端，疫苗接种被认为是控制Hp感染最有效的方法。 目前，Hp疫苗研究的热点主要集中在尿素酶(Ure)，空泡毒素(VacA)和细胞相关毒素(CagA)，热休克蛋白60(HSP60)等亚单位保护性抗原上。尿素酶难以诱导出全面而稳定的保护性免疫应答；CagA和VacA变异较大，且CagA是否为Hp感染所必须还存在有一定争议；HSP60和人体自身蛋白有比较高的同源性，用HSP60免疫小鼠只能诱导局部保护性免疫应答，并会导致小鼠肠胃炎，其作为疫苗的安全性和有效性尚需进一步验证。在幽门螺杆菌的体内清除过程中，黏膜免疫发挥主要作用。因此，筛选出合适的候选疫苗，诱导抗Hp的保护性黏膜免疫，仍是Hp疫苗研究的主要方向之一。 黏附素在Hp感染定植过程中不可缺少的，且定位于Hp表面，具有良好的抗原性。特异性的抗黏附素抗体能有效的抑制Hp定植并将其清除。因此，黏附素已成为Hp疫苗候选抗原研究的主要方向之一。幽门螺杆菌黏附素研究热点主要集中在两类主要黏附素上：①可与人细胞表面的Le~b抗原结合的血型抗原结合黏附素(blood group antigen-binding adhesin, BAb)；②可与人的红细胞紧密结合的N-乙酰神经氨酰乳糖结合原纤维血凝素(N-acetylneuraminyl-lactose-binding haemagglutinin，NLBH)。NLBH长约1.4kb，分为3个阅读框(ORF)，其中ORF2亦即hpaA基因，长783bp，，编码幽门螺杆菌黏附素A(Helicobacter pylori adhesin A，HpaA)为Hp黏附定植过程所必须，hpaA突变的Hp菌株无法在小鼠体内定植。hp0410是hpaA基因家族同系成员，长为750bp，很可能为Hp黏附因子的一种，具体功能尚不明确。hp0231是对Hp基因组阅读框分析所预测出的基因，长为789bp，表达产物可能为分泌型的蛋白。目前，有关HP0410和HP0231的免疫原性以及其作为候选疫苗的前景尚未阐明。 本研究的主要目的是：①通过生物信息学分析，预测hp0231和hp0410编码蛋白的性质特征和免疫原性；②在大肠杆菌原核表达系统中克隆表达hp0410，并纯化获得重组蛋白；③用重组蛋白筛查胃溃疡患者和健康人血清，检测HP0410能否在体内诱导出特异性抗体，并进行免疫组化实验，探讨该蛋白是否可作为一种新的检测Hp感染的方法；④建立噬菌体肽展示文库，对HP0410的抗原表位进行研究；⑤建立细胞模型，初步探讨HP0410与胃上皮细胞的相互作用。通过以上研究，为构建多嵌合表位重组疫苗，或以嗜酸乳杆菌为载体的活疫苗的研制提供理论依据和工作基础。 二、研究方法 1、提取幽门螺杆菌标准株NCTC11639的基因组DNA，按照GenBank中幽门螺杆菌标准株22695的序列设计引物PCR，扩增hp0231和hp0410基因，克隆入pMD18-T载体中测序，进行生物信息学分析。 2、hp0231和hp0410克隆入表达载体pGEX-4T-1中，诱导表达，SDS-PAGE电泳鉴定。大量表达HP0410，GST柱纯化重组蛋白。 3、利用纯化HP0410从29株小鼠抗幽门螺杆菌单抗中筛选出抗HP0410单抗，特异性—敏感性实验及免疫组化鉴定，进行酶联免疫吸附(ELISA)实验，筛查85份胃溃疡患者血清和90份健康人血清，分析ELISA结果。 4、筛选出抗HP0410单抗E018，利用该单抗和M13噬菌体12肽随机肽库，构建HP0410抗原表位的抗原展示库，阳性噬菌体测序，分析HP0410的抗原表位。 5、建立HP0410和细胞相互作用的模型，通过测定不同浓度、不同时间HP0410作用下SGC7901细胞IL-8和GRO-a分泌量的变化，以及细胞ELISA探讨HP0410和SGC7901细胞的相互作用。 三、研究结果 1、生物信息学分析表明，HP0231和HP0410均为外膜蛋白，有跨膜结构域和信号肽序列，具有良好的抗原性。hp0231和hp0410在Hp中普遍存在，BLAST分析表明这2个蛋白有很高的种属特异性，为Hp所特有。 2、构建重组菌株，高水平表达可溶性重组蛋白HP0410。利用纯化的重组HP0410蛋白筛查29株单抗，获得高特异性单抗一株(E018)。 3、HP0410筛查85份胃溃疡患者血清，阳性94.1％，结果表明抗HP0410抗体在患者血清中普遍存在；筛查90份正常人血清，阳性74.4％，阳性检出率与我国Hp感染率接近，表明Hp携带者体内也存在抗HP0410抗体；免疫组化实验表明利用抗HP0410单抗检测HP0410，可作为一个有效的检测Hp感染的方法。 4、分析噬菌体测序结果获得HP0410的模拟空间表位，其中1个为模拟HP0410与E018特异性结合的抗原决定簇的候选表位。展示的抗原表位在HP0410上的分布符合生物信息学预测结果，且多处于的β转角结构中，这些表位的HP0410同源区与预测的HLAⅡ类分子结合结构域一致。 5、HP0410不能显著性的上调胃腺癌细胞SGC7901表达IL-8，也不能刺激细胞产生GRO-a，提示HP0410并非HP的炎性因子，符合其主要功能为介导黏附的预测；细胞ELISA结果呈阴性，提示E018针对的抗原决定簇即为HP0410的黏附结构域亦可能HP0410不能直接与SGC7901细胞结合。 四、结论 1、生物信息学分析表明HP0410有良好的抗原性，可以诱导出特异性的体液免疫应答反应，提示HP0410可作为Hp的候选疫苗。 2、免疫组化实验表明检测HP0410可成为检测Hp感染的新手段。 3、噬菌体肽库展示的抗原表位与HP0410同源性不高，而与生物信息学预测的位置相符，提示获得的表位为HP0410高抗原性位点的模拟空间表位，并且可被HLAⅡ类分子识别并递呈，从而激发保护性免疫应答。竞争-抑制实验表明候选表位为E018所识别的抗原决定簇的模拟表位。 4、细胞实验提示HP0410没有明显的致炎作用，利用其构建的嵌合疫苗安全性较高。
[Abstract]:I. background and purpose
Medical professionals have long believed that stress and lifestyle are the main causes of chronic gastritis and peptic ulcer. In 1983, Australian scientists J. Robin Warren and Barry J. Marshall first isolated and cultured Helicobacter pylori (Hp), and confirmed that Hp infection can cause gastritis and peptic ulcer. Helicobacter pylori is the most common pathogen of chronic gastritis and peptic ulcer in the world. It is closely related to the occurrence of gastric adenocarcinoma and gastric mucosa-associated B-cell lymphoma. The focus.
At present, triple drugs (such as bismuth + metronidazole + antibiotics) are commonly used in the treatment of gastritis and peptic ulcer at home and abroad. Effective methods.
At present, the research hotspots of HP vaccine mainly focus on the protective antigens of urease, vacuole toxin (VacA), cell-related toxin (CagA), heat shock protein 60 (HSP60), etc. Urease is difficult to induce a comprehensive and stable protective immune response; CagA and VacA have great variation, and whether CagA is necessary for HP infection still exists. It is controversial that HSP60 has a high homology with human autoproteins. Mice immunized with HSP60 can only induce local protective immune responses and cause gastroenteritis in mice. The safety and efficacy of HSP60 as a vaccine need to be further verified. Mucosal immunity plays a major role in the process of clearance of Helicobacter pylori in vivo. It is still one of the main directions in the study of Hp vaccine to find suitable candidate vaccines and induce protective mucosal immunity against Hp.
Adhesin is indispensable in the process of H.pylori infection and is localized on the surface of H.pylori and has good antigenicity.Specific anti-adhesin antibodies can effectively inhibit and eliminate H.pylori colonization.Adhesin has become one of the main research directions of candidate antigens of H.pylori vaccine. Major adhesins included: 1) blood group antigen-binding adhesin (BAb), 2) N-acetylneuraminyl-lactose-binding haemagglutinin (NLBH), which binds tightly to human erythrocytes. B, divided into three reading frames (ORFs), of which ORF2, also known as hpaA gene, is 783 B P long and encodes Helicobacter pylori adhesin A (HpaA), which is necessary for the process of Hp adhesion and colonization. HpaA mutant HP strains can not be colonized in mice. Hp0410 is a member of the hpaA gene family, 750 B P long, and may be one of the Hp adhesion factors. Hp0231 is a 789 BP long gene predicted by reading frame analysis of the Hp genome. The expressed product may be a secretory protein. At present, the immunogenicity of HP0410 and HP0231 and their prospects as candidate vaccines have not been clarified.
The main objectives of this study were: (1) to predict the properties and immunogenicity of the proteins encoded by hp0231 and hp0410 by bioinformatics analysis; (2) to clone and express hp0410 in E. coli prokaryotic expression system and purify the recombinant protein; and (3) to screen the serum of patients with gastric ulcer and healthy people with recombinant protein and detect whether HP0410 can be detected in vivo Specific antibodies were induced and immunohistochemical assays were carried out to explore whether the protein could be used as a new method for detection of Hp infection; 4. phage peptide display library was established to study the antigen epitope of HP0410; 6. cell model was established to explore the interaction between HP0410 and gastric epithelial cells. The recombinant vaccine with multiple chimeric epitopes or the live vaccine with Lactobacillus acidophilus as the carrier will provide theoretical basis and work basis.
Two, research methods.
1. The genomic DNA of standard strain NCTC11639 of Helicobacter pylori was extracted, and primers PCR was designed according to the sequence of standard strain 22695 of Helicobacter pylori in GenBank. The genes of hp0231 and hp0410 were amplified and cloned into pMD18-T vector for sequencing and bioinformatics analysis.
2, hp0231 and hp0410 were cloned into expression vector pGEX-4T-1, induced expression and identified by SDS-PAGE electrophoresis. HP0410 was expressed in large quantities and purified by GST column.
3. Anti-HP0410 monoclonal antibodies were screened from 29 strains of mouse anti-Helicobacter pylori monoclonal antibodies by purified HP0410. The specificity-sensitivity test and immunohistochemical identification were carried out. Enzyme-linked immunosorbent assay (ELISA) was used to screen 85 sera from patients with gastric ulcer and 90 sera from healthy volunteers.
4. The anti-HP0410 monoclonal antibody E018 was screened out. Using this monoclonal antibody and M13 phage 12 peptide random peptide library, the antigen display library of HP0410 epitope was constructed. The positive phage was sequenced and the antigen epitope of HP0410 was analyzed.
5. The interaction model between HP0410 and SGC7901 cells was established. The changes of IL-8 and GRO-a secretion in SGC7901 cells under different concentrations and different time of HP0410 were measured. The interaction between HP0410 and SGC7901 cells was investigated by cell ELISA.
Three, research findings
1. Bioinformatics analysis showed that HP0231 and HP0410 were both outer membrane proteins with transmembrane domain and signal peptide sequence, and had good antigenicity.
2. The recombinant strain was constructed and the soluble recombinant protein HP0410 was highly expressed. 29 monoclonal antibodies were screened by purified recombinant HP0410 protein, and a high specific monoclonal antibody (E018) was obtained.
3, HP0410 screened 85 sera of gastric ulcer patients, positive 94.1%, the results showed that anti-HP0410 antibody was prevalent in the sera of patients; screening 90 normal sera, positive 74.4%, positive detection rate was close to the infection rate of Hp in China, indicating that the carrier of Hp also had anti-HP0410 antibody in vivo; immunohistochemical test showed that the use of anti-HP0410 monoclonal antibody detection. HP0410 can be used as an effective method to detect Hp infection.
4. By analyzing the results of phage sequencing, the simulated spatial epitopes of HP0410 were obtained, one of which was a candidate epitope for the antigenic determinant that mimicked the specific binding of HP0410 to E018. The distribution of the displayed epitopes on HP0410 was in accordance with the bioinformatics prediction, and most of them were in the beta-corner structure. The homologous regions of these epitopes were in accordance with the predicted HLA. Class II molecules are consistent with domains.
5. HP0410 could not significantly up-regulate the expression of IL-8 and stimulate the production of GRO-a in gastric adenocarcinoma cell line SGC7901, suggesting that HP0410 was not an inflammatory factor of HP, which accorded with the prediction of HP-mediated adhesion; the results of cell ELISA were negative, suggesting that the epitope of HP0410 targeted by E018 might not be a direct binding domain of HP0410. And then combined with SGC7901 cells.
Four. Conclusion
1. Bioinformatics analysis showed that HP0410 had good antigenicity and could induce specific humoral immune response, suggesting that HP0410 could be used as a candidate vaccine for Hp.
2, immunohistochemistry showed that detection of HP0410 could be a new way to detect Hp infection.
3. The antigenic epitope displayed by phage peptide library was not homologous to HP0410, but was consistent with the predicted position of bioinformatics. The obtained epitope was a simulated spatial epitope of HP0410 high antigenic site and could be recognized and presented by HLA class II molecule, thus stimulating protective immune response. Competition-inhibition test showed that the candidate epitope was E018. The mimic epitopes of the identified epitopes are identified.
4, cell experiments suggest that HP0410 has no obvious inflammatory effect, and the chimeric vaccine constructed by using it has high safety.
【学位授予单位】：南方医科大学
【学位级别】：硕士
【学位授予年份】：2007
【分类号】：R378
本文编号：2222837
[Abstract]:I. background and purpose
Medical professionals have long believed that stress and lifestyle are the main causes of chronic gastritis and peptic ulcer. In 1983, Australian scientists J. Robin Warren and Barry J. Marshall first isolated and cultured Helicobacter pylori (Hp), and confirmed that Hp infection can cause gastritis and peptic ulcer. Helicobacter pylori is the most common pathogen of chronic gastritis and peptic ulcer in the world. It is closely related to the occurrence of gastric adenocarcinoma and gastric mucosa-associated B-cell lymphoma. The focus.
At present, triple drugs (such as bismuth + metronidazole + antibiotics) are commonly used in the treatment of gastritis and peptic ulcer at home and abroad. Effective methods.
At present, the research hotspots of HP vaccine mainly focus on the protective antigens of urease, vacuole toxin (VacA), cell-related toxin (CagA), heat shock protein 60 (HSP60), etc. Urease is difficult to induce a comprehensive and stable protective immune response; CagA and VacA have great variation, and whether CagA is necessary for HP infection still exists. It is controversial that HSP60 has a high homology with human autoproteins. Mice immunized with HSP60 can only induce local protective immune responses and cause gastroenteritis in mice. The safety and efficacy of HSP60 as a vaccine need to be further verified. Mucosal immunity plays a major role in the process of clearance of Helicobacter pylori in vivo. It is still one of the main directions in the study of Hp vaccine to find suitable candidate vaccines and induce protective mucosal immunity against Hp.
Adhesin is indispensable in the process of H.pylori infection and is localized on the surface of H.pylori and has good antigenicity.Specific anti-adhesin antibodies can effectively inhibit and eliminate H.pylori colonization.Adhesin has become one of the main research directions of candidate antigens of H.pylori vaccine. Major adhesins included: 1) blood group antigen-binding adhesin (BAb), 2) N-acetylneuraminyl-lactose-binding haemagglutinin (NLBH), which binds tightly to human erythrocytes. B, divided into three reading frames (ORFs), of which ORF2, also known as hpaA gene, is 783 B P long and encodes Helicobacter pylori adhesin A (HpaA), which is necessary for the process of Hp adhesion and colonization. HpaA mutant HP strains can not be colonized in mice. Hp0410 is a member of the hpaA gene family, 750 B P long, and may be one of the Hp adhesion factors. Hp0231 is a 789 BP long gene predicted by reading frame analysis of the Hp genome. The expressed product may be a secretory protein. At present, the immunogenicity of HP0410 and HP0231 and their prospects as candidate vaccines have not been clarified.
The main objectives of this study were: (1) to predict the properties and immunogenicity of the proteins encoded by hp0231 and hp0410 by bioinformatics analysis; (2) to clone and express hp0410 in E. coli prokaryotic expression system and purify the recombinant protein; and (3) to screen the serum of patients with gastric ulcer and healthy people with recombinant protein and detect whether HP0410 can be detected in vivo Specific antibodies were induced and immunohistochemical assays were carried out to explore whether the protein could be used as a new method for detection of Hp infection; 4. phage peptide display library was established to study the antigen epitope of HP0410; 6. cell model was established to explore the interaction between HP0410 and gastric epithelial cells. The recombinant vaccine with multiple chimeric epitopes or the live vaccine with Lactobacillus acidophilus as the carrier will provide theoretical basis and work basis.
Two, research methods.
1. The genomic DNA of standard strain NCTC11639 of Helicobacter pylori was extracted, and primers PCR was designed according to the sequence of standard strain 22695 of Helicobacter pylori in GenBank. The genes of hp0231 and hp0410 were amplified and cloned into pMD18-T vector for sequencing and bioinformatics analysis.
2, hp0231 and hp0410 were cloned into expression vector pGEX-4T-1, induced expression and identified by SDS-PAGE electrophoresis. HP0410 was expressed in large quantities and purified by GST column.
3. Anti-HP0410 monoclonal antibodies were screened from 29 strains of mouse anti-Helicobacter pylori monoclonal antibodies by purified HP0410. The specificity-sensitivity test and immunohistochemical identification were carried out. Enzyme-linked immunosorbent assay (ELISA) was used to screen 85 sera from patients with gastric ulcer and 90 sera from healthy volunteers.
4. The anti-HP0410 monoclonal antibody E018 was screened out. Using this monoclonal antibody and M13 phage 12 peptide random peptide library, the antigen display library of HP0410 epitope was constructed. The positive phage was sequenced and the antigen epitope of HP0410 was analyzed.
5. The interaction model between HP0410 and SGC7901 cells was established. The changes of IL-8 and GRO-a secretion in SGC7901 cells under different concentrations and different time of HP0410 were measured. The interaction between HP0410 and SGC7901 cells was investigated by cell ELISA.
Three, research findings
1. Bioinformatics analysis showed that HP0231 and HP0410 were both outer membrane proteins with transmembrane domain and signal peptide sequence, and had good antigenicity.
2. The recombinant strain was constructed and the soluble recombinant protein HP0410 was highly expressed. 29 monoclonal antibodies were screened by purified recombinant HP0410 protein, and a high specific monoclonal antibody (E018) was obtained.
3, HP0410 screened 85 sera of gastric ulcer patients, positive 94.1%, the results showed that anti-HP0410 antibody was prevalent in the sera of patients; screening 90 normal sera, positive 74.4%, positive detection rate was close to the infection rate of Hp in China, indicating that the carrier of Hp also had anti-HP0410 antibody in vivo; immunohistochemical test showed that the use of anti-HP0410 monoclonal antibody detection. HP0410 can be used as an effective method to detect Hp infection.
4. By analyzing the results of phage sequencing, the simulated spatial epitopes of HP0410 were obtained, one of which was a candidate epitope for the antigenic determinant that mimicked the specific binding of HP0410 to E018. The distribution of the displayed epitopes on HP0410 was in accordance with the bioinformatics prediction, and most of them were in the beta-corner structure. The homologous regions of these epitopes were in accordance with the predicted HLA. Class II molecules are consistent with domains.
5. HP0410 could not significantly up-regulate the expression of IL-8 and stimulate the production of GRO-a in gastric adenocarcinoma cell line SGC7901, suggesting that HP0410 was not an inflammatory factor of HP, which accorded with the prediction of HP-mediated adhesion; the results of cell ELISA were negative, suggesting that the epitope of HP0410 targeted by E018 might not be a direct binding domain of HP0410. And then combined with SGC7901 cells.
Four. Conclusion
1. Bioinformatics analysis showed that HP0410 had good antigenicity and could induce specific humoral immune response, suggesting that HP0410 could be used as a candidate vaccine for Hp.
2, immunohistochemistry showed that detection of HP0410 could be a new way to detect Hp infection.
3. The antigenic epitope displayed by phage peptide library was not homologous to HP0410, but was consistent with the predicted position of bioinformatics. The obtained epitope was a simulated spatial epitope of HP0410 high antigenic site and could be recognized and presented by HLA class II molecule, thus stimulating protective immune response. Competition-inhibition test showed that the candidate epitope was E018. The mimic epitopes of the identified epitopes are identified.
4, cell experiments suggest that HP0410 has no obvious inflammatory effect, and the chimeric vaccine constructed by using it has high safety.
【学位授予单位】：南方医科大学
【学位级别】：硕士
【学位授予年份】：2007
【分类号】：R378
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