幽门螺杆菌napA基因的克

发布时间：2018-06-29 01:23

本文选题：幽门螺杆菌 + NAP　；参考：《第一军医大学》2007年硕士论文

【摘要】： 一、研究背景和目的幽门螺杆菌(Helicobacter pylori，Hp)是一种微需氧革兰阴性螺旋杆菌，可导致胃炎和消化道溃疡，并与胃腺癌、胃黏膜相关性淋巴样组织(gastric mucosal-associated lymphoid tissue lymphoma，MALT)淋巴瘤病的发生密切相关，全球人口感染率超过50％，1994年被国际癌症研究中心定为Ⅰ类致癌物。目前，临床上多采用质子泵抑制剂和抗生素联合治疗，但存在耐药性、病人依从性差、易复发等副问题，效果不甚理想。因此，疫苗是防治Hp感染的有效手段，筛选免疫保护力强的无毒抗原是研制疫苗的关键。研究较多的抗原主要有尿素酶B(Urease B)、空泡毒素(Vacuolatint cytotoxin A，VacA)、细胞毒素相关蛋白(Cytotoxin associated protein A，CagA)等，在动物实验中虽显示一定的免疫保护作用，但都具有一定的局限性，迄今未有理想的疫苗问世。中性粒细胞激活蛋白(Neutrophil-activating protein，NAP)是近期发现的与黏附和炎症相关的重要毒力因子，由napA基因编码，分子量为15kDa，4股螺旋结构的单体构成十二聚体铁蛋白，NAP与细菌DNA保护性蛋白(Dps)及铁蛋白(Flp)具有高度同源性，存在于所有菌株中。NAP能持续趋化和激活嗜中性粒细胞，促进中性粒细胞对胃上皮细胞的黏附，刺激胃黏膜上皮细胞快速反应，通过上调VCAM-1、ICAM-1、E选择素和前炎症细胞因子及趋化因子促进白细胞的募集，并促使中性粒细胞释放反应性氧代谢物，引发胃黏膜炎症病变。多数Hp感染者血清中存在抗NAP抗体，动物实验证实NAP免疫小鼠可抵抗Hp的感染，保护率达80％。可见，NAP是Hp重要的毒力因子及候选抗原。本研究在克隆表达NAP的基础上，展开NAP的表位组学研究，利用噬菌体肽库展示技术，寻找NAP精确的抗原表位，为筛选更多的保护性抗原表位及制备多价合成肽亚单位疫苗奠定基础；展开胃癌、消化道溃疡、胃炎病人与正常人群抗NAP抗体水平的调查，探讨NAP与胃癌之间的相关性，并对获得的3株抗NAP单克隆抗体(monoclonal antibodies，mAb)进行鉴定，探讨其临床应用价值。二、研究方法 1、根据GenBank提供的Hp 26695株基因组序列设计引物，以NCTC 11639基因组为模板，扩增napA基因，连接pMD18-T载体，构建napA／pMD18-T重组克隆，PCR及双酶切鉴定后测序，将重组质粒napA／pMD18-T与表达载体pGEX-4T-1用EcoRⅠ和XholⅠ双酶切后连接，转化宿主菌Top10，构建融合表达载体napA／pGEX-4T-1，双酶切后测序。对测序结果进行生物信息学分析：利用BLAST工具进行同源性搜索；利用DNAMAN软件分析napA的DNA序列和编码蛋白的特性；利用harvard大学的在线软件进行抗原表位预测；应用Signal P3.0 server分析氨基酸序列中是否存在信号肽。 2、利用IPTG诱导表达目的蛋白，优化表达条件，实现融合蛋白的可溶性表达，通过GST亲和层析柱纯化NAP蛋白，SDS-PAGE电泳及Western Blot鉴定表达蛋白。 3、利用间接ELISA法，以纯化蛋白NAP与Hp阳性胃癌、消化道溃疡、胃炎患者血清及正常人群血清进行反应，检测NAP的免疫反应性，比较NAP在胃病人群及正常人群的抗体水平，探讨NAP与胃癌的相关性。 4、以天然的Hp全菌蛋白为抗原免疫BALB／C小鼠，利用杂交瘤技术制备mAb。利用间接ELASA法，鉴定mAb与肠道致病菌交叉免疫反应，以NAP蛋白为抗原，从不与肠道致病菌发生交叉免疫反应的mAb中筛选抗NAP mAb。利用鼠mAb亚类检测试剂盒，检测阳性杂交瘤细胞培养上清，鉴定抗NAP mAb的亚类；采用间接ELISA法测定抗NAP mAb抗体效价；选用间接ELISA方法，测定抗NAP mAb浓度，按公式Kaff=(n-1)／2(n[Ab']t-[Ab]t)计算亲和常数。将抗NAP mAb与10株常见肠道致病菌进行免疫组化试验，鉴定抗体反应的特异性；将NAP mAb与Hp涂片样本及Hp感染患者胃黏膜标本进行免疫组化试验，鉴定mAb反应的敏感性。 5、利用噬菌体随机7肽库，以抗NAP mAb(E019)为固化抗原，通过ELASA法筛选阳性噬菌体克隆，竞争结合抑制试验鉴定NAP的抗原表位。三、研究结果 1、成功扩增及克隆NCTC11639株napA基因，基因全长435bp。结果已登录GenBank，登录号为DQ341279。 2、BLAST同源检索显示该基因与GenBank中公布的其它20株菌株的napA基因的核酸序列相比有高度同源性(94％～98％)；napA基因编码蛋白与细菌DNA保护性蛋白(Dps)及铁蛋白具有高度同源性，与人及哺乳动物基因组DNA同源性很低(＜10％)。 3、经二级结构、亲水性、抗原性指数、信号肽、三维结构分析显示有4个高抗原性肽段，分别是第4～24，55～77，95～103，118～140氨基酸序列，平均抗原指数为1.0236，其中118～140长22个氨基酸残基的肽段抗原性指数1.15，亲水性强、含有1个β转角，表明该肽段可能含有良好的抗原表位。 4、PCR证实napA在Hp菌株中普遍存在，与国内外文献报道相同，说明该基因高度保守。 5、构建napA-pGEX-4T-1-E. coli Top 10高效可溶性原核表达系统，通过优化表达条件，确定最佳表达条件为1mmol／L IPTG诱导浓度，诱导温度30℃，诱导时间4 h。 6、表达产物经GST亲和层析纯化，获得纯度较高且有生物活性的目的蛋白，分子量是44Kda。重组NAP与患者血清及购买的商品化兔抗全菌Hp抗体之间可发生特异性免疫反应，不与正常人血清反应，说明重组NAP具有良好的免疫反应性。 7、Hp IgG抗体试剂盒检测结果表明：64％正常人群Hp感染阳性，均值为0.46±0.27；70％胃癌患者Hp感染阳性，均值为0.55±0.13；68％胃溃疡患者Hp感染阳性均值为0.43±0.21；64％胃炎患者Hp感染阳性，均值为0.68±0.13。 8、NAP抗体水平检测结果表明：97.5％胃癌患者血清中NAP抗体检测结果阳性，均值为1.01±0.13；92.9％胃溃疡患者血清中NAP抗体检测结果阳性，均值为0.98±0.17；85.7％胃炎患者血清中NAP抗体检测结果阳性，，均值为0.89±0.07；正常人群血清中60％NAP抗体检测结果阳性，均值为0.61±0.14。20～29岁与40～49岁年龄段抗体水平有明显差异(~*P＜0.05)，其它年龄段NAP抗体水平无明显差异。 9、从29株小鼠抗Hp全菌mAb中筛选获得3株抗NAP的mAb，mAb经亚类鉴定显示全部为IgG1，抗体效价为1：16至1：32，抗体亲和力为1×10~(-10)至5.2×10~(-12)mol／L。Western blot鉴定表明，NAP蛋白可与NAP mAb之间发生免疫学反应。免疫组化结果表明，3株mAb特异性较高，敏感性强，能与Hp纯培养物及Hp感染胃黏膜标本发生强阳性反应，不与其他肠道菌发生反应。 10、利用噬菌体肽库技术，初步筛选到NAP的线性表位(FAHLATQ)。四、结论 1、本研究首次从国际标准株NCTC 11639基因组DNA中克隆中性粒细胞激活蛋白基因napA，Genbank登录号DQ341279。 2、成功构建napA-pGEX-4T-1-E. coli Top 10高效可溶性原核表达系统。重组NAP经鉴定具有良好的免疫原性和免疫反应性，可作为Hp基因工程亚单位疫苗的候选组分，并为研究Hp致病机制、免疫保护机制及诊断试剂奠定基础。 3、NAP是高度保守的基因，是良好的免疫原。初步预测NAP有4个高抗原性肽段，其中118～140肽段可能含有良好的抗原表位。 4、成功制备高亲和力抗NAP全菌mAb，3株mAb(E006、E019、E023)特异性高，敏感性强，具有良好的应用前景。 5、Hp在广东地区的胃病及正常人群中感染率较高；NAP抗体表达水平高显示NAP与胃癌有一定关联，NAP是胃癌发生高风险因子；患者年龄与NAP抗体水平无明显相关性，此项研究为NAP病原学研究提供了理论基础，为Hp感染相关胃癌的诊断、治疗和疫苗研究打下基石。 6、首次筛选到NAP的线性表位(FAHLATQ)，位于预测肽段(118～140)内，为下一步开展多价合成肽疫苗及蛋白质组学研究奠定良好基础。
[Abstract]:First, research background and purpose
Helicobacter pylori (Hp) is a micro aerobic gram negative spiral bacilli, which can lead to gastritis and peptic ulcers, and is closely related to gastric adenocarcinoma and gastric mucosa associated lymphoid tissue (gastric mucosal-associated lymphoid tissue lymphoma, MALT) lymphoma. The global population infection rate is over 50%, 1994. It is designated as type I carcinogen by the International Center for cancer research.
At present, the clinical use of proton pump inhibitors and antibiotics combined treatment, but the existence of drug resistance, patient compliance, easy to relapse and other problems, the effect is not ideal. Therefore, the vaccine is an effective means to prevent and control Hp infection, the screening of immunoprotective non toxic antigen is the key to the development of vaccines. The major antigen mainly includes the urease B (U Rease B), vacuolated toxin (Vacuolatint cytotoxin A, VacA), cytotoxin related protein (Cytotoxin associated protein A, CagA) and so on. Although it shows certain immune protection in animal experiments, it has some limitations, so far no ideal vaccine has been published.
Neutrophil-activating protein (NAP) is an important virulence factor associated with adhesion and inflammation. It is encoded by the napA gene, the molecular weight is 15kDa, the monomer of the 4 Strand spiral structure constitutes twelve polybody ferritin, and NAP is highly homologous to the bacterial DNA protective protein (Dps) and the ferritin (Flp). In all strains,.NAP can continue to chemotaxis and activate neutrophils, promote the adhesion of neutrophils to gastric epithelial cells, stimulate the rapid reaction of gastric epithelial cells, promote the recruitment of leukocytes by up regulation of VCAM-1, ICAM-1, E selectin and pro-inflammatory cytokines and chemokines, and promote the release of reactive oxygen generation by neutrophils. Most Hp infected people have anti NAP antibodies in the serum of most Hp infected people. Animal experiments confirm that NAP immune mice can resist Hp infection, the protection rate is 80%., and NAP is an important virulence factor and candidate antigen of Hp.
On the basis of cloning and expression of NAP, this study carried out the study of NAP's epitopes, using phage peptide library display technology to find the exact epitopes of NAP, laying the foundation for screening more protective epitopes and preparing polyvalent peptide subunit vaccines, developing gastric cancer, alcic ulcer, and anti NAP antibody in gastritis patients and normal people. The correlation between NAP and gastric cancer was investigated by a level survey, and 3 anti NAP monoclonal antibodies (monoclonal antibodies, mAb) were identified and their clinical value was discussed.
Two, research methods
1, according to the Hp 26695 genome sequence provided by GenBank, the primers were designed, the napA gene was amplified by the NCTC 11639 genome, the pMD18-T vector was connected, the recombinant clone of napA / pMD18-T was constructed, PCR and double enzyme digestion were sequenced, and the recombinant plasmid napA / pMD18-T was connected with the EcoR I and the double enzyme. Top10, a fusion expression vector napA / pGEX-4T-1 was constructed and sequenced after double enzyme digestion. Bioinformatics analysis of the sequencing results: using BLAST tool for homologous search; using DNAMAN software to analyze the characteristics of DNA sequence and encoded protein of napA; use Harvard University's line software to predict antigen epitopes; use Signal. P3.0 server analyzed whether there was a signal peptide in the amino acid sequence.
2, the expression of the target protein was induced by IPTG, the expression conditions were optimized, the soluble expression of the fusion protein was realized, the NAP protein was purified by GST affinity chromatography column, and the expression protein was identified by SDS-PAGE electrophoresis and Western Blot.
3, the indirect ELISA method was used to purify protein NAP and Hp positive gastric cancer, alimentary tract ulcer, serum of gastritis patients and normal population serum, to detect the immunoreactivity of NAP, compare the antibody level of NAP in the patients with gastric disease and normal population, and to explore the correlation between NAP and gastric cancer.
4, the BALB / C mice were immunized with the natural Hp whole bacteria protein as antigen, and the indirect ELASA method was used to prepare mAb. by hybridoma technology. The cross immunoreaction of mAb and intestinal pathogenic bacteria was identified, NAP protein was used as antigen, and the anti NAP mAb. using mAb subclass detection kit was screened and tested positive for anti NAP mAb. using mAb in mAb. Hybridoma cell culture supernatant, identification of anti NAP mAb subclass, indirect ELISA assay to determine the titer of anti NAP mAb antibody, indirect ELISA method, determination of anti NAP mAb concentration, and formula Kaff= (n-1) / 2 (n[Ab']t-[Ab]t) to calculate affinity constant. Specificity, NAP mAb and Hp smear samples and Hp infected patients gastric mucosal specimens were immunohistochemical test to identify the sensitivity of mAb reaction.
5, using the phage random 7 peptide library and the anti NAP mAb (E019) as the curing antigen, the positive phage clones were screened by the ELASA method, and the competitive binding inhibition test was used to identify the epitopes of NAP.
Three, the results of the study
1, the napA gene of NCTC11639 strain was successfully amplified and cloned, and the full-length 435bp. gene was registered in GenBank. The accession number was DQ341279..
2, BLAST homologous retrieval showed that the gene was highly homologous compared to the nucleotide sequences of the other 20 strains of napA published in GenBank, and the napA gene encoded protein was highly homologous to the bacterial DNA protective protein (Dps) and ferritin, and was very homologous to the human and mammalian genome DNA (< 10%).
3, through the two stage structure, hydrophilicity, antigenicity index, signal peptide and three-dimensional structural analysis, there are 4 high antigenic peptide segments, which are fourth ~ 24,55 ~ 77,95 to 103118~140 amino acid sequences, and the average antigen index is 1.0236. The peptide antigenicity index of 118~140 long 22 amino acid residues is 1.15, and the hydrophilic property is strong and contains 1 beta angles. The peptide segment may contain a good epitope.
4, PCR confirmed that napA is ubiquitous in Hp strains, which is similar to that reported both at home and abroad, indicating that the gene is highly conserved.
5, the napA-pGEX-4T-1-E. coli Top 10 high efficiency soluble prokaryotic expression system was constructed. By optimizing the expression conditions, the optimum expression condition was 1mmol / L IPTG induced concentration, the induction temperature was 30, and the induction time was 4 h..
6, the expression product was purified by GST affinity chromatography to obtain a purer protein with higher purity and bioactivity. The molecular weight of the recombinant NAP was a specific immune response between the recombinant NAP and the serum of the patient and the commercialized Rabbit anti whole Hp antibody, which did not react with the normal human serum, indicating that the recombinant NAP has a good immune response.
7, the detection results of Hp IgG antibody kit showed that 64% normal population Hp infection was positive, the mean value was 0.46 + 0.27, 70% gastric cancer patients were positive for Hp infection, the mean value was 0.55 + 0.13, the mean value of Hp infection in 68% gastric ulcer patients was 0.43 + 0.21, and the Hp infection was positive in the patients with gastric cancer, the mean was 0.68 + 0.13..
8, NAP antibody level detection results showed that 97.5% gastric cancer patients' serum NAP antibody test results were positive, the mean value was 1.01 + 0.13, and the serum NAP antibody test results of 92.9% gastric ulcer patients were 0.98 + 0.17, and the NAP antibody test results in the serum of 85.7% gastritis patients were 0.89 + 0.07, and the normal population serum was 60. The results of%NAP antibody test were positive. There was a significant difference between 0.61 0.14.20 to 29 years and 40~49 years old age antibody levels (~*P < 0.05). There was no significant difference in the level of NAP antibody in other age groups.
9, 3 strains of anti NAP mAb were screened from 29 mice resistant to Hp whole bacteria. MAb was identified as IgG1, and the antibody titer was from 1:16 to 1:32. The antibody affinity was 1 x 10~ (-10) to 5.2 * 10~ (-12) mol / L.Western. The immunological results showed that 3 strains were specific. It is highly sensitive and sensitive. It can react strongly with Hp pure culture and Hp infected gastric mucosa specimens, and does not react with other intestinal bacteria.
10, phage peptide library technology was used to preliminarily screen the linear epitope (FAHLATQ) of NAP.
Four. Conclusion
1, this study was the first to clone neutrophil activating protein gene napA, Genbank accession number DQ341279. from genomic DNA of international standard strain NCTC 11639.
2, successful construction of napA-pGEX-4T-1-E. coli Top 10 high efficiency soluble prokaryotic expression system. The recombinant NAP has good immunogenicity and immune response, which can be used as a candidate component of the Hp gene engineering subunit vaccine, and lay the foundation for the study of the pathogenesis of Hp, the mechanism of immune protection and the diagnostic reagents.
3, NAP is a highly conserved gene and a good immunogen. It is preliminarily predicted that NAP has 4 highly antigenic peptides, of which 118~140 peptides may contain good epitopes.
4, successful preparation of high affinity NAP resistant whole strain mAb and 3 mAb (E006, E019, E023) are highly specific, sensitive and promising.
5, Hp has high infection rate in gastric disease and normal population in Guangdong area; high expression of NAP antibody shows a certain association between NAP and gastric cancer, NAP is a high risk factor for gastric cancer, and there is no significant correlation between the age of the patients and the level of NAP antibody. This study provides a theoretical basis for the study of NAP pathogeny and the diagnosis of Hp infection related gastric cancer. Treatment and vaccine research are the cornerstone.
6, the linear epitope (FAHLATQ) of NAP was screened for the first time, located in the predicted peptide segment (118~140), and laid a good foundation for the next step to carry out polyvalent peptide vaccine and proteomics.
【学位授予单位】：第一军医大学
【学位级别】：硕士
【学位授予年份】：2007
【分类号】：R378

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