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首页> 外文期刊>World Journal of Gastroenterology >Construction of an oral recombinant DNA vaccine from H pylori neutrophil activating protein and its immunogenicity
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Construction of an oral recombinant DNA vaccine from H pylori neutrophil activating protein and its immunogenicity

机译：幽门螺杆菌嗜中性粒细胞活化蛋白口服重组DNA疫苗的构建及其免疫原性

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AIM: To construct a live attenuated Salmonella typhimurium (S. typhimurium) strain harboring the H pylori neutrophil activating protein (HP-NAP) gene as an oral recombinant DNA vaccine, and to evaluate its immunogenicity. METHODS: By genetic engineering methods, the genomic DNA of H pylori was extracted as a template. The total length of the HP-NAP gene was amplified by poly-merase chain reaction (PCR) and cloned into pBT vector for sequencing and BLAST analysis, then subcloned into a eukaryotic expression vector pIRES followed by PCR identification and restriction enzyme digestion. The identified recombinant plasmid pIRES-NAP was transfected into COS-7 cells for target fusion protein expression, and its antigenicity was detected by Western blotting. Then the recombinant plasmid was transformed into a live attenuated S. typhimurium strain SL7207 as an oral vaccine strain, and its immunogenicity was evaluated with animal experiments. RESULTS: A 435 bp product was cloned using high homology with HP-NAP gene in GenBank (more than 98%). With identification by PCR and restriction enzyme digestion, a recombinant eukaryotic expression plasmid pIRES-NAP containing the HP-NAP gene of H pylori was successfully constructed. The expressed target protein had a specific reaction with H pylori whole cell antibody and showed a single strip result detected by Western blotting. Oral immunization of mice with recombinant DNA vaccine strain SL7207 (pIRES-NAP) also induced a specific immune response. CONCLUSION: The successful construction of HP-NAP oral DNA vaccine with good immunogenicity may help to further investigate its immunoprotection effects and develop vaccine against H pylori infection.
机译：目的：构建携带幽门螺杆菌嗜中性粒细胞活化蛋白（HP-NAP）基因的减毒活鼠伤寒沙门氏菌（S. typhimurium）菌株作为口服重组DNA疫苗，并评估其免疫原性。方法：通过基因工程方法，提取幽门螺杆菌的基因组DNA作为模板。通过聚合酶链反应（PCR）扩增HP-NAP基因的全长，并将其克隆到pBT载体中进行测序和BLAST分析，然后再亚克隆到真核表达载体pIRES中，然后进行PCR鉴定和限制性酶切。将鉴定出的重组质粒pIRES-NAP转染到COS-7细胞中用于靶融合蛋白的表达，并通过Western印迹检测其抗原性。然后，将重组质粒转化为减毒活的鼠伤寒沙门氏菌SL7207口服疫苗株，并通过动物实验评估其免疫原性。结果：与GenBank中的HP-NAP基因高度同源，克隆了一个435 bp的产物（98％以上）。通过PCR鉴定和限制性内切酶消化，成功构建了含有幽门螺杆菌HP-NAP基因的重组真核表达质粒pIRES-NAP。表达的靶蛋白与幽门螺杆菌全细胞抗体有特异性反应，并通过蛋白质印迹法检测到单条结果。用重组DNA疫苗株SL7207（pIRES-NAP）进行的小鼠口服免疫也诱导了特异性免疫反应。结论：成功构建具有良好免疫原性的HP-NAP口服DNA疫苗可能有助于进一步研究其免疫保护作用，并开发针对幽门螺杆菌感染的疫苗。

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