幽门螺杆菌中性粒细胞激活蛋白DNA疫苗的构建及其免疫保护作用

Construction of an oral DNA vaccine carrying H. pylori neutrophil-activating protein and its immunoprotection effect

目的:构建携带幽门螺杆菌中性粒细胞激活蛋白(Hp neutrophil-activating protein,Hp-NAP)基因(napA)活减毒鼠伤寒沙门菌重组DNA疫苗,初步观察其对慢性Hp感染的免疫保护作用。方法:应用基因工程技术扩增全长napA,测序并经同源性分析后,将其亚克隆入真核表达载体pIRES,鉴定正确后将重组质粒转化活减毒鼠伤寒沙门菌构建Hp-NAP口服DNA疫苗。口服Hp-SS1建立SS1长期感染小鼠模型,30周后随机均分为3组,每组各5只。治疗组予10 9 cfu／0.4 ml疫苗菌灌胃,1次／周×3周;2个对照组分别予等体积生理盐水或空白质粒。末次免疫4周后行快速尿素酶检测,ELISA测定血清抗体效价。结果:重组真核表达质粒pIRES-napA成功转化活减毒鼠伤寒沙门菌SL7207;所克隆napA与GenBank中SS1-napA核苷酸和蛋白质的同源性均>98％。免疫后4周治疗组75％(3／4)小鼠快速尿素酶检测阴性,对照组均阳性,差异显著(P<0.05);治疗组血清抗Hp-NAP抗体效价明显升高。结论:成功构建了具有较好免疫保护作用的Hp-NAP口服重组DNA疫苗,为进一步研制多价抗Hp核酸疫苗奠定了基础。

Objective: To construct a live attenuated Salmonella typhimurium (S. typhimurium) strain carrying Heli cobacter pylori (H. pylori) neutrophil-activating protein (Hp-NAP) gene as an oral recombinant DNA vaccine, and to observe its immunotherapy effect against chronic H. pylori infection. Methods: By genetic engineering method, a 435 bp nap A gene (encoding Hp-NAP) was subcloned into an eukaryotic expression vector pIRES. After sequencing and BLAST analysis, the identified recombinant plasmid was transformed into a live attenuated S. typhimurium strain SL7207, and then lavaged into a long-term (30 weeks) model of mice infected by Sydney strain (SS1). Results: Using polymerase chain reaction (PCR) and restriction enzyme digestion, a recombinant eukaryotic expression plasmid pIRES-napA harboring nap A gene of H. pylori was constructed, and the recombinant plasmid was successfully transformed into the live attenuated S. typhimurium strain SL7207. After 4 weeks of immunization, 75% of mice treated with DNA vaccine were rapid urease test negtive, while those treated with vacant plasmid or normal saline alone were all positive (P<0. 05). And the litre of serum Hp-NAP antibody was significantly elevated in treated group. Conclusion: An effective recombinant live attenuated S. typhimurium strain carrying Hp-NAP gene is successfully constructed,which may help to develop polyvalent DNA vaccine against H. pylori infection.