幽门螺杆菌中性粒细胞激活蛋白核酸疫苗实验研究

目的 构建幽门螺杆菌(Hp)中性粒细胞激活蛋白(Hp-NAP)口服DNA疫苗，初步评价其免疫治疗作用，为Hp疫苗研制奠定基础。方法 PCR扩增全长Hp-NAP基因(napA)，测序并同源性分析后，亚克隆入真核表达载体pIRES，双酶切并PCR鉴定。将重组质粒plRES-napA转化减毒鼠伤寒沙门菌SL7207，口服接种长期感染Hp之BALB／c小鼠，观察疗效。结果PCR扩增出-435bp产物，序列分析表明，所克隆序列与CenBank中SSl-napA核苷酸及蛋白质同源性均＞98％。PCR及双酶切证实，成功构建了携带napA的重组减毒鼠伤寒沙门菌DNA疫苗。疫苗接种后4周，治疗组3／4小鼠快速尿素酶检测阴性，对照组均阳性(P=0．0476)；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治疗作用的Hp—NAP口服重组DNA疫苗，为进一步研制多价抗HpDNA疫苗奠定了基础。

Preliminary study on immunotherapy of an oral recombinant DNA vaccine of Helicobacter pylori neutrophil activating protein

Objective To construct an oral recombinant DNA vaccine of Helicobacter pylori(H. pylori) neutrophil activating protein (Hp-NAP), and to evaluate its immunotherapeutic effects. Methods The napA gene (encoding Hp-NAP) was amplified by poly mera se chain reaction(PCR) and cloned into TA cloning vector pBT. After nucleotide s equencing and sequence analysis, the target sequence was subcloned into an eukar yot ic expression vector pIRES. Then the identified recombinant plasmid, pIRES-napA , was transformed into a live attenuated Salmonella typhimurium(S. typhimurium ) strain SL7207, and lavaged into a long-term(30 weeks) model of BALB/c mice infected by Sydney strain(SS1) of H. pylori. Results A 435 bp target gene of napA was amplified by PCR. Seq uenci ng and BLAST analysis showed that most of the cloned napA sequence was homologou s with that of SS1 strain of H. pylori. provided by GenBank, and the homolog y of neucleotide and protein was over 98%, respectively. PCR and restriction enzyme digestion id entification indicated that a recombinant live attenuated S. typhimurium DNA vaccine strain carrying Hp-napA gene was successfully constructed. After 4 wee ks of oral immunization, 75% of mice treated with DNA vaccine were rapid urease test negative, while those with vacant plasmid or normal saline alone were all p ositive (P= 0.0476). The titer of serum Hp-NAP antibody was signific antly elevated in treatment group. Conclusions The successful construction of an effective oral recom binant DNA vaccine of Hp-NAP may be helpful for the further development of polyvalent DNA vaccine against H. pylori infection.