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目的 构建含幽门螺杆菌 (H .pylori)hpaA基因的重组减毒鼠伤寒沙门菌核酸疫苗。方法 应用PCR方法从H .pylori标准菌株基因组DNA中扩增hpaA基因 ,克隆入 pUCmT载体 ,检测hpaA基因序列 ,经过酶切、连接反应将其克隆入真核表达载体pIRES,转入大肠杆菌 ,筛选阳性克隆 ,通过PCR和酶切反应鉴定。重组载体pIRES hpaA转化入减毒鼠伤寒沙门菌LB5 0 0 0 ,抽提质粒 ,再转化入SL72 0 7,反复传代 ,鉴定重组核酸疫苗菌的稳定性。重组载体pIRES hpaA通过脂质体法转染COS 7细胞 ,SDS PAGE及Western印迹法检测pIRES hpaA表达HpaA蛋白的免疫原性。结果 成功扩增出长约 75 0bp的hpaA基因 ,测序结果表明扩增出的hpaA基因与H .pylorihpaA序列一致 ,PCR和酶切鉴定结果证实hpaA基因克隆入真核表达载体pIRES,成功构建了含hpaA基因的幽门螺杆菌核酸疫苗pIRES hpaA ,并成功构建了幽门螺杆菌hpaA基因的减毒鼠伤寒沙门菌核酸疫苗 ,重组核酸疫苗稳定 ,Western印迹法检测到了特异性的蛋白条带。结论 构建了具有免疫反应性的H .pylorihpaA减毒鼠伤寒沙门菌核酸疫苗 ,为进一步探索其免疫作用奠定了基础。
Objective To construct a recombinant attenuated Salmonella typhimurium DNA vaccine containing hpaA gene of Helicobacter pylori. Methods The hpaA gene was amplified by PCR from the genomic DNA of H. pylori strain and cloned into pUCmT vector. The hpaA gene was sequenced and cloned into the eukaryotic expression vector pIRES by restriction enzyme digestion and ligation. Positive clones were identified by PCR and enzyme digestion. The recombinant vector pIRES hpaA was transformed into attenuated Salmonella typhimurium LB5 0 0 0 and the plasmid was extracted and transformed into SL72 0 7 for repeated passages to confirm the stability of the recombinant nucleic acid vaccine. The recombinant vector pIRES hpaA was transfected into COS 7 cells by lipofectamine. The immunogenicity of HpaA protein expressed by pIRES hpaA was detected by SDS PAGE and Western blot. Results The hpaA gene of about 75 bp in length was amplified successfully. Sequencing results showed that the amplified hpaA gene was consistent with H. pylori hpaA. PCR and restriction enzyme digestion confirmed that hpaA gene was cloned into eukaryotic expression vector pIRES. hpaA gene of Helicobacter pylori nucleic acid vaccine pIRES hpaA, and an attenuated Salmonella typhimurium nucleic acid vaccine of Helicobacter pylori hpaA gene was successfully constructed. The recombinant nucleic acid vaccine was stable and a specific protein band was detected by Western blotting. Conclusion The construction of an immunoreactive H. pylori hpaA attenuated Salmonella typhimurium nucleic acid vaccine laid the foundation for further exploring its immunological function.