幽门螺杆菌分子内佐剂三价疫苗的构建、纯化及活性研究  

Construction, purification, and immunogenicity of a trivalent inner adjuvant vaccine of Helicobacter pylori

作　　者：高原[1] 张卫军[1] 邹全明[1] 

机构地区：[1]第三军医大学临床微生物学及临床免疫学教研室暨重庆市生物制药工程技术研究中心,重庆400038

出　　处：《免疫学杂志》2006年第6期690-693,697,共5页Immunological Journal

基　　金：国家十五"863"资助项目(2003AA215020);国家"十五"重大科技专项课题(2003AA2Z3C64)资助

摘　　要：目的构建幽门螺杆菌(helicobacterpylori,Hp)中性粒细胞激活蛋白基因napA、黏附素hpaA、尿素酶B亚单位活性功能片段ureB414与大肠杆菌不耐热肠毒素B亚单位(heat-labileenterotoxinBsubunit,LTB)的分子内佐剂三价融合蛋白疫苗,并通过口服免疫小鼠研究其抗原性,为疫苗的开发奠定基础。方法利用分子克隆技术构建携带napA-hpaA-ureB414-LTB融合基因的重组原核表达质粒pET-22b-NHUL,转化E.coliBL21(DE3),进行原核表达,重组表达蛋白采用HK26/60Superdex-75分子筛和亲和层析柱ChelatingSepharose进行纯化,Tris-Tricine电泳和Westernblot鉴定,GM1-ELISA检测rNHUL与神经节苷脂的结合。口服免疫Balb/c小鼠,检测重组融合蛋白的抗原性。结果重叠延伸PCR扩增出了约1590bp的NHUL融合基因;其核苷酸序列与GenBank公布的相关序列一致。重组融合蛋白以可溶和包涵体两种形式表达,表达量约占细菌总蛋白的25%,Tris-Tricine初步测定目的蛋白的Mr约59000,纯化后纯度大于90%。Westernblot检测其具有良好的抗原性。该重组蛋白保持了与神经节苷脂GM1结合的生物学活性。动物实验表明,多亚单位疫苗口服免疫小鼠后血清特异性IgG、IgA和胃黏液、肠黏液的抗原特异性sIgA抗体显著高于单一亚单位疫苗。结论所获得重组融合蛋白具有良好的抗原性和黏膜佐剂活性,为进一步的疫苗研究奠定了基础。Objective To construct a recombinant trivalent inner adjuvant vaccine of Helicobacter priori （Hp） neutrophil-activa-ting protein A （napA）, HpaA, UreB414, and heat-labile enterotoxin B subunit （LTB）, and study its antigenicity so as to lay a foundation for developing a Hp vaccine. Methods The fusion gene of napA, hpaA, ureB414, and LTB was cloned by SOE PCR （ splicing by overlap extension）, and transfected into an expression vector （pEY-22b）. The fusion gene was expressed in E. coli BL21 （DE3） and purified with HK 26/60 Superdex-75 chromatography and Chelating Sepharose affinity chromatography. The fusion protein was isolated and identified with Tris- Tricine and Western blotting, respectively. The binding activity to GM1 ganglioside was analyzed by GM1-ELISA. Balb/c mice were administered the fusion protein via the oral route in order to investigate its mucosal immunoactivity. Results The fusion gene was successfully cloned into pET-22b. The gene segment inserted into the recombinant vector was identified by using restriction enzyme digestion and sequencing methods and was consistent with that in Genbank. The relative molecular weight of expressed product was Mr 59 000, and the protein was expressed with dissoluble and inclusion body. The purity of rNHUL was up to 90% after HK 26/60 Superdex-75 chromatography and Chelating Sepharose attlnity chromatography. The purified rNHUL kept GM1 binding ability, immunogenicity, and immunoreactivity. After immunization with multi-subunit vaccine, the serum special IgG, IgA antibodies, and mucosal sIgA antibodies in the stomach and small intestine of mice were significantly increased as compared with mice immunized with mono-subunit alone. Conclusion Recombinant antigen NHUL is great potential as a practical genetic engineering vaccine to prevent Hp infection.

关 键 词：幽门螺杆菌 中性粒细胞激活蛋白 大肠杆菌不耐热肠毒素B亚单位 疫苗 分子内佐剂 

分 类 号：R392[医药卫生—免疫学]