幽门螺杆菌中性粒细胞激活蛋白分子内佐剂融合疫苗的构建、纯化及活性研究

目的构建幽门螺杆菌（HP）中性粒细胞激活蛋白基因（napA）与大肠杆菌不耐热肠毒素B亚单位（heat-labile enterotoxin B subunit，LTB）的分子内佐剂融合蛋白疫苗，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naDA—LTB融合基因的重组原核表达质粒pET-22b—napA—LTB，转化E、coli BL21（DE3），进行原核表达，重组表达蛋白采用亲和层析柱Chelating Sepharose进行纯化，Tris—Tricine电泳和Western blot鉴定，GM1-EUSA检测rNAP-LTB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760bp的napA—LTB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30％，Tris—Tricine初步测定目的蛋白的相对分子质量（Mr）约29000，纯化后纯度大于90％。Western blot检测其具有良好的抗原性。该重组蛋白保持了与GM1神经节苷脂结合的生物学活性。动物实验表明，分子内佐剂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抗体显著高于无佐剂单一亚单位疫苗。结论所获得的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。

Study on Construction,Purification and Immunogenicity of Inner Adjuvant Vaccine of Helicobacter pylori Neutrophil Activating Protein

Objective To construct a recombinant inner adjuvant DNA vaccine of Helicobacter pylori (HP) neutrophil activating protein A(NapA) and heat-labile enterotoxin B subunit(LTB),and to study its antigenicity so as to lay a foundation for developing a HP vaccine.Methods The fusion gene of napA and LTB was cloned with SOE PCR (splicing by overlap extension),and constructed into an expression vector (pET-22b).The fusion gene was expressed in E.coli BL21(DE3) and purified with Chelating Sepharose affinity chromatography.The fusion protein were isolated and identified with Tris-Tricine and Western blot.The binding activity with GM1 ganglioside was analyzed by GM1-ELISA.Administered BALB/c mice via the oral route with fusion protein 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 those papers reported before.The molecular weight of expressed product was 29 000,and the protein was expressed with dissoluble and inclusion body respectively.The purity of rNAP-LTB was up to 90% after Chelating Sepharose affinity chromatography.The purified rNAP-LTB keeps GM1 binding ability immunogenicity and immunoreactivity.After immunization with inner adjuvant vaccine,serum special IgG,IgA antibodies and mucosal sIgA antibodies in the stomach and small intestine were significantly increased as compared with that after rNAP immunization alone.Conclusion Recombinant antigen NAP-LTB is a great potential as a practical genetic engineering vaccine to prevent HP infection.