重组质粒pYTA/Aβ-HBcAg的构建及其在大肠杆菌中的表达

目的 :研究 β 淀粉样肽 (Aβ)与HBcAg的融合基因Aβ HBcAg的原核表达 ,并检测表达的融合蛋白Aβ HBcAg的免疫原性。方法 :从重组质粒 7Z/Aβ HBcAg中 ,切下含Aβ HBcAg的基因片段 ,将其融合于质粒pYTA1的lac启动子之后 ,构建重组表达质粒pYTA/Aβ HBcAg。以此重组质粒转化大肠杆菌DH5α,在IPTG诱导下表达。超声破碎表达的细菌 ,通过SDS PAGE及考马斯亮蓝染色 ,检测融合蛋白Aβ HBcAg的表达。采用ELISA法检测融合蛋白的反应原性。融合蛋白经饱和硫酸铵盐析法分离和纯化后免疫BALB/c小鼠 ,用ELISA法检测血清中抗 Aβ抗体的滴度。 结果 :融合蛋白以可溶性形式存在于细菌裂解液的上清中 ,其表达量为菌体总蛋白量的 7%。表达的融合蛋白既有Aβ的反应原性 ,又有与HBcAg的反应原性。经 3次免疫后 ,BALB/c小鼠血清中抗 Aβ抗体的滴度最高可达为 1∶16 0 0 0。结论 :融合基因Aβ HBcAg可在大肠杆菌DH5α中表达。表达产物为可溶性蛋白 ,存在于细菌裂解液的上清中 ,具有较强的免疫原性。本研究为正在进行的AD基因工程疫苗的动物实验打下了基础

Construction of recombinant plasmid pYTA/Aβ-HBcAg and its expression in E.coli

AIM: To study the expression of fusion gene Abeta-HBcAg in E.coli and detect the immunogenicity of fusion protein Abeta-HBcAg. METHODS: The Abeta-HBcAg fusion gene was cut from recombinant plasmid pBV220/Abeta-HBcAg, and was inserted into the plasmid pYTA1 to construct recombinant plasmid pYTA/Abeta-HBcAg. The recombinant plasmid was transformed into E.coli DH5alpha, and expressed under IPTG induction. The expression of the fusion protein Abeta-HBcAg was detected by SDS-PAGE. The antigenicity of the fusion protein was detected by ELISA. BALB/c mice were immunized intraperitoneally with the fusion protein purified by salting out with saturate ammonium sulfate. The titers of anti- Abeta antibodies of the immunized mice was evaluated by ELISA. RESULTS: Fusion protein existed in supernatant of the bacteria lysate and its expression level was about 7% of the total bacteria protein. The fusion protein reacted with both Abeta and HBcAg. The highest titer of anti-Abeta antibodies could reach to 1:16 000 after immunization for 3 times. CONCLUSION: Recombinant gene Abeta-HBcAg can be expressed in E.coli DH5alpha. The expression protein exists in supernatant of the bacteria lysate and has good immunogenicity. This study lays the foundation for the experimental animal study of AD gene-engineering vaccine.