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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
Objective: To study the prokaryotic expression of Aβ HBcAg fusion gene of β-amyloid peptide (Aβ) and HBcAg and to detect the immunogenicity of the expressed fusion protein Aβ HBcAg. Methods: The gene fragment containing Aβ HBcAg was excised from the recombinant plasmid 7Z / Aβ HBcAg and fused with the lac promoter of plasmid pYTA1 to construct the recombinant expression plasmid pYTA / Aβ HBcAg. The recombinant plasmid was transformed into E. coli DH5α and expressed under the induction of IPTG. The expressed bacteria were sonicated and the expression of the fusion protein Aβ HBcAg was detected by SDS PAGE and Coomassie brilliant blue staining. The reaction of the fusion protein was detected by ELISA. The fusion protein was isolated and purified by ammonium sulfate salting and immunized BALB / c mice. The titer of anti-Aβ antibody in serum was detected by ELISA. Results: The fusion protein was soluble in the supernatant of the bacterial lysate and expressed at 7% of the total bacterial protein. The expressed fusion protein not only has the reactogenicity of Aβ, but also has the reactivity with HBcAg. After three immunizations, the highest anti-Aβ antibody titer in BALB / c mice serum was 1:16 0 0 0. Conclusion: The fusion gene Aβ HBcAg can be expressed in Escherichia coli DH5α. The expression product is a soluble protein, which is present in the supernatant of the bacterial lysate and has strong immunogenicity. This study lays the foundation for the ongoing animal experiments of AD genetic engineering vaccines