抗阿尔茨海默病重组蛋白疫苗的克隆、表达和免疫学鉴定-东南大学学报医学版

抗阿尔茨海默病重组蛋白疫苗的克隆、表达和免疫学鉴定

单位：中国药科大学生命科学与技术学院,微基因药学实验室,江苏,南京,210009

分类号：Q78, R392

出版年·卷·期（页码）：2006·25·第四期（232-235）

摘要：

目的:制备抗阿尔茨海默病的重组蛋白疫苗.方法:将β淀粉样蛋白42肽N端表位Aβ115基因,通过来源于破伤风毒素的辅助性T细胞表位多肽TTP基因片段,与大肠杆菌L-门冬酰胺酶Ⅱ(AnsB)基因的C末端融合,构建表达质粒pET28a-AnsB-TTP-Aβ115.转化至E. coli BL-21,TBYE培养基(Kanr)培养,乳糖诱导表达,通过渗透休克、DEAE52-cellulose和Sephadex G-100柱层析制备融合蛋白rAnsB-TTP-Aβ115,通过酶活力和抗原性测定鉴定融合蛋白.结果:获得的融合蛋白rAnsB-TTP-Aβ115保留了AnsB 71%的催化活力,具有与抗Aβ142抗体特异性结合的能力.结论:获得了在AnsB多聚酶分子表面呈现有Aβ115表位的融合蛋白rAnsB-TTP-Aβ115,为抗阿尔茨海默病疫苗研究奠定了基础.

Objective To develop a novel recombinant vaccine against Alzheimers disease.Methods The expression plasmid of pET28a-AnsB-TTP-Aβ_{1-15}encoding a fusion protein composed of L-asparaginase B,a tetanus toxin peptide(TTP) spacer(831-854 fragment),and the B cell epitope(Aβ_{1-15}) of Aβ_{1-42} peptide was constructed.It was transformed into E.coli and the fusion protein of rAnsB-TTP-Aβ_{1-15} was expressed and targeted to the periplasm of bacteria after inducing by lactose.The periplasm fusion protein was extracted by osmic shock and furthermore purified by DEAE52-cellulose and Sephadex G-100.This purified fusion protein will be detected with asparaginase activity and ELISA assay.Result The purified rAnsB-TTP-Aβ_{1-15} didnt only exhibit approximately 71% activity of the native enzyme,but also could bind to anti-Aβ_{1-42} antibody. Conclusion The study showed that the fused B cell epitope of Aβ_{1-42} could be displayed on the surface of rAnsB-TTP-Aβ_{1-15},which is hope for develop to a future vaccine a gainst Alzheimers disease.

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