抗阿尔茨海默病重组蛋白疫苗的克隆、表达和免疫学鉴定

A novel recombinant vaccine against Alzheimer's disease:cloning,expression,purification and antigenicity evaluation

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Aβ1-15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pET28 a-AnsB-TTP-Aβ1-15。转化至E.coliBL-21,TBYE培养基(Kanr)培养,乳糖诱导表达,通过渗透休克、DEAE52-cellu lose和Sepha-dex G-100柱层析制备融合蛋白rAnsB-TTP-Aβ1-15,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-Aβ1-15保留了AnsB 71%的催化活力,具有与抗Aβ1-42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Aβ1-15表位的融合蛋白rAnsB-TTP-Aβ1-15,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-A1-15 encoding a fusion protein composed of L-asparaginase B, a tetanus toxin peptide （TTP） spacer （831 -854 fragment）, and the B cell epitope （Aβ1-15） of Aβ1-42 peptide was constructed. It was transformed into E. coli and the fusion protein of rAnsB-TTP-Aβ1-15 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-Aβ1-15 didn＇t only exhibit approximately 71% activity of the native enzyme, but also could bind to anti-Aβ1- 42 antibody. Conclusion The study showed that the fused B cell epitope of Aβ1-42 could be displayed on the surface of rAnsB-TTP-Aβ1-15 , which is hope for develop to a future vaccine against Alzheimer＇s disease.