Department of Human Anatomy and Histoembryology, School of Medicine, Xi'an Jiaotong University, 76(#) West Yanta Road, Xi'an, Shaanxi 710061, People's Republic of China.
J Neurosci Methods. 2013 Aug 15;218(1):48-54. doi: 10.1016/j.jneumeth.2013.05.006. Epub 2013 May 20.
β-Amyloid peptide (Aβ) immunization is regarded as the most promising therapy to Alzheimer' s disease. The full length Aβ as antigen might induce meningoencepholontis adverse effect since the middle and C-terminal fragments of Aβ contain T cell epitopes. While N-terminal fragment of Aβ, containing B cell epitope, has weak or no immunogenicity. To improve the immunogenicity, we used HBV core antigen as carrier to make fusion protein containing 2 Aβ1-15. The fusion protein was expressed in Escherichia coli harboring the recombinant plasmid pET/c-2Aβ15-c. Transmission electron microscope (TEM) showed that fusion protein could form virus-like particles (VLPs). After 7-weeks immunization with Aβ-HBc VLPs through subcutaneous injection, the titer of anti-Aβ antibody in sera of BALB/c mice reached up to 10(5), higher than Aβ peptide immunization. Aβ-HBc VLPs immunization did not elicit Aβ-specific T cell proliferation. The main isotypes of antibody in mice immunized with Aβ-HBc VLPs were IgG1 and IgG2b, while isotype in mice immunized with Aβ1-42 was IgG2a. When the antisera from mice immunized with Aβ-HBc VLPs were co-incubated for 1 week at 37°C with Aβ, fibers of aggregated Aβ was reduced or diminished. The antibodies also prevented PC12 cells from injury by toxicity of Aβ. In conclusion, recombinant c-2Aβ15-c gene can be expressed in E. coli. The expressed protein could form VLPs and has strong immunogenicity. The antisera prevented Aβ fiber formation and protected the PC12 cells against toxicity of Aβ. This study lays the foundation for the experimental study of AD gene engineering vaccine.
β-淀粉样肽（Aβ）免疫被认为是治疗阿尔茨海默病最有前途的疗法。全长 Aβ作为抗原可能会引起脑膜脑炎的不良反应，因为 Aβ 的中、C 端片段含有 T 细胞表位。而 Aβ 的 N 端片段，含有 B 细胞表位，免疫原性较弱或没有。为了提高免疫原性，我们使用乙肝核心抗原作为载体，构建了含有 2 个 Aβ1-15 的融合蛋白。融合蛋白在携带重组质粒 pET/c-2Aβ15-c 的大肠杆菌中表达。透射电子显微镜（TEM）显示融合蛋白可以形成病毒样颗粒（VLPs）。通过皮下注射 Aβ-HBc VLPs 对 BALB/c 小鼠进行 7 周免疫后，血清中抗 Aβ 抗体的滴度达到 10(5)，高于 Aβ 肽免疫。Aβ-HBc VLPs 免疫没有引起 Aβ 特异性 T 细胞增殖。用 Aβ-HBc VLPs 免疫的小鼠的主要抗体类型是 IgG1 和 IgG2b，而用 Aβ1-42 免疫的小鼠的抗体类型是 IgG2a。当用 Aβ-HBc VLPs 免疫的小鼠的抗血清在 37°C 下共孵育 1 周与 Aβ 混合时，聚集的 Aβ纤维减少或消失。该抗体还可防止 Aβ 毒性对 PC12 细胞造成损伤。综上所述，重组 c-2Aβ15-c 基因可在大肠杆菌中表达。表达的蛋白可以形成 VLPs，具有很强的免疫原性。抗血清可以防止 Aβ 纤维形成，并保护 PC12 细胞免受 Aβ 的毒性。本研究为 AD 基因工程疫苗的实验研究奠定了基础。
