重组四价Aβ_(1-15)蛋白的表达及免疫原性鉴定  

Expression of recombinant quadrivalent Aβ_(1-15) and its immunogenicity identification

作　　者：邹俊涛[1] 姚志彬[1] 张革[2] 汪华侨[1] 徐杰[1] 谢瑶[1] 袁群芳[1] 

机构地区：[1]中山大学中山医学院人体解剖学教研室脑研究室,广东广州510080 [2]中山大学药学院分子生物学实验室,广东广州510080

出　　处：《中国病理生理杂志》2009年第1期79-83,共5页Chinese Journal of Pathophysiology

基　　金：国家重点基础研究发展计划(973计划)资助项目(No.2006cb500700);粤港关键领域重点突破项目资助(No.20054982210);国家自然科学基金资助项目(No.30400512);广东省社会发展计划资助项目(No.2006B36004001);广州市科技计划应用基础研究资助项目(No.2006J1-C0101);中国博士后基金资助项目(No.20080440120);广东省科技社会发展基金资助项目(No.2008B030301320)

摘　　要：目的:制备重组人四价串联Aβ1-15老年性痴呆二代蛋白疫苗,探讨其免疫原性。方法:以本室前期制备的含四价Aβ1-15基因(4×Aβ15)的重组质粒pcDNA-4×Aβ15为模板,PCR扩增4×Aβ15基因并克隆至pGEX-4T-2质粒中,转化大肠杆菌,诱导表达GST-4×Aβ15。经thrombin酶切去除GST后得到纯化的4×Aβ15蛋白。将4×Aβ15免疫BALB/c小鼠,观察其诱导体液免疫反应的能力。结果:经鉴定、测序,获取重组质粒pGEX-4×Aβ15,诱导表达后Western blotting显示在相对分子质量约35×103处有GST-4×Aβ15表达带,thrombin酶切去除GST后得到相对分子质量约9×103的4×Aβ15蛋白。4×Aβ15蛋白免疫小鼠诱导出(964.6±401.3)mg/L抗Aβ抗体。结论:成功构建了pGEX-4×Aβ15原核表达质粒并表达人重组4×Aβ15蛋白,免疫动物证实该蛋白疫苗有较强的免疫原性。AIM： To prepare the second - generation Alzheimer disease vaccine of quadrivalent tandem Aβ1-15 and to explore its immunogenicity. METHODS： The 4×Aβ15 gene was amplified by PCR with the recombinant plasmid pcDNA -4×Aβ15 as a template, which was prepared by our research group before. The 4×Aβ15 gene was then cloned to the pGEX -4T- 2 plasmid. The fused GST- 4×Aβ15 protein was expressed when the bacteria containing the recombinant plasmid was induced. The purified 4×Aβ15 protein was then obtained after the GST - 4×Aβ15 protein was digested by thrombin. Meanwhile, BALB/c mice were used to confirm whether they could generate A13 specific humoral immunity when immunized with the 4 ×Aβ15 protein. RESULTS： After identification and DNA sequence analysis, the recombinant plasmid pGEX -4×Aβ15 was constructed. Expression of recombinant 35 kD GST -4×Aβ15 was verified by SDS - PAGE and Western blotting. The 9 kD 4×Aβ15 protein was produced after thrombin digestion of GST - 4×Aβ15. Immunizing mice with the 4×Aβ15 protein have induced high titre anti - Aβ antibodies. CONCLUSION： The recombinant prokaryotic vector pGEX -4 ×Aβ15 was constructed successfully, and the purified 4 ×Aβ15 protein was obtained after prokaryotic expression. It is demonstrated that the 4×Aβ15 protein is a strong immunogen, confirmed by immunization of BALB/c mice.

关 键 词：阿尔茨海默病 疫苗 β淀粉样蛋白 原核表达 免疫力 

分 类 号：R363[医药卫生—病理学]