β淀粉样肽与HbcAg/MIR融合蛋白的免疫血清在AD转基因细胞模型中的生物效应  

Biological Effects of Immune Serum from Fusion Protein of β-amyloid Peptide and HbcAg/MIR on AD Transgeneic Cells

作　　者：刘朝晖[1] 冯改丰[1] 李月英[1] 马延兵[1] 胡海涛[1] 

机构地区：[1]西安交通大学医学院人体解剖与组织胚胎学系环境与相关疾病教育部重点实验室,西安710061

出　　处：《四川大学学报（医学版）》2007年第4期608-612,共5页Journal of Sichuan University(Medical Sciences)

基　　金：陕西省自然科学基金(批准号2001SM57);西安交通大学科学研究基金(批准号2002003)资助

摘　　要：目的研究β淀粉样肽(Aβ)与乙肝核心抗原主要免疫优势区(HbcAg/MIR)的融合蛋白(c-Aβ-c)在BL21/pET28原核表达体系的表达并进行腹腔内注射表达的融合蛋白免疫小鼠,检测免疫血清的免疫原性以及体外的生物学效应。方法应用分子克隆技术构建原核表达质粒pET-28a/c-Aβ-c并进行c-Aβ-c融合基因在BL21菌的IPTG诱导表达,SDS-PAGE分析表达的融合蛋白,用纯化的c-Aβ-c融合蛋白腹腔注射免疫动物,ELASA检测其抗Aβ抗体效价,MTT法和流式细胞术检测它应用于Alzheimer’sdisease(AD)转基因细胞的生物效应。结果表达的c-Aβ-c融合蛋白主要以包涵体形式存在于细菌的沉淀中,表达水平占细菌总蛋白量的30%以上。免疫小鼠血清中抗Aβ抗体效价达1∶16000,抗血清抑制了AD转基因细胞中Aβ的神经细胞毒性,明显降低了细胞凋亡的比率。结论c-Aβ-c融合蛋白具有良好的Aβ免疫原性,其动物免疫血清有效抑制Aβ肽的细胞毒性。Objective To investigate the expression of fusion protein c-Aβ-c, a fusion protein from the β amyloid peptide （Aβ） and the Hepatitis B core antigen/ Major immunodominant region （HbcAg/MIR）, in the BL21/pET28 prokaryotic expression system and to immunize mice with the expressed fusion protein and evaluate the immunogenicity and biological effects of the serum in vitro. Methods The recombinant prokaryotic expression plasmid PET-28a /c-Aβ-c was constructed by molecular cloning technique and the c-Aβ-c fusion gene expression was induced in E. coli BL21 by isopropyl-1-thio-b-Dgalactopyranoside （IPTG）. The expressed fusion protein was analyzed by SDS-PAGE. Intraperitoneal injection （i. p） of the purified c-Aβ-c fusion protein was given to the BALB/c mice. The anti-Aβ effect of the immune serum was detected by indirect ELISA. The biological effect of the immune serum on Alzheimer＇s disease （AD） transgeneic cells was assessed by MTT assay and lfow cytometer. Results The c-Aβ-c fusion protein was found in the sediment of the isolated bacteria. The expressed protein comprised more than 30% of the total proteins in the bacteria sediment. The anti-Aβ antibody in the serum of the immunized mice reached 1 ： 16000. The antiserum reduced the cytotoxicity of Aβ peptide in the AD transgeneic cells and significantly decreased the apoptosis of cells. Conclusion The c-Aβ-c fusion protein has good Aβ immunogenicity and the animal immune serum efficiently inhibits the cytotoxicity of Aβ peptide.

关 键 词：Β淀粉样肽 主要免疫优势区 融合蛋白 免疫血清 

分 类 号：R749.1[医药卫生—神经病学与精神病学]