CTFβ表达纯化及生物学性质鉴定  

Expression, purification and the biological activites of C-terminal fragment CTFβ

作　　者：宋琳琳[1] 杨海强[1] 张莹[1] 何金生[1] 古应彩 侯明旭[1] 刘楠楠[1] 

机构地区：[1]北京交通大学理学院生命科学与生物工程研究院,100044

出　　处：《中华实验和临床病毒学杂志》2014年第5期395-397,共3页Chinese Journal of Experimental and Clinical Virology

基　　金：国家自然科学基金（81100809和81271417）;北京交通大学2014年大学生创新训练计划项目

摘　　要：目的 表达和纯化淀粉样前体蛋白（APP）的羧基末端水解片段CTFβ,并鉴定其生物学性质,为在阿尔茨海默病（AD）抗体筛选中的应用奠定基础.方法 以APP基因为模板,克隆CTFβ的基因并测序鉴定;将CTFβ基因克隆到表达载体pET-30a（＋）上,构建重组表达质粒pET30a-CTFβ;转化大肠埃希菌BL21,IPTG诱导表达,利用Ni-NTA亲和层析对重组蛋白进行纯化,并用Western Blot和ELISA检测其免疫反应性,并初步探索其作为检测抗原的实验条件.结果 重组蛋白在大肠埃希菌中可溶性表达,Western Blot结果,用抗组氨酸标签抗体做为一抗,（10～25） ×103处显示与预计相对分子质量大小一致的条带;此外,在80×103以上的位置尚有较粗的蛋白条带.用抗Aβ的单抗进一步分析发现,（10～25） ×103及80×103以上的条带可以被抗Aβ（17-24）单抗4G8识别,而80×103以上的条带还可以被Aβ寡聚体单抗识别,说明表达产物还形成了高相对分子质量的聚集体,位于80×103以上.间接ELISA结果表明CTFβ用于AD抗体检测的最佳包被剂量是1 nv孔.结论 本研究成功表达和纯化了CTFβ,并鉴定了其单体和聚集体的免疫反应性,为其在AD检测中的应用提供实验依据.Objective Proteolysis of the C-terminal fragment （CTFβ） of the amyloid precursor protein （APP） generates the Aβ peptides associated with Alzheimer＇ s disease （AD）.The metabolism of CTFβ may play key roles in early stage of AD before Aβ generation.The aim of this study was to express,identify and purify the CTFβ,so as to provide evidence for its application in the development of AD detection system.Methods APP gene was used as the template,and the gene of CTFβ was cloned to pMD18-T vector through PCR.After sequencing,the CTFβ gene was cloned into the expression vector pET-30a（＋） to construct the recombinant expression plasmid pET30a-CTFβ.The expression plasmid was transformed into Escherichia coli BL21 and the expression of CTFβ was induced by Isopropyl-β-D-thiogalactoside （IPTG）.The effect of expression was confirmed by Western blottng.The recombinant protein was purified by using Ni-NTA affinity chromatography column,and the immunoreactivity of recombinant protein was detected by Western blotting and indirect ELISA.Results Western Blot results showed that recombinant protein was solubly expressed in E.coli and its molecular weight was about 10 × 103 to 25 × 103,and the size of fusion protein was consistent with prediction.In addition,the data of Western blotting showed that there were still some thick bands above the position of 80 × 103.Furthermore,the immunoblotting demonstrated that the 10× 103 to 25 × 103 of monomer of fusion protein was recognized by anti-histidine （his） tag and anti-Aβ （17-24） （4G8） antibody,while the high molecular aggregates were above 80 × 103 which were detected respectivly by anti-his,anti-Aβ antibody NU1,NU4 and A8.However,our data suggested that the bands above 80 × 103 were poorly recognized by fibril specific antibody NU6.These results demonstrated that the purified recombinant protein showed a specific immunoreactivity.Finally,indirect ELISA showed that the optimal concentration of CTFβ to coat the ELISA plate was 1 ng/well

关 键 词：阿尔茨海默病 蛋白质构像 原核表达 

分 类 号：R749.16[医药卫生—神经病学与精神病学]