CAJ | 학술논문

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.
목적 표체화순화정분양전체단백(APP)적최기말단수해편단CTFβ,병감정기생물학성질,위재아이자해묵병(AD)항체사선중적응용전정기출.방법 이APP기인위모판,극륭CTFβ적기인병측서감정;장CTFβ기인극륭도표체재체pET-30a(+)상,구건중조표체질립pET30a-CTFβ;전화대장애희균BL21,IPTG유도표체,이용Ni-NTA친화층석대중조단백진행순화,병용Western Blot화ELISA검측기면역반응성,병초보탐색기작위검측항원적실험조건.결과 중조단백재대장애희균중가용성표체,Western Blot결과,용항조안산표첨항체주위일항,(10～25) ×103처현시여예계상대분자질량대소일치적조대;차외,재80×103이상적위치상유교조적단백조대.용항Aβ적단항진일보분석발현,(10～25) ×103급80×103이상적조대가이피항Aβ(17-24)단항4G8식별,이80×103이상적조대환가이피Aβ과취체단항식별,설명표체산물환형성료고상대분자질량적취집체,위우80×103이상.간접ELISA결과표명CTFβ용우AD항체검측적최가포피제량시1 nv공.결론 본연구성공표체화순화료CTFβ,병감정료기단체화취집체적면역반응성,위기재AD검측중적응용제공실험의거.
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 when it was used to detect Aβ antibody.Conclusion In this study,one of the aggregate-prone fragment of APP,CTFβ,was successfully expressed,purified,and identified,which would provide experimental clue to futher application in biochemical diagnosis of AD.