CAJ | 학술논문

目的探讨一种在常用真核细胞系中表达、纯化和鉴定阿尔茨海默病(AD)相关蛋白-β-分泌酶(BACE1)的方法. 方法在从人脑基因库中获取BA CE1序列并成功扩增的基础上,将其插入带有增强绿色荧光蛋白(EGFP)的表达载体pEGFP-c3中.将BACE1/pEGFP-c3重组质粒用脂质体转染至HEK293细胞中表达,再通过TALON亲和色谱法分离纯化及酶切得到BACE1蛋白.用Western blotting及荧光共振能量共振转移(FRET)法鉴定BACE1的表达和体外活性.同时将BACE1/pEGFP-c3质粒与表达淀粉样前体蛋白(APP)的重组质粒(APP/pDsRed-Monomer-N1)共转染HEK293细胞,再以Western blotting法检测BACE1切割底物APP的效果. 结果 (1)实验获得的BA CE1基因与GenBank中的序列一致.(2)活性测定表明,空白对照组、标准BACE1阳性对照组、纯化的BACE1实验组的荧光强度分别为55.013 ±3.597、2639.548±207.190和1836.629±154.195,差异有统计学意义(F=78.681,P=0.000),其中后2组差异无统计学意义(P＞0.05).(3)Western blotting结果显示转染的BACE1在细胞内可切割底物APP并产生CTF-APP条带. 结论本实验方法可成功获取BA CE1基因片段并在HEK293细胞中高效表达,同时具备生物学活性,可为AD临床药物的开发提供物质基础.
목적 탐토일충재상용진핵세포계중표체、순화화감정아이자해묵병(AD)상관단백-β-분비매(BACE1)적방법. 방법 재종인뇌기인고중획취BA CE1서렬병성공확증적기출상,장기삽입대유증강록색형광단백(EGFP)적표체재체pEGFP-c3중.장BACE1/pEGFP-c3중조질립용지질체전염지HEK293세포중표체,재통과TALON친화색보법분리순화급매절득도BACE1단백.용Western blotting급형광공진능량공진전이(FRET)법감정BACE1적표체화체외활성.동시장BACE1/pEGFP-c3질립여표체정분양전체단백(APP)적중조질립(APP/pDsRed-Monomer-N1)공전염HEK293세포,재이Western blotting법검측BACE1절할저물APP적효과. 결과 (1)실험획득적BA CE1기인여GenBank중적서렬일치.(2)활성측정표명,공백대조조、표준BACE1양성대조조、순화적BACE1실험조적형광강도분별위55.013 ±3.597、2639.548±207.190화1836.629±154.195,차이유통계학의의(F=78.681,P=0.000),기중후2조차이무통계학의의(P＞0.05).(3)Western blotting결과현시전염적BACE1재세포내가절할저물APP병산생CTF-APP조대. 결론 본실험방법가성공획취BA CE1기인편단병재HEK293세포중고효표체,동시구비생물학활성,가위AD림상약물적개발제공물질기출.
Objective To introduce a practical method that can be used to efficiently express,purify and identify Alzheimer's disease (AD) related beta-site app-cleaving enzyme 1 (BACE1) in common eukaryotic cells.Methods BACE1 cDNA was fished out from human brain cDNA library and ligated into the pEGFP-c3 expression vector,and then,the recombinant plasmid was transfected into the HEK293 cells.The BACE1 protein was purified with TALON Mental Affinity Resins column.The target protein was identified by Western blotting and fluorescence resonance energy transfer (FRET).BACE1 Activity Assay Kit was employed to test the activity of purified BACE1 in vitro.The recombinant BACE1/pEGFP-c3 plasmid and amyloid precusor protein (APP)/pDsRed-Monomer-N1 plasmid were co-transfected to the HEK293 cells and the cleavage activity of BACE1 in the cells was identified by Western blotting.Results The sequencing data of the obtained BACE1 gene were identical with those in GenBank.Activity test showed that the fluorescent values of blank controls,expressed BACE1 and standard BACE1 were 55.013±3.597,1836.629±154.195 (n=3) and 2639.548±207.1901 (n=3),respectively;as compared with the control group,significant differences were noted in both of the two groups (F=78.681,P=0.000);however,there is no significant difference between expressed BACE1 and standard BACE1 groups (P＞0.05).Westem blotting showed the co-transfected BACE1 could cleave APP in HEK293 cells and the CTF-APP band was detectable.Conclusion A practical protocol is established for high expression,purification and identification of BACE1 in HEK293 cells,which is helpful to obtain BACE1,an important molecular target in AD research and treatment.