摘要
用已构建的表达载体pPELB-B3,在大肠杆菌Rosetta中可溶性表达人源抗谷胱甘肽(GSH)单链抗体B3(scFv-B3),经N i2+螯合亲和层析纯化后,用点印迹法验证了其与GSH结合的特异性.将水相合成的半导体纳米粒子(半导体量子点,QD s)在N-羟基琥珀酰亚胺(NHS)和1-乙基-3-(3-二甲基氨基丙基)碳酰二亚胺盐酸盐(EDC)的作用下,与scFvs连接.光谱分析和膜印迹结果表明,scFvs成功地共价连接到QD s表面,所得的QD-scFvs复合物能够较好地识别GSH.荧光显微镜观察QD-scFvs与人乳腺癌细胞MCF-7的作用结果,初步判断QD-scFvs能够跨膜进入细胞.
In order to generate catalytic antibodies with glutathione peroxidase （GPX） activity, the clone B3 that bound specifically to glutathione （GSH） was selected from the phage display antibody library[human syn-thetic VH ＋ VL single-chain Fv fragment （scFv） library ] and the expression vector of scFv-B3 was constructed in previous study. The expression vector pPELB-B3 constructed was transformed into the Escherichia eoli Rosetta to express the human anti-GSH single chain fragments variable （scFvs） antibodies. The scFv-B3 was purified by Ni^2＋ -immobilized metal affinity chromatography （IMAC）. 3-Mercaptopropyl acid-stabilized CdTe quantum dots synthesized in aqueous solution were used to conjugate with scFvs. Spectra analysis show that the fluorescent of QD-seFvs undergo a blue-shift in the emission peak, which suggest that the seFvs have been ef- fectively bound to the scFvs, via covalent conjugation. It is also suggested by the result of the simple new method of dot blot. The resuhs of membrane dot indicate that the QD-scFvs can specifically recognize the GSH. The status of the QD-seFvs acting on the MCF-7 cells was observed under fluorescence microscope. The results show that QD-seFvs can enter the cytoplasm across the membrane.
出处
《高等学校化学学报》
SCIE
EI
CAS
CSCD
北大核心
2009年第3期506-509,共4页
Chemical Journal of Chinese Universities
基金
吉林省科学技术厅基金(批准号:200705380和20070726)
中国博士后基金(批准号:2004036395)
吉林大学本科生创新性实验计划资助
关键词
量子点
人源单链抗体
谷胱甘肽过氧化物酶(GPX)
共价结合
印迹
Quantum dot
Human single chain fragments variable （scFvs）
Glutathione peroxidase （GPX）
Covalent conjugation
Dot blot
