【摘要】 目的:制备抗人黑素瘤细胞单链抗体与碲化镉量子点(CdTe guantun dot,CdTe)连接的纳米探针,观察其对恶性黑素瘤细胞的特异性结合效果。方法:将抗人黑素瘤神经节苷脂单链抗体(anti-human melanoma ganglioside single chain varia-ble fragment antibody,GD/ScFvMEL)基因克隆到pET32a(+)载体中,然后在BL21(DE3)细菌中进行诱导表达,采用变性法纯化表达的蛋白,再用改良的透析法复性目的蛋白,SDS-PAGE分析表达的GD/ScFvMEL抗体蛋白。制备的GD/ScFvMEL抗体与量子点连接制备成纳米探针GD/ScFvMEL-QDs,然后与黑素瘤A375细胞株及胃癌MGC-803细胞株孵育,观察纳米探针与肿瘤细胞结合的特异性。结果:PCR、双酶切和序列测定证实重组子的拼接完全正确,SDS-PADE结果显示,GD/ScFvMEL抗体的表达量达40%。纯化复性后的GD/ScFvMEL抗体连接量子点制备GD/ScFvMEL-QDs纳米探针,经扫描电镜、X衍射分析、荧光光谱和SDS-PAGE分析证实纳米探针的成功制备。GD/ScFvMEL-QDs纳米探针能特异性结合A375黑素瘤细胞,不与胃癌细胞MGC-803细胞结合。结论:成功制备抗人黑素瘤神经节苷脂单链抗体-量子点纳米探针,该探针可特异性结合黑素瘤细胞。更多还原

【Abstract】 Objective:To prepare a nanoprobe,anti-human melanoma ganglioside single chain variable fragment (GD/ScFvMEL) antibody conjugated with CdTe quantum dot,and to observe its ability to specifically bind human malignant melanoma cells.Methods:The GD/ScFvMEL gene was cloned into pET32a (+),and the plasmid was then transformed into E.coli BL21 (DE3) for GD/ScFvMEL protein antibody expression.The expressed GD/ScFvMEL antibody was purified by denaturing method and further refolded by modified dialysis method.The purified GD/ScFvMEL antibody was analyzed by SDS-PAGE.The GD/ScFvMEL-QDs nanoprobe was prepared by conjugating GD/ScFvMEL antibody with CdTe quantum dot,and its specificity was observed by incubating with MGC-803 cells and melanoma A375 cells.Results:The recombinant pET32a-GD/ScFvMEL was constructed and confirmed by PCR,restriction endonuclease analysis and DNA sequencing.The proportion of expressed GD/ScFvMEL antibody in total bacteria proteins was about 40% as detected by SDS-PAGE.The purified-and refolded-GD/ScFvMEL antibody was effectively conjugated with CdTe quantum dot,and the resulting GD/ScFvMEL-QDs nanoprobe was successfully prepared.The GD/ScFvMEL-QDs nanoprobe could specifically bind melanoma A375 cells,but could not bind stomach cancer MGC-803 cells.Conclusion:We have successfully prepared an anti-human melanoma ganglioside single-chain antibody-CdTe quantum dot nanoprobe,which can specifically bind melanoma cells.更多还原