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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。
OBJECTIVE: To prepare nano-probes with single-chain anti-human melanoma cells linked with CdTe guantun dots (CdTe) and observe their specific binding to malignant melanoma cells. Methods: The gene of anti-human melanoma ganglioside single chain variable fragment (GD / ScFvMEL) was cloned into pET32a (+) vector and then transformed into BL21 (DE3) The expressed protein was purified by denaturation method, then the target protein was refolded by modified dialysis method, and the expressed GD / ScFvMEL antibody protein was analyzed by SDS-PAGE. The prepared GD / ScFvMEL antibody was connected with the quantum dot to prepare the nano-probe GD / ScFvMEL-QDs, and then incubated with the melanoma A375 cell line and the gastric cancer MGC-803 cell line to observe the specificity of the nano-probe binding to tumor cells. Results: PCR, double digestion and sequencing confirmed that the recombinants were completely spliced. The results of SDS-PAGE showed that the expression of GD / ScFvMEL antibody reached 40%. The GD / ScFvMEL-QDs nanodialysis probes were prepared by the connection of the purified renaturation GD / ScFvMEL antibody, and the successful preparation of nanoprobe was confirmed by scanning electron microscopy, X-ray diffraction analysis, fluorescence spectroscopy and SDS-PAGE analysis. The GD / ScFvMEL-QDs nanoprobe can specifically bind A375 melanoma cells without binding to gastric cancer cell line MGC-803. CONCLUSION: Anti-human melanoma ganglioside single chain antibody-quantum dot nanoprobe has been successfully prepared, which can specifically bind to melanoma cells.