【摘要】 利用大肠杆菌表达系统制备了重组融合蛋白antiEGFR/MEL,并用Ni2+层析柱对其进行了纯化.该重组蛋白中抗表皮生长因子受体(antiEGFR)单链抗体(scFv)主要靶向喉癌细胞中的EGFR,而蜂毒肽(MEL)主要抑制肿瘤细胞增殖.采用SDS-PAGE和Westernblot检测证明了antiEGFR/MEL的有效表达.共聚焦显微镜和流式细胞术实验结果表明,antiEGFR/MEL可与Hep-2肿瘤细胞有效结合,而几乎不与EGFR阴性的Jurkat细胞结合.噻唑蓝(MTT)检测结果说明,antiEGFR/MEL可有效抑制人喉癌细胞Hep-2的增殖.以上结果表明,antiEGFR/MEL能够有效靶向EGFR阳性肿瘤细胞,并有效抑制肿瘤细胞增殖,有望应用于EGFR靶向肿瘤治疗.更多还原

【Abstract】 Laryngeal carcinoma is poor prognosis and patients with laryngeal carcinoma usually present late leading to the reduced treatment efficacy and high rate of recurrence. Incidence and mortality rates of laryngeal carcinoma are equivalent,suggesting a failure of current therapies,despite the variety of combined modality approaches which have been introduced to complement surgical treatment. A successful cancer therapeutic should target tumours specifically with limited systemic toxicity. Herein,the recombinant antiEGFR / MEL protein was expressed in Escherichia coli( E. coli),refolded and purified on an immobilized Ni2 +-affinity chromatography column. In the protein,anti epidermal growth factor receptor( EGFR) single chain antibody was used to target the EGFR in the laryngeal cancer cell,and the melittin was used to mediate inhibition of cell growth.Sodium dodecyl sulfate-polyacrylamide gel electrophoresis( SDS-PAGE) and Western blotting analysis revealed that antiEGFR / MEL was sufficiently expressed. Confocal microscopy and flow cytometry demonstrated that antiEGFR / MEL bound specifically to Hep-2 cells,as almost no binding to Jurkat cells was observed under identical time and dosage conditions. MTT assay showed that antiEGFR / MEL suppressed the growth of Hep-2cells effecitively. Collectively,these results suggest that antiEGFR / MEL is biologically active and specific toward EGFR-positive tumor cells and may represent an effective EGFR-targeted cancer therapy.更多还原