靶向喉癌的蜂毒肽重组抗体的构建及鉴定

doi:10.7503/cjcu20140421

高等学校化学学报 ›› 2014, Vol. 35 ›› Issue (12): 2551.doi: 10.7503/cjcu20140421

靶向喉癌的蜂毒肽重组抗体的构建及鉴定

孙丽丽^1,², 王浩然^2,³, 徐一鸣³, 费丹^2,⁴, 付婷婷^2,⁵, 管国芳²()

1. 吉林省肿瘤医院头颈外一科, 长春 130012
2. 吉林大学第二临床医院耳鼻咽喉科, 长春 130041
3. 吉林大学动物医学学院, 长春 130062
4.吉林大学中日联谊医院超声科, 长春 130033
5. 日照市东港区人民医院, 日照 276800

收稿日期:2014-05-05 出版日期:2014-12-10 发布日期:2014-11-29
作者简介:联系人简介: 管国芳, 女, 博士, 教授, 主要从事喉癌分子生物学及基因治疗研究. E-mail:guan-guo@163.com
基金资助:
国家自然科学基金(批准号: 81101140)、国家“八六三”计划项目(批准号: 2012AA02A407)和吉林省科技发展计划重点项目(批准号: 20130206041NY)资助

Synthesis and Characterization of a Melttin Expressing Recombinant Antibody Targeting Human Laryngeal Cancer^†

SUN Lili^1,², WANG Haoran^2,³, XU Yiming³, FEI Dan^2,⁴, FU Tingting^2,⁵, GUAN Guofang^2,^*()

1. Head and Neck Surgery, Tumor Hospital of Jilin Province, Changchun 130012, China
2. Department of Otolaryngology, Head and Neck Surgery, Second Hospital of Jilin University, Changchun 130041, China
3. College of Veterinary Medicine, Jilin University, Changchun 130062, China
4. Department of Ultrasound, China-Japan Union Hospital of Jilin University, Changchun 130033, China
5. People’s Hospital of Donggang District of Rizhao, Rizhao 276800, China

Received:2014-05-05 Online:2014-12-10 Published:2014-11-29
Contact: GUAN Guofang E-mail:guan-guo@163.com
Supported by:
† Supported by the National Natural Science Foundation of China(No.81101140), the National High Technology Research and Development Program of China(No.2012AA02A407) and the Key Technologies Research and Development Program of Jilin Province, China(No.20130206041NY)

摘要/Abstract

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Western blot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.

关键词: 喉癌, 表皮生长因子受体, 单链抗体, 蜂毒肽, 原核表达

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-2 cells 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.

Key words: Laryngeal carcinoma, Epidermal growth factor receptor, Single chain antibody, Melittin, Prokaryotic expression

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孙丽丽, 王浩然, 徐一鸣, 费丹, 付婷婷, 管国芳. 靶向喉癌的蜂毒肽重组抗体的构建及鉴定. 高等学校化学学报, 2014, 35(12): 2551.

SUN Lili, WANG Haoran, XU Yiming, FEI Dan, FU Tingting, GUAN Guofang. Synthesis and Characterization of a Melttin Expressing Recombinant Antibody Targeting Human Laryngeal Cancer^†. Chem. J. Chinese Universities, 2014, 35(12): 2551.

图/表 5

Fig.1 Schematic illustration of the plasmid design

Fig.2 Western blot analysis of target protein M: low molecular weight protein marker; 1. antiEGFR/MEL induction in BL21(DE3) induced by 1.4 mmol/L IPTG; 2. antiEGFR/MEL expressed in the inclusion body fraction; 3. SDS-PAGE assay of purified antiEGFR/MEL; 4. Western blot assay of purified antiEGFR/MEL.

Fig.3 Identification of cellular binding activity of antiEGFR/MEL (A) Negative control of IFA analysis of the Hep-2 cells; (B) IFA analysis of the Hep-2 cells incubated with the antiEGFR/MEL; (C) negative control of IFA analysis of the Jurkat cells; (D) IFA analysis of the jurkat cells incubated with the antiEGFR/MEL; (E) negative control of confocal analysis of the Hep-2 cells; (F) confocal analysis of the Hep-2 cells incubated with the antiEGFR/MEL; (G) negative control of confocal analysis of the jurkat cells; (H) confocal analysis of the jurkat cells incubated with the antiEGFR/MEL.

Fig.4 Identification of cellular binding activity of antiEGFR/MEL (A) Negative control of flow cytometric analysis of the Hep-2 cells; (B) flow cytometric analysis of the Hep-2 cells incubated with the antiEGFR/MEL; (C) negative control of flow cytometric analysis of the Jurkat cells; (D) flow cytometric analysis of the jurkat cells incubated with the antiEGFR/MEL.

Fig.5 Specific cytotoxicity activity of antiEGFR/MEL

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靶向喉癌的蜂毒肽重组抗体的构建及鉴定

Synthesis and Characterization of a Melttin Expressing Recombinant Antibody Targeting Human Laryngeal Cancer^†

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靶向喉癌的蜂毒肽重组抗体的构建及鉴定

Synthesis and Characterization of a Melttin Expressing Recombinant Antibody Targeting Human Laryngeal Cancer†

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Synthesis and Characterization of a Melttin Expressing Recombinant Antibody Targeting Human Laryngeal Cancer^†