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

doi:10.7503/cjcu20140421

Abstract

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

CLC Number:

TrendMD:

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^†[J]. Chem. J. Chinese Universities, 2014, 35(12): 2551.

Figures/Tables 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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