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Construction and characterization of an anti‐asialoglycoprotein receptor single‐chain variable‐fragment‐targeted melittin
Author(s) -
Zhao Xiaorong,
Yu Zhen,
Dai Wentao,
Yao Zhenjiang,
Zhou Wei,
Zhou Weiping,
Zhou Junli,
Yang Yonggang,
Zhu Yinchang,
Chen Sidong,
Cao Limin
Publication year - 2011
Publication title -
biotechnology and applied biochemistry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.468
H-Index - 70
eISSN - 1470-8744
pISSN - 0885-4513
DOI - 10.1002/bab.57
Subject(s) - asialoglycoprotein receptor , immunotoxin , melittin , recombinant dna , fusion protein , microbiology and biotechnology , cytotoxicity , biology , flow cytometry , antibody , cytolysis , single chain variable fragment , in vitro , biochemistry , peptide , immunology , hepatocyte , gene
Antibody–therapeutic agent conjugation to be delivered specifically to tumor cells is required for many target‐based therapeutic strategies. In the present study, a recombinant immunotoxin was constructed by which melittin was fused to an anti‐asialoglycoprotein receptor (ASGPR) single‐chain variable fragment antibody (C1), and targeting ability and cytolytic efficacy of the fusion protein were studied. Our results suggested that the recombinant 29.4 kDa protein C1M was expressed in Escherichia coli as a soluble style. Binding of C1M to the surface of hepatocellular carcinoma (HCC) cells was confirmed by both immunohistochemistry and flow cytometry assays. C1M kept the hemolytic activity of melittin and exhibited cytolytic capacity to HepG2 cells at a concentration of 1.5 µg/mL, under which erythrocytes would not be lysed. The effects were greatly inhibited by coadministration with asialoorosomucoid, a natural ligand for ASGPR. These results suggested that C1M conferred targeting and ASGPR‐specific cytotoxicity to HCC cells. This work makes it possible to further investigate its antihepatoma efficacy in vivo .