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Recombinant h H sc F v– RC ‐ RN ase protein derived from transgenic tobacco acts as a bifunctional molecular complex against hepatocellular carcinoma
Author(s) -
Cui Lijie,
Peng Huizhen,
Zhang Ran,
Chen Yuhui,
Zhao Lingxia,
Tang Kexuan
Publication year - 2012
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.1039
Subject(s) - nicotiana tabacum , microbiology and biotechnology , recombinant dna , biology , fusion protein , cell culture , transgene , western blot , hepatocellular carcinoma , blot , biochemistry , gene , cancer research , genetics
Hepatocellular carcinoma ( HCC ) is a common clinical primary malignant tumor; however, efficient drugs for the treatment of HCC are still lacking at the present time. To develop a new approach for liver cancer therapy, we designed a chimeric gene ( his‐HR ) encoding a single‐chain variable fragment of human HA b25 (h H sc F v) fused to a cytotoxic ribonuclease from Rana catesbeiana ( RC ‐ RN ase) and expressed the corresponding fusion protein in transgenic tobacco ( Nicotiana tabacum ). Eleven positive transgenic plant lines were identified from 204 regenerated tobacco plants by PCR and S outhern blot analysis, and the immunocompetence of the recombinant his‐ HR protein was confirmed by W estern blotting. The expression levels of his‐ HR protein ranged from 0.75 to 1.99 µg/g in the fresh tobacco leaves. To characterize the bifunction of the expressed his‐ HR protein in tobacco, binding specificity and cell toxicity to several cell lines were examined by the indirect immunocytochemical streptavidin–biotin complex method and 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyl‐2 H ‐tetrazolium bromide assay. Data indicated that the his‐ HR protein had stronger specific binding affinity to H ep G 2 (human liver HCC cell line) than to the other tumor cell lines and normal liver cell line, and the capacity to kill the HCC cell lines SMMC 7721 and H ep G 2 with an half maximal inhibiting concentration of 2.0 and 2.4 nM, respectively. The results suggest that recombinant bifunctional his‐ HR protein derived from transgenic plants may provide a novel strategy to treat HCC in the future.