Recombinant hHscFv-RC-RNase protein derived from transgenic tobacco acts as a bifunctional molecular complex against hepatocellular carcinoma.

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 HAb25 (hHscFv) fused to a cytotoxic ribonuclease from Rana catesbeiana (RC-RNase) 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 Southern blot analysis, and the immunocompetence of the recombinant his-HR protein was confirmed by Western 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-2H-tetrazolium bromide assay. Data indicated that the his-HR protein had stronger specific binding affinity to HepG2 (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 SMMC7721 and HepG2 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.