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

Area of Science:

• Biotechnology
• Molecular Biology
• Oncology

Background:

• Hepatocellular carcinoma (HCC) is a prevalent malignancy with limited effective therapeutic options.
• Developing novel strategies for HCC treatment is a critical unmet medical need.

Purpose of the Study:

• To design and express a novel bifunctional fusion protein, his-HR, in transgenic tobacco for potential HCC therapy.
• To evaluate the binding specificity and cytotoxic activity of the recombinant his-HR protein against HCC cell lines.

Main Methods:

• A chimeric gene (his-HR) encoding a single-chain variable fragment (hHscFv) and a cytotoxic ribonuclease (RC-RNase) was constructed and expressed in transgenic tobacco (Nicotiana tabacum).
• Positive transgenic lines were confirmed using PCR and Southern blot analysis.
• Protein expression, immunocompetence, binding affinity, and cytotoxicity were assessed using Western blotting, indirect immunocytochemistry, and MTT assays.

Main Results:

• Eleven positive transgenic tobacco lines were generated, expressing the his-HR protein at levels ranging from 0.75 to 1.99 µg/g.
• The his-HR protein demonstrated stronger specific binding to the HepG2 HCC cell line compared to other tested cell lines.
• The protein exhibited potent cytotoxicity against SMMC7721 and HepG2 HCC cell lines with half maximal inhibiting concentrations of 2.0 nM and 2.4 nM, respectively.

Conclusions:

• Recombinant his-HR protein expressed in transgenic plants possesses specific binding affinity and significant cytotoxic effects on HCC cells.
• This plant-derived bifunctional protein represents a novel and promising strategy for future hepatocellular carcinoma treatment.