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Human interferon (IFN) is a type of cytokine that regulates the immune system’s response to viral and bacterial infections. Recombinant IFN-α has been approved for use in the treatment of a variety of viral infections as well as an anticancer medication for various forms of leukemia. The objective of the current study is to produce a functionally active recombinant human IFN-α2a from transgenic <i>Raphanus sativus</i> L. plants. Therefore, a binary plant expression construct containing the IFN-α2a gene coding sequence, under the regulation of the cauliflower mosaic virus 35SS promoter, was established. <i>Agrobacterium</i>-mediated floral dip transformation was used to introduce the IFN-α2a expression cassette into the nuclear genome of red and white rooted <i>Raphanus sativus</i> L. plants. From each genotype, three independent transgenic lines were established. The anticancer and antiviral activities of the partially purified recombinant IFN-α2a proteins were examined. The isolated IFN-α2a has been demonstrated to inhibit the spread of the Vesicular Stomatitis Virus (VSV). In addition, cytotoxicity and cell apoptosis assays against Hep-G2 cells (Human Hepatocellular Carcinoma) show the efficacy of the generated IFN-α2a as an anticancer agent. In comparison to bacterial, yeast, and animal cell culture systems, the overall observed results demonstrated the efficacy of using <i>Raphanus sativus</i> L. plants as a safe, cost-effective, and easy-to-use expression system for generating active human IFN-α2a.