الفهرس 
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Abstract
The plant expression constructs were used for the nuclear transformation of Raphanus sativus and Eruca sativa plants via Agrobacterium-mediated floral dip transformation and the bacterial expression construct was used for the production of recombinant hu.IFNα2a protein from BL21(DE3) bacteria. In order to express hu.IFNα2a protein in bacteria, it was decided to clone the coding sequence of the hu.IFNα2a gene into the bacterial expression vector pET22b(+).
A bacterial expression construct named pET-IFNα2a.His was generated and transformed into BL21(De3)competent bacteria. Recombinant IFNα2a protein was expressed in bacteria via IPTG induction of protein expression. The bacterial protein extracts were then characterized by SDS-PAGE and Western blot for the expresseion of IFNα2a protein. The results of SDS-PAGE and western blot analysis showed that IFNα2a was expressed in bacteria. Bioactivity assays using this bacterial protein extract (ELISA) showed that IFNα2a is functionally expressed in bacteria. Moreover, the IFNα2a coding sequence was also cloned into the plant expression vector pTRA-K (resistant to Kanamycin) and pTRA-PT (resistant to phosphinotricin). pTRA-K. IFNα2a was used for wild type white and red radish transformation events. However, pTRA-PT- IFNα2a was used for Eruca sativa transformation. At least three independent transgenic lines from each genotype were generated. The existence and expression of IFNα2a in transgenic plants were assessed by PCR and RT-PCR analyses, respectively. It was indicated that IFNα2a protein is expressed in planta. Enzyme Linked Immunosorbant Assay (ELISA) showed that IFNα2a is functionally expressed and produced in transgenic plants.
Total and partially pyrified IFNα2a protein extracts from transgenic plants were used for evaluating the efficiency of the recombinantly produced IFNα2a protein as antiviral and antitumor agent. The cytotoxicity assays using infected Vero cell lines with vesicular somatitis virus (VSV) and the produced interferon showed that IFNα2a produced from plants have high enhanced activity as antiviral agent. Moreover, the evaluation of recombinant IFNα2a protein activity using HePG2-cells revealed that the IFNα2a produced from all transgenic lines has high activity in reducing cell growth and cause apoptosis of these cell lines. Taken together, IFNα2a can be safely produced from plants and used as antiviral and antitumor agent.