| Human interferonα2b(hIFN-α2b)processes a variety of biological functions, including broad-spectrum antiviral, anti-cell division and immunomodulatory activities. In the clinic, it′s mainly for the treatment of viral hepatitis and cancer diseases. At present, the clinical application of interferon preparations are mainly obtained from the E.coli expression system, and the main administration is injection. Because the half-life is short, interferon would soon disappeare from the blood after the intravenous injection. It is difficult to maintain a high plasma concentration in clinical applications. The problem can only be solved through increasing the frequency and dosage. So that it is easy to produce interferon-induced antibody and increase the toxic side effects. The injected administration is inconvenient and expensive for patients. Therefore, it was need to develop new formulations of interferon.Cholera toxin B subunit (CTB) is a non-toxic binding subunit of cholera toxin, it can specific bind with the gangliosides GM1 receptor which placed on the membrane of nucleated cells. CTB can be used for carrier protein, so exogenous peptides may be combined with CTB in order to increase the opportunity to contact with the cell membrane receptor. Based on these characteristics of CTB, today it has been applied to the oral vaccines and oral drugs research.In recent years, the use of transgenic plants producing pharmaceutical proteins are able to in-depth study. Compared with the traditional system, the plant expression system is efficient, economical and convenient and so on. In addition, the most important edible plants also have characteristics. Therefore, the use of transgenic plants as bioreactors production of oral pharmaceutical proteins are being increasingly subject to national attention. It was report that plants can express high levels of functional recombinant proteins.For transgenic plant material can be divided into plants, explant and callus cells, and so on. Plant tissue cell culture studies have decades of history. Compared with conventional agricultural production, it is not impacted by the ecological environment and the pests. Ginseng is a precious medicinal plants, has a unique nutritional and medicinal value. The common receptors of transgenic plants are tobacco, rice, tomato, potato and so on. The expression of human interferon in ginseng has not been reported.In this study, human interferonα2b and cholera toxin B subunit gene was fused by gene fusion, which CTB was located at the N-terminal and hIFN-α2b was placed at the C-terminal. They were linked by a flexible peptide chain. First, the fusion gene was constructed into the pMD18-T vector to form the pMDCI. The pMDCI and pBI121 was digested with BamH I and Sac I. The target gene fragment and vector fragments were recovered by agarose gel electrophoresis and connected to receive expression vector. The recombinant plasmid was confirmed by PCR and restriction digestion, finally obtaining the plant expression vector pBICI.The pBICI was transformed into Agrobacterium tumefaciens stain LBA4404 directly by the freeze-thaw method. Subsequently, Agrobacterium tumefaciens carrying pBICI was used to transform ginseng cells. Ginseng cells were co-cultivated for 48～72 h with Agrobacterium tumefaciens carrying pBICI. Ginseng cells were then rinsed with sterilized water added 500 mg/L cefotaxime to kill the Agrobacterium tumefaciens on the surface of cells, and blotted dry on a sterilized paper towel, and placed onto a 67-V medium added 500 mg/L cefotaxime for recovery. After recovery period of 7 days, the ginseng cells were transferred to a 67-V medium added 500mg/L cefotaxime and 50mg/L G418 to select transgenic progeny. About 3～4 months later, G418-resistant ginseng cells regenerants were removed to a 67-V medium containing 25 mg/L G418. We have obtained the cell line of ginseng which carrying CTB-hIFN-α2b fusion gene. Transgenic cells were checked for the presence of target gene using PCR and RT-PCR. Samples containing the target gene showed a clear band in site of 880 bp by agarose electrophoresis analysis. The results demonstrated that an amplified product with expected size was present in G418 resistant cells and absent in non-transformed cells. Expression products were carried out Western blot, anti-viral activity and GM1 binding assay analysis, the results showed that the expression protein of a human interferon and CTB protein properties biological activity.From these results, it was concluded that fusion proteins can be expressed in transgenic ginseng callus cells and are able to keep the activity.
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