The Study Of Human Interferon-alpha 2b Expressed By Ginseng Callus Cells

Objective To construct the eukaryotic expression vector pBIFNincluding huIFN-α2b gene and to study the integration and expression ofhuman interfern-alpha 2b in Panax ginseng callus cells.Methds Human interferon-alpha 2b gene was amplified from therecombinant plasmid pBV889 by PCR and cloned into eukaryotic expressionplasmid pBI121 by DNA recombinant technology. Thus, the eukaryoticexpression vector pBIFN including huIFN-α2b gene was constructed. Andthen the pBIFN was transformed into Agrobacterium tumefaciens strainLBA4404. After that, huIFN-α2b gene was introduced into Ginseng calluscells via Agrobacterium-mediated transformation. The positive cells werescreened by 50 mg/L G418 67-V substrate. The integrated exogenous gene inthe isolated and purified genome of antibiotic resistance cells was amplified byPCR and identifeied through sequence analysis. RT-PCR was applied to detectthe transcription of human interferon-alpha 2b gene. Western blot detected theexpression of the huIFN-α2b. WISH-VSV system analysed the bioactivity ofthe expressed huIFN-α2b.Results The genome of the antibiotic resistance cells was extracted andthe objective gene was amplified by PCR. The result of sequence analysissuggested that the huIFN-α2b gene has been integrated into the Ginseng calluscells'genome. RT-PCR analysis shows that there are transcription products.WISH-VSV system assay shows that the expressed huIFN-α2b possessesrelatively lower bioactivity.Cnclusin I have successfully constructed the eukaryotic expressionvector pBIFN including huIFN-α2b gene and obtained the Ginseng callus cellswhich can express huIFN-α2b.