Expression And Biological Activity Detection Of Recombinant Porcine IL-2

【Objectiv】Interleuk in 2 (IL-2) is an important cytokine released from activated T lymphocytes, which activated lymphokine actived killer (LAK) cells and natural killer (NK) cells , and promoted cytokine production of helper T Cells and natural killer cells , and stimulated T cell growth in vitro and so on . It plays a important role in body immune regulator.The clone of IL-2 gene and detection of its biological activities is very useful. This study aims to product recombinant porcine interleukin 2 and construct a new type gene-engineering vaccine.【Method】The total RNA in the lymphocytes stimulated with concanavalin A from the peripheral blood of porcine as a template, used the reverse transcription chain reaction (RT-PCR) amplified and cloned cDNA of porcine interleukin 2,subcloned mature protein gene of porcine interleukin 2 into eukaryotic expression vector pPICZαA, constructed recombinant eukaryotic expression vector, established recombinant porcine interleukin 2 express system after transformed into Pichia Pastoris X-33 yeast strain by electroporation, detected bioactivity of production with MTT assay.【Results】With the reverse transcription chain reaction (RT-PCR), the gene of porcine interleukin 2 was amplified (approximately 500bp), from the total RNA in the lymphocytes stimulated with concanavalin A from the peripheral blood of porcine. Then the amplified fragment was inserted into pMD-18-T vector and transformed into DH5α.The positive clone contained recombinant plasmid pMD18-T-PoIL-2 was obtained after detected by picked up plaques, agarose gel electrophoresis, digested with EcoRⅠ/SalⅠand sequenced. Then used the recombinant plasmid as a template to amplify mature protein gene of porcine interleukin 2,and cloned into eukaryotic expression vector pPICZαA and transformed into DH5α,detected by picked up plaques and digested with XhoⅠ/XbaⅠ,it is showed eukaryotic expression vector pPICZαA-PoIL-2 contained mature protein gene of porcine interleukin 2.The resulted recombinant expression vector was transformed into Pichia Pastoris X-33 yeast strain by electroporation obtained engineering Pichia Pastoris strain X-33/pPICZα-A-PoIL-2 by screened and PCR. The transformants were induced to secretive express and SDS-PAGE showed that expression product was about 18ku. All results above showed recombinant porcine interleukin 2 express system was established successfully. The detection of MTT verified recombinant porcine interleukin 2 have some bioactivities to lymphocytes of the peripheral blood of porcine.【Conclusion】This study established porcine interleukin 2 express system successfully and recombinant porcine interleukin 2 have some bioactivities to lymphocytes of the peripheral blood of porcine.