Expression of Human Interferon α-2b Gene in Tobacco(Nicotiana tabacum L.)
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摘要: 应用PCR的技术从质粒pAIFN中扩增人干扰素α-2b(Human interferon α-2b,HuIFN α-2b)编码基因,将其连接到pBI121双元载体构建植物真核表达载体pBIFN;用冻融法将该载体转染根癌农杆菌LBA4404;并用叶盘浸染法转化烟草叶片,经转化的烟草叶片的组织培养,诱导愈伤获得再生植株。通过应用PCR,RT-PCR,Wes-tern blot和WISH/VSV方法检测获得的烟草再生植株,结果表明HuIFN α-2b基因已成功整合进烟草核基因组并表达出具有活性的HuIFN α-2b蛋白。本文对HuIFN α-2b基因在烟草核系统中的表达进行了研究,为进一步在烟草叶绿体系统中该基因的表达研究奠定了基础。Abstract: Polymerase Chain Reaction(PCR)amplifies the human interferon α-2b(HuIFN α-2b) gene encoding region from the plasmid pAIFN.The encoding region sequence was liga-sed into the pBI121 vector to construct plant eukaryotic expression vector pBIFN.The freeze-thaw method was used to transfect the vector pBIFN into Agrobacterium tumefaciens LBA4404 and then the leaf disc dip method was used to transform the A.tumefaciens LBA4404 embo-died with pBIFN into the tobacco(Nicotiana tabacum L.)leaf explants and plants were regene-rated from cultured transformed tobacco-leaf explants.The molecular analysis methods of PCR,RT-PCR,Western blot and the WISH/VSV were applied to confirm the regenerated tobacco plants and the results showed that the HuIFN α-2b gene was successfully integrated into the tobacco nuclear genome and HuIFN α-2b protein was expressed with activity.This paper documents the expression of the HuIFN α-2b protein in tobacco nuclear transformation system and implies that the potential application of HuIFN α-2b gene in tobacco chloroplast transformation system is feasible.
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