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浙江大学学报（农业与生命科学版）
浙江大学学报(农业与生命科学版)  2011, Vol. 37 Issue (4): 380-386    DOI: 10.3785/j.issn.1008-9209.2011.04.004
生物科学与技术     
鹅α干扰素基因在COS-7细胞中的瞬时表达及抗病毒功能初探
刘 菲1，2,唐卫杰1,程安春1，2,汪铭书1，2,赵婷婷1,高丽芹1
１ .四川农业大学动物医学院,四川 雅安 ６２５０１４; ２ .四川农业大学动物医学院动物疫病与人类健康四川省重点实验室,四川 雅安 ６２５０１４
Transient expression of goose IFN‐α gene in COS-7 cells and primary investigation of its antiviral function
LIU Fei１, ２ , TANG Wei-jie１ , CHENG An-chun１ ,２ , WANG Ming-shu１ ,２ , ZHAO Ting-ting１ , GAO Liqin １
（1 . College of Veterinary Medicine , Sichuan A gricultural University , Y a′an , Sichuan 625014 , China ; 2 . Key L aboratory o f A nimal Diseases and Human Health of Sichuan Prov ince , College of Veterinary Medicine , Sichuan A gricultural University , Y a′an , Sichuan 625014 , China）
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摘要： 为了研究鹅α 干扰素( goIFN‐α) 基因的特性和功能,将天府肉鹅的α 干扰素基因克隆到pcDNA３ .１( ＋ )真核表达载体上,构建重组质粒pcDNA３ .１‐goIFN‐α ,脂质体介导将其转入COS‐７ 细胞中;应用间接免疫荧光、酶联免疫吸附测定( enzyme‐linked immunosorbent assay ,ELISA) 和免疫印迹(Western‐blot)法检测goI FN‐α 基因在COS‐７ 细胞中的转录和表达情况,并对COS‐７ 细胞表达上清液进行抗病毒活性检测.结果表明:目的蛋白在转染１２ h 后就可以被检测出,３６ h 前目的蛋白在上清液中快速增加,之后增加速度变慢;重组质粒能够在COS‐７ 细胞内得到高效表达,表达的蛋白分子质量为３８ku ,比预测的分子质量(约２１ ku)大;间接免疫荧光显示,细胞内出现黄绿色荧光,胞核附近最亮;表达产物经细胞病变抑制法测定,显示出具有明显的抗猪水泡性口炎病毒(VSV)及小鹅瘟病毒(GPV)的活性.
Abstract: To elucidate the character and functions of goose interferon‐alpha gene ( I FN‐α ) , the gene IFN‐α of Tianfu meat goose open reading frame ( ORF) was cloned into eukaryotic expression vector pcDNA３ .１ ( ＋ ) to generate the recombinant plasmid pcDNA３ .１‐goIFN‐α , which was then transfected into the COS‐７ cells by lipofectin transfection . Then , its expression and localization in COS‐７ cells were determined by indirect immunofluorescence , enzyme‐linked immunosorbent assay and Western‐blot assay , and the antiviral activity of supernatant from transfected COS‐７ cells was also determined . The results indicated that goIFN‐α protein was fist detected at hour １２ post‐transfection , which was rapid increase in the supernatant before ３６ h , thereafter it became slow . The recombinant plasmid could be
highly expressed in COS‐７ cells , and a protein of ３８ ku in transfected COS‐７ cells was also detected , which was larger than the predicted molecular mass of ２１ ku . The indirect immunofluorescence showed that the cells appeared yellow‐green fluorescence , the fluorescent intensity in which was the most highest nearby nucleus . The antiviral activities of supernatant against vesicular stomatitis virus ( VSV) and gosling plague virus (GPV) were obvious , as verified by method of cytopathic effect inhibition test .
出版日期: 2011-07-20
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刘 菲
唐卫杰
程安春
汪铭书
赵婷婷
高丽芹

引用本文:

刘 菲,唐卫杰,程安春,汪铭书,赵婷婷,高丽芹. 鹅α干扰素基因在COS-7细胞中的瞬时表达及抗病毒功能初探[J]. 浙江大学学报(农业与生命科学版), 2011, 37(4): 380-386.

LIU Fei,TANG Wei-jie,CHENG An-chun,WANG Ming-shu,ZHAO Ting-ting,GAO Li-qin. Transient expression of goose IFN‐α gene in COS-7 cells and primary investigation of its antiviral function. Journal of Zhejiang University: Agric. & Life Sci., 2011, 37(4): 380-386.

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http://www.zjujournals.com/agr/CN/10.3785/j.issn.1008-9209.2011.04.004    或    http://www.zjujournals.com/agr/CN/Y2011/V37/I4/380

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