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当前位置: 首页 > 文章 > 新疆野生盘羊ISG15基因的克隆表达 畜牧兽医学报 2013,44 (7) 1023-1029
Position: Home > Articles > Cloning and Expression of the ISG15 Gene of Xinjiang Wild Argali Chinese Journal of Animal and Veterinary Sciences 2013,44 (7) 1023-1029

新疆野生盘羊ISG15基因的克隆表达

作  者：
鲁海富;沈文;刘恺;崔茹鹏;杨文;姜方配;孙延鸣
单  位：
石河子大学动物科技学院;乌鲁木齐市动物园
关键词：
野生盘羊;ISG15;克隆表达;活性检测
摘  要：
本研究旨在克隆表达新疆野生盘羊ISG15基因,并对其表达产物的活性进行检测。首先,利用RT-PCR和RACE技术克隆盘羊ISG15基因的cDNA全长序列,再将ISG15基因克隆至真核表达载体pPIC9K,经电击转化至GS115中并用甲醇进行诱导表达,用Ni 2+螯合亲和层析法对表达的蛋白进行纯化,最后通过淋巴细胞转化试验来检测表达蛋白的活性。结果表明:克隆得到的新疆野生盘羊ISG15基因cDNA全长为642bp,开放阅读框为474bp,编码157个氨基酸。经酵母重组菌株GS115/pPIC9K-ISG15表达,SDS-PAGE结果显示表达的蛋白约为33ku,表达的蛋白可用Ni 2+螯合亲和层析方法纯化,淋巴细胞增殖试验结果显示,表达的ISG15蛋白能够显著地刺激淋巴细胞增殖(P<0.05),表明表达的蛋白具有生物学活性。
译  名：
Cloning and Expression of the ISG15 Gene of Xinjiang Wild Argali
作  者：
LU Hai-fu1,SHEN Wen1,LIU Kai 2,CUI Ru-peng1,YANG Wen1,JIANG Fang-pei 1,SUN Yan-ming1*(1.College of Animal Science and Technology,Shihezi University,Shihezi 832003,China;2.Zoo of Urumqi,Urumqi 830094,China)
关键词：
Wild argali;ISG15;cloning and expression;activity detection
摘  要：
The aim of this study was to clone and express the ISG15 gene of Xinjiang Wild argali,and to detect the activity of the expression product.The full length cDNA of Xinjiang Wild argali ISG15 gene was cloned by RT-PCR and RACE,then ISG15 gene was cloned into eukaryotic expression vector pPIC9K,the pPIC9K-ISG15 was transformed into Pichia Pastoris GS115 yeast genome by electroporation and the expression was induced by methanol,the expressed protein was purified by Ni2+ chelate affinity chromatography,finally the activity of the expressed protein was detected by lymphocyte transformation test.The result showed that the full length cDNA of Xinjiang Wild argali ISG15 was 642bp,the open reading frame was 474bp,encoded 157 amino acids.The yeast recombinant strains GS115/pPIC9K-ISG15 was used to express ISG15,SDSPAGE analysis showed the expressed protein was 33ku,the expressed protein could be purified by Ni 2+chelate affinity chromatography.The lymphocyte proliferation test results showed that the expressed product could significantly stimulate lymphocyte proliferation(P<0.05),which proved the expressed protein had biological activity.

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