牦牛IFN-β基因在毕赤酵母中的分泌表达  

Secretion expression of yak IFN-β gene in the Pichia pastoris

作　　者：王生富[1,2] 孙晓林[1] 段风云[2] 何延华[1] 闫鸿斌[2] 景志忠[1,2] 

机构地区：[1]甘肃农业大学动物医学院,甘肃兰州730070 [2]中国农业科学院兰州兽医研究所,家畜疫病病原生物学国家重点实验室,甘肃省动物寄生虫病重点实验室,甘肃兰州730046

出　　处：《甘肃农业大学学报》2010年第5期6-10,共5页Journal of Gansu Agricultural University

基　　金：国家高新技术“863”项目(2006AA10A203);国家支撑计划项目(2006BAD31B03)

摘　　要：用真核表达引物从pGEM-yakIFN-β重组质粒中扩增出牦牛IFN-β基因,将目的基因和真核表达载体pPIC9K连接转入E.coli的JM109中,得到pPIC9K-yakIFN-β重组表达质粒.通过电击法将经SalⅠ线性化的pPIC9K-yakIFN-β质粒转化到巴斯德毕赤酵母GS115感受态细胞中,采用G418抗性梯度法筛选得到多拷贝重组菌株,利用甲醇诱导表达,分泌表达产物用MTT法检测其生物学活性.经SDS-PAGE电泳分析表明:表达出约22ku大小分泌于胞外的牦牛IFN-β蛋白分子量,比理论值稍大;MTT结果表明:纯化的重组yak-IFN-β蛋白具有促进淋巴细胞增殖的活性.The yak IFN-β gene was amplified from pGEM-yak IFN-β with expression primer,the yak IFN-β gene was inserted into the expression vector pPIC9K and transformed into E.coli.Positive recombinants were selected,sequenced and named pPIC9K-yak IFN-β.It was linearized by SalⅠ,then the linear DNA transformed into Pichia pastoris GS115by electroporation,multi-copy recombinant strains were screened by G418.The expression of recombinant fusion protein was induced by methanol and bioactivity of yak IFN-β was measured by MTT assays.The secreted supernatant was tested by SDS-PAGE.The results showed that the secreted yak IFN-β protein was about 22ku and had been expressed successfully in Pichia pastoris through the shake culture respectively.The high-level expression of yak IFN-β in the Pichia pastoris was observed in the supernantant with a little larger molecular weight than that of natural yak IFN-β.The results of MTT showed that the recombinant yak IFN-β with obvious biological activity.

关 键 词：牦牛 IFN-β基因 巴斯德毕赤酵母 分泌表达 

分 类 号：S823.85[农业科学—畜牧学]