鸡生长激素在毕赤酵母中的表达、纯化和活性鉴定  

Expression,Purification and Biological Activity Identification of Chicken Growth Hormone in Pichia pastoris

作　　者：郁建锋[1,2] 何赛[1] 金泽楷 周欢庆[1] 董小敏[1] 龚道清 顾志良[1] YU Jian-feng HE Sai JIN Ze-kai ZHOU Huan-qing DONG Xiao-min GONG Dao-qing GU Zhi-liang(Changshu Institute of Technology , Changshu 215500, China Yangzhou University, Yangzhou 225009, China)

机构地区：[1]常熟理工学院,常熟215500 [2]扬州大学,扬州225009

出　　处：《中国畜牧兽医》2016年第11期2970-2975,共6页China Animal Husbandry & Veterinary Medicine

基　　金：国家自然科学基金(31072025);苏州市科技计划项目(SYN201416)

摘　　要：生长激素(growth hormone,GH)与受体结合调节靶细胞的功能在机体的生长发育及代谢过程中起着极其重要的作用。为获得高纯度具有生物学活性的鸡生长激素(chicken growth hormone,cGH),试验将C端融合了6个组氨酸的cGH成熟片段基因序列克隆入毕赤酵母表达载体pPIC9K中,使用电转法将重组质粒导入毕赤酵母GS115,以1%的甲醇诱导重组菌(GS115-pPIC9K-cGH)96h,表达大小约为25ku的目的蛋白cGH,经Ni离子亲和层析和聚丙烯酰胺葡聚糖凝胶层析获得95%以上的cGH重组蛋白。实时荧光定量检测结果表明,纯化的cGH重组蛋白可刺激鸡肝癌细胞LMH内的胰岛素样生长因子Ⅰ(insulin-like growth factorⅠ,IGF-Ⅰ)基因的mRNA表达上调,表明所发酵纯化的重组蛋白cGH具有生物学活性,这为其结构和功能的研究及生产应用奠定了基础。The growth hormone（GH）regulated diverse functions of the target cells through binding its receptor and played important roles in the process of metabolism of the body＇s growth and development.In order to obtain high purity chicken growth hormone（cGH）with biological activity,the gene segment of the mature cGH with six histidine at the C terminus was cloned into the expression vector pPIC9 K.The recombinant plasmid（pPIC9K-cGH）was transformed to Pichia pastoris（GS115）by electroporation to construct the recombinant Pichia pastoris（GS115-pPIC9K-cGH）.The GS115-pPIC9K-cGH secreted the recombinant protein（cGH）whose molecular weight was about 25 ku induced by 1% methanol induction for 96 h.The purity of cGH attained at least 95%through using Ni affinity chromatography and polyacrylamide glucan gel chromatography to purify the recombinant protein.The results of Real-time quantitative-PCR showed that the purified cGH recombinant proteins could up-regulate its target gene insulin-like growth factorⅠ（IGF-Ⅰ）mRNA in chicken liver cancer cell（LMH）.It certified that the recombinant cGH protein had biological activity.This study laid a foundation for research of the structure,function and application of cGH.

关 键 词：鸡生长激素 毕赤酵母 表达 纯化 生物学活性 

分 类 号：S831.2[农业科学—畜牧学]