摘要
中性粒细胞蛋白酶抑制剂elafin是中性粒细胞弹性蛋白酶特异性抑制剂。为建立中性粒细胞蛋白酶抑制剂elafin毕赤酵母表达系统,通过逆转录PCR扩增获取elafincDNA,双酶切后定向克隆至穿梭质粒pPIC9K上。SalI酶切线性化后,电穿孔法转至毕赤酵母GS115菌株中,PCR筛选出阳性克隆,接种至最小甘油缓冲培养基中,甲醇诱导表达后,取上清阳离子交换层析纯化靶标蛋白。分别以western-blot、弹力纤维蛋白-聚丙烯酰胺凝胶电泳检测重组elafin免疫原性、抗蛋白酶活性。重组elafin在毕赤酵母系统中呈分泌性表达,产量及活性高,分离纯化简便。此表达系统的建立为其后续研究及临床应用奠定了基础。
Elafin is a specific inhibitor of neutrophil elastase. To establish expression system of neutrophil proteinases inhibitor elafin in Pichia pastoris, Elafin cDNA was obtained by reverse transcription PCR （RT-PCR）. With restricted enzyme digestion, gene of interest was cloned into shuttle vector pPICgK. Then the construct was linearized and transformed into Pichia pastoris competent cells by electroperation. Selected positive clone was inoculated into buffered minimal glycerol （BMG） culture media. Add 100% methanol to a final concentration of 1. 0% every 24h to maintain induction. At several different time points, transfer 1 ml of expression cuhrure to centrifuge to analyze the expression level by Coomassie-stained SDS-PAGE. Concentrated supernatnat was loaded onto a S6 column using a chromatographic system to purify secreted reconhinant elafin（ rec -elafin）. Rec-elafin immunogen and activity against proteinases was investigated by western blot and elastin-PAGE respectively. The rec-elafin expression level was up to 12 mg/L. Secreted elafin in Pichia pastoris was easily purified by one-step cationic exchange chromatography and retained its activity.
出处
《中国生物工程杂志》
CAS
CSCD
北大核心
2006年第12期29-33,共5页
China Biotechnology
基金
重庆市应用基础研究所资助项目(2003032)
关键词
中性粒细胞蛋白酶抑制剂
表达
纯化
毕赤酵母
Neutrophil proteinases inhibitor Elafin Expression Purification Pichia pastoris
