直组人嗜中性白细胞活化蛋白-1/白细胞介素-8的纯化与鉴定  

Purification and Characterization of Recombinant Human Neutrophil Activating Protein-1/Interleukin-8

作　　者：周圆[1] 金冬雁[1] 徐荣辉[2] 侯云德[1] 

机构地区：[1]中国预防医学科学院病毒学研究所病毒基因工程国家重点实验室,北京100052 [2]广州医学院微生物质学教研室

出　　处：《生物化学杂志》1992年第6期719-723,共5页

摘　　要：本文利用SDS-PAGE及蛋白质电泳印迹技术,从带有相应表达质粒的重组大肠杆菌裂解液中,将所表达的重组人嗜中性白细胞活化蛋白-1/白细胞介素-8(NAP-1/IL-8)转移至聚偏二氟乙烯膜上,直接进行N-末端15个氨基酸的序列分析,从而确证该目标蛋白得到高效表达和正确加工。随后采用Bio-Gel P30凝胶过滤层析和Mono-S阳离子交换层析对重组人NAP-1/IL-8进行了分离纯化,纯化产品达到SDS-PAGE纯。利用琼脂糖平板法测定了纯化产品的嗜中性白细胞趋化活性,推算其比活为2.8×10~5U/mg蛋白。又利用SDS-PAGE测出重组NAP-1/IL-8的分子量约为8.5kD,但根据凝胶过滤层析的洗脱时间推定,在溶液中确实存在分子量稍大于14.4kD的NAP-1/IL-8二聚体。Recombinant human neutrophil activating protein-1/interleukin-8 (NAP-1/IL-8) from E.coli lyzate was electroblotted from the SDS-PAGE gel onto the PVDF-type problott membrane and its N-terminal amino acid sequence was determined directly.The efficient expression and processing of the target protein were confirmed by the sequencing result.The recombinant NAP-1/IL-8 was then purified through gel filtration (Bio-Gel P30) and cation exchange (Mono-S) chromatography.The purified protein with a specific chemotactic activity of 2.8**********x105U/mg,as assayed by the agarose plate method, appeared as a single band on SDS-PAGE gel.Based on the fact that NAP-1/IL-8 was eluted earlier in gel filtration than the 14kD internal standard molecule lysozyme, it was estimated that NAP-1/IL-8 dimers were present in solution.

关 键 词：NAP-1/IL-8 提纯 鉴定 

分 类 号：Q78[生物学—分子生物学]