重组人IL-6/IL-2融合蛋白的高密度发酵和纯化  

High Density Fermentation and Purification of Recombinant Human IL-6/2 Fusion Protein

作　　者：李董[1] 袁文肃[1] 马小彤[1] 蔡辉国[1] 明宇[1] 李静[1] 赵春华[1] 

机构地区：[1]中国医学科学院中国协和医科大学血液学研究所实验血液学国家重点实验室,天津300020

出　　处：《高技术通讯》2003年第11期27-30,共4页Chinese High Technology Letters

基　　金：863计划(2001AA215301);973规划(001CB5099)资助项目

摘　　要：克隆的IL-6/IL-2融合蛋白编码基因插入pBV220载体,转化BL21(DE3)菌株中表达。发酵过程中,30℃扩增生长6小时,42℃诱导培养4小时,控制溶解氧在30％～50％。发酵液中最终菌体密度(OD600)达29.17(相当于每升发酵液含55克湿菌体),表达的融合蛋白呈包涵体形式。表达蛋白占菌体总蛋白的30％左右。菌体经过超声破碎后反复洗涤包涵体,融合蛋白纯度达到70％,进一步用离子交换层析和凝胶过滤层析纯化使其纯度达95％以上。IL-6/2融合蛋白中的IL-6和IL-2活性分别为1×107和2×105 U/mg。重组人IL-6,/IL-2融合蛋白在大肠杆菌中达到了中试水平的高表达。The gene of interleukin-6 and interleukin-2 fusion protein obtained by PCR ligation was inserted into plas-mid pBV220, which was transformed into E. coli BL21(DE3). In a 10 liters bioengineering fermenter, the bacteria grew for 6 hours at 30 ℃ and raised to 42℃ in 15 minutes for 4 hours. A feeding-in-batch process was used to keep glucose at low level and dissolved oxygen at 30 % - 50 %. The final density of bacterial reached to OD600 = 29. 17, approximately equaled to 55g wet weight per liter. The fusion protein presented as inclusion body and accounted for about 30% of total bacterial protein. The purity of fusion protein was about 70% after ultrasonic decomposition and washing repeatedly, and reached above 95 % after purification by ion exchange and gel-filtration chromatography. The final fusion protein demonstrated a IL-6 activity of 1 × 10 U/mg and IL-2 activity of 2× 105 U/mg. In conclusion, expression of recombinant human IL-6/2 fusion protein is achieved in E. coli by high density fermentation.

关 键 词：白细胞介素 纯度 离子交换层析 凝胶过滤层析 大肠杆菌 

分 类 号：TQ920[轻工技术与工程—发酵工程]