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.
出处
《高技术通讯》
EI
CAS
CSCD
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
