重组人IL-4大肠杆菌表达与纯化  

Expression and Purification of Recombinant Human Interleukin 4 in Escherichia coli

作　　者：邱燕[1] 孙九如 黄阳滨 黄智华 陈霖杰 刘勇 林跃鑫[1] 

机构地区：[1]福建师范大学生命科学学院,福州350007 [2]上海新生源生物医药有限公司,上海201203

出　　处：《生物工程学报》2006年第6期962-967,共6页Chinese Journal of Biotechnology

摘　　要：根据大肠杆菌密码子偏爱性优化并合成人白细胞介素4基因,以pET30a(+)为载体构建了重组表达质粒pET30a(+)/rhIL-4,将重组质粒转化大肠杆菌BL21(DE3)感受态细胞,诱导表达并超声破菌检测重组蛋白的表达形式。采用5L发酵罐培养工程菌,发酵液OD600为0.6时诱导3.5h收集菌体,检测目的蛋白的表达量。收集的菌体经压榨破菌获得包涵体,通过包涵体变性、层析、透析复性等方法对rhIL-4进行纯化。采用人红细胞白血病细胞(TF-1)测定纯化的rhIL-4的生物活性。测序表明目的基因已插入载体pET30a(+)中,重组蛋白以包涵体形式表达,单位体积重组蛋白的表达量达200mg/L发酵液,建立了对包涵体形式表达的rhIL-4纯化方法,最终得率为40mg/L发酵液,纯度大于98%,回收率为20%以上。免疫印迹法检测诱导表达的重组蛋白和纯化的蛋白为IL-4,N端氨基酸序列测定结果与理论相符,生物活性检测纯化的蛋白比活性达2.5×106AU/mg。这为rhIL-4进一步产业化研究建立了基础。Human interleukin 4 （IL-4） cDNA was optimized and synthesized according to E. coli preferred codon. A recombinant expression plasmid pET-30a（ ＋ ）/rhIL-4 was constructed with the target cDNA inserted between Nde Ⅰ and EcoR Ⅰ sites, which can translate the mature IL-4 protein with an extra methionine residue at N-terminal. The expression vector was transformed into E. coli BL21 （DE3）. The rhIL-4 protein was expressed in the inclusion body. By using the optimized fermentation conditions, the high expression level was achieved with the expression level as high as 35% of total protein obtained. A purification＇ strategy has been designed which includes Q-Sepharose and SP-Sepharose ion-exchange chromatography and dialysis renaturation. The rhIL-4 was purified with the purity more than 98 % and the yield of 40 mg per liter fermentation culture achieved. Western blot proved that the purified protein is IL-4. Amino acid sequencing revealed that N-terminal 16 residue sequence is identical to the theoretical sequence. Biological activity assay on TF-1 cells demonstrated that the rhIL-4 is active with an activity of 2.5 ×10^6 AU/mg. This study promises large scale production of rhIL-4.

关 键 词：rhIL-4 大肠杆菌 发酵 纯化 

分 类 号：Q78[生物学—分子生物学]