人IL-18的基因克隆、表达及纯化

Gene Cloning,Expression and Purification of Human IL-18

目的 :克隆人IL 1 8基因 ,并构建高表达人IL 1 8的工程菌 ,纯化获得重组人IL 1 8。方法 :从人肿瘤组织内提取总RNA ,利用逆转录聚合酶链反应扩增人IL 1 8基因 ,在基因的 5′和 3′端分别加上NcoI和EcoRI酶切位点 ,酶切后直接克隆至质粒表达载体pET2 8a( + )内 ,转化大肠杆菌BL2 1 (DE3) ,挑选转化子 ,IPTG诱导后SDS PAGE筛选高表达人IL 1 8的工程菌。提取表达菌质粒进行DNA序列分析。表达菌大量培养及IPTG诱导后 ,超声破菌收集包涵体 ,十二烷基肌苷酸钠溶解后用阳离子柱和分子筛纯化。结果 :克隆的人IL 1 8基因序列完全正确 ,工程菌表达的人IL 1 8约占菌体蛋白 30 % ,以包涵体形式存在 ,经阳离子柱和分子筛纯化获得了较纯的人IL 1 8。结论 :可用构建的工程菌大量制备人IL 1 8,为开展人IL 1 8药物的临床前研究奠定了基础。

Objective:To clone the cDNA of human IL-18,construct the engineered Escherichia coli expressing human IL\|18,and purify the expressed human IL\|18.Methods:Total RNAs were purified from human pulmonary carcinoma,intestine carcinoma and womb cancer respectively.With the total RNAs as templates,the cDNA of IL\|18 was cloned by RT\|PCR,and inserted into plasmid pET28a(+) between NcoI and EcoRI sites for expressing the recombinant human IL\|18 in Escherichia coli BL21(DE3),the recombinants were screened by PCR and SDS\|PAGE.The expressed recombinant human IL\|18 was purified with S\|Sepharose FF cation column and Sephacryl S\|200 filtration column.Results:The engineered Escherichia coli expressing human IL\|18 was established,the expressed IL\|18 exists in the form of inclusion body and accounts for about 30% of the total soluble proteins of Escherichia coli BL21(DE3),and the purified IL\|18 has high purity.Conclusion:The engineered E.coli and purification methods are suitable for preparing plenty of human IL\|18,which has laid base for basic studies,pre\|clinical and clinical tests of human IL-18.