人白细胞介素11的基因克隆及其在大肠杆菌中的表达  

The Gene Cloning of IL-11 and Its Expression in E.coli

作　　者：杜秋江[1] 马雁冰[1] 刘红岩[1] 陈尔佳[1] 周丽[1] 姬秋彦[1] 李智华[1] 徐维明[1] 孙茂盛[1] 戴长柏[1] 

机构地区：[1]中国医学科学院

出　　处：《中国生物化学与分子生物学报》1999年第6期861-865,共5页Chinese Journal of Biochemistry and Molecular Biology

摘　　要：从人胚肺二倍体细胞ＫＭＢ１７抽提总ＲＮＡ，经ＲＴ－ＰＣＲ扩增到人ＩＬ－１１ｃＤＮＡ，克隆于ｐＢＳ－ＳＫ，以双脱氧链终止法进行序列分析；扩增去除Ｎ 端第一个氨基酸的ＩＬ－１１ｃＤＮＡ，克隆于硫氧还蛋白融合表达载体ｐＴｈｉｏＨｉｓＡ，经过对表达质粒的改造，使融合表达的ＩＬ－１１能被肠激酶精确切割；表达产物经金属鏊合亲合层析后经肠激酶切，再通过阳离子交换层析纯化后，用其依赖细胞株７ＴＤ１检测活性．结果表明，克隆的ＩＬ－１１ｃＤＮＡ 序列与文献报道一致；表达的融合蛋白占菌体总蛋白的３０％ 左右，以可溶性形式存在；纯化产物具有维持依赖细胞株生长的能力．由此，获得了较高纯度的具有生物学活性的ｒｈＩＬ－１１，为中试生产奠定了基础．Total RNA was extracted from a human diploid embryo lung cell line,KMB 17 ,and IL 11 cDNA was obtained by RT PCR.The cDNA was cloned into the His patch Thiofusion TM expression vector pThioHisA.The cDNA sequence was determined by Sanger's method.The expression plasmid was reconstructed to fit the precise cut by Enterokinase.After metal chelation chromatography,the expression product was subjected to Enterokinase digestion and cation exchange chromatography.The biological activity of purified rhIL 11 was determined using a dependent cell line 7TD1.The results show that the DNA sequence of cloned IL 11 cDNA is the same as that reported.The expressed fusion protein accounts for approximately 30% of the total cell protein and is soluble.Purified product has the ability to sustain the growth of dependent cell line.Recombinant human IL 11 with high purity and biological activity has been obtained through this research,which has laid a good foundation for Biopilot scale production trial of rhIL 11.

关 键 词：人 白细胞介素-11 基因克隆 表达 大肠杆菌 

分 类 号：TQ464[化学工程—制药化工] Q78[生物学—分子生物学]