人白细胞介素－3在大肠杆菌中的高效表达  

High-Level Expression of the Gene Encoding Human Interleukin-3 in E.coli

作　　者：杨立宏[1] 陈常庆[1] 周斌[1] 陈南春[1] 药立波[1] 苏成芝[1] 

机构地区：[1]中国科学院上海生物工程研究中心,第四军医大学生物化学教研室

出　　处：《生物化学与生物物理学报》1994年第5期477-484,共8页

摘　　要：人白细胞介素－３（ｈｕｍａｎＩｎｔｅｒｌｅｕｋｉｎ３，ｈＩＬ－３）是一种造血前体细胞早期分化的关键调节因子。用ＰＣＲ方法从人Ｔ淋巴细胞ｃＤＮＡ文库中扩增出０．４４ｋｂ的ＤＮＡ片段，并克隆入ｐＵＣ１９载体中。经ＤＮＡ序列测定，确定０．４４ｋｂ的ＰＣＲ产物合完整的编码人白细胞介素－３成熟蛋白ｃＤＮＡ序列，并在信号肽与成熟蛋白编码序列之间通过突变引入了限制性内切酶位点和ＡＴＧ起始密码。构建的ＰＬ启动子控制下的ｈＩＬ－３ｃＤＮＡ表达质粒，转入大肠杆菌Ｔａｐ１０６，经４２℃热诱导，获得ｈＩＬ－３的表达产物。ＳＤＳ－ＰＡＧＥ电泳显示表达产物为１５ｋｄ，约占细菌总蛋白的１５％。表达产物经ＥＬＩＳＡ和Ｗｅｓｔｅｒｎ－ｂｌｏｔ验证。ｈＩＬ－３表达产物在细胞内形成包涵体，纯化包涵体，使产物纯度提高到７０．８％，产物复性后，能明显促进ｈＩＬ－３依赖细胞株生长，具有明显的生物活性。产物转移到ＰＶＤＦ膜后进行Ｎ端序列分析，Ｎ端１６个氨基酸正确。Human interlenkln 3(hIL-3) is a key regulatory molecule in the early differentiation of hematopoietic precursor cell. To study the structure-function relation ship of hIL-3,we have amplified a 0.44kb DNA fragment from cDNA Librarian ofT cell by PCR and aloned it into the plasmid vector pUC19. DNA sequencing showed that this DNA fragment encompassed the complete hIL-3 coding region and had Nco I site and ATG code at the 5'end,and BamHI site atthe 3' end.The expression plasmid of hIL-3 gene nuder the COntrol of λ PL promoter was constructed and transformed to E. coli Tap106.The hlL-3 protein product was obtained from the cells induced at 42℃ and its molecular weight was appronimately 15kd in SDS-PAGE. The induced hIL-3 protein existed as inclusion bodies with a yield of about 15% of the total cellular proteins.The product can be isolate in theform of inclusion bodies with more than 70.8%purify.The partially purified hIL-3 protein was subjected to SDS-PAGE and transfered to PVDF membrane.The peptides sequence analysis showed that the first 16 amino acids of the hIL-3product were correct.After refolding the partially purified hIL-3 showed high activity.

关 键 词：人白细胞介素-3 基因 大肠杆菌 基因表达 

分 类 号：Q78[生物学—分子生物学]