人白细胞介素-3基因翻译起始区的改造提高其在大肠杆菌中的表达水平  

Increased Expression of Human Interleukin 3 Gene in Escherichia coli by Translation Initiation Region Sequence Modification

作　　者：杨立宏[1] 陈常庆[2] 高冕[2] 苏成芝[1] 

机构地区：[1]第四军医大学生物化学教研室,西安710032 [2]中国科学院上海生物工程研究中心,上海200233

出　　处：《生物工程学报》1995年第4期297-303,共7页Chinese Journal of Biotechnology

摘　　要：为了提高人白细胞介素-3(hIL-3)在大肠杆菌中的表达,在计算机辅助下,设计合成了PCR突变引物,用于改造起始密码AUG上下游序列,并在不改变5＇端氨基酸编码的前提下,尽可能选用大肠杆菌高频使用的密码子。将经改造后的hIL-3cDNA和翻译起始区置于P_L启动子之下,转入大肠杆菌Tap106,经42℃热诱导后,获得表达产物,提高表达水平近一倍,表达量达到菌体总蛋白量的30％左右。表达产物经Western blot验证,经PVDF膜转移后进行N端顺序分析,证明前15个氨基酸正确,产物经包涵体纯化后,纯度提高至80％以上,初步复性后能明显促进hIL-3依赖细胞的生长。In order to increase expression lever of human interleukin 3 (hIL3) in E. coli. , this paper designed PCR mutation oligonucleotide primers in aid of computer and synthesized it. These primers were used to modify the translation initiation region sequence at two sides of AUG initiation codon, simultaneously to modify some codons into preferred codons for E. coll. , but not to change amino acid sequence. After it was modified, the hIL 3 gene was cloned into expressed plasmid at the down stream of PL promoter and transfected into E. coli. Tap 106. SDS-PAGE analysis revealed that expression of modified hIL 3 was raised up to about 30% of total cell protein, twice as much that of wild type. Western-blot and N terminal 15 amino acid analysis showed that this recombinant protein was consistent with the natural hIL 3. By the isolution of inclusion bodies, hIL 3 could be obtained with purify of more than 80%, After refolding inclusion bodies, it was able to obviously stimulate proliferation of hIL 3 dependent cell TF-1.

关 键 词：人白细胞介素-3 基因 大肠杆菌 表达 基因工程 

分 类 号：R392-33[医药卫生—免疫学] R394.8[医药卫生—基础医学]