人干细胞因子在大肠杆菌中的表达研究  

The Expression Study of Human Stem Cell Factor (hSCF) in E. coli

作　　者：毛华[1] 赵峻 张劲秋 狄旭[1] 陈卫东[1] 陈松森[1] 梁植权[1] 

机构地区：[1]中国医学科学院中国协和医科大学 基础医学研究所,北京100005

出　　处：《中国医学科学院学报》1998年第4期279-284,共6页Acta Academiae Medicinae Sinicae

基　　金：国家"九五"科技攻关项目(96-C02-01-10)

摘　　要：目的 经过改造重组人干细胞因子(human stem cell factor,hSCF),提高其表达量。方法 应用人工合成寡核苷酸引物及PCR技术,改变人干细胞因子hSCF cDNA起始密码子ATG与SD序列之间的距离及在翻译起始区(即N端氨基酸)部分采用大肠杆菌(E.coli)喜用型密码子的策略,使重组表达质粒pBV220-hSCF在E.coli中获得高效稳定表达。结果 DNA序列测定证明基因的阅读框架完全正确。重组人干细胞因子的表达量从改造前占E.coli总蛋白的12%提高到40%左右。纯化的重组hSCF N端17个氨基酸测序结果表明,除第一个氨基酸为甲硫氨酸外,其余与天然hSCF序列完全一致。纯化的重组hSCF经Western blot证明,与标准hSCF一致。结论 提示hSCF cDNA的5′侧翼区结构对其基因表达有显著的影响。Objective To improve the expression level of recombinant human stem cell factor (rhSCF). Methods With PCR techniques and synthesized oligonucleotide as primers, the number of nucleotides between the SD sequence and the starting codon ATG of human stem cell factor cDNA was altered, and the preferable codons of E. coli in the N-terminal amino acid sequence were selected. Results SDS-PAGE electrophoresis indicated that the ratio of expressed rhSCF to total E. coli proteins increased from 12% to 40% by thermal inducing. With DNA sequencing, the reading frame of the gene was proved to be correct. The sequence of 17 N-terminal amino acids of purified recombinant hSCF has been proved to be identical to the natural hSCF, except the first amino acid-Met. Conclusions These studies suggest that the 51-flanking region of hSCF cDNA plays an important role in its gene expression.

关 键 词：重组 高效稳定表达 SD序列 干细胞因子 

分 类 号：Q784[生物学—分子生物学]