重组人干细胞因子在原核系统中高效表达的策略  

Strategies of high-level expression of human recombinant stem cell factor in E.coli

作　　者：郭斯启[1] 奚永志[1] 崔建武[1] 习彩霞[1] 刘楠[1] 梁飞[1] 孔繁华[1] 

机构地区：[1]军事医学科学院附属医院免疫学研究室国家生物医学分析中心免疫分析实验室,北京100039

出　　处：《中国输血杂志》2004年第4期227-231,共5页Chinese Journal of Blood Transfusion

基　　金：国家自然科学基金课题 ( 3 980 0 0 5 6)

摘　　要：目的　探索并建立人干细胞因子 (SCF)的原核高效表达系统。方法　设计特异性引物 ,以RT PCR从人胎肝组织中扩增出干细胞因子的膜外区活性片段 ,克隆进入pGEM T载体。以Goldkey软件进行翻译起始序列RNA结构和自由能优化 ;通过合成寡核苷酸和PCR技术将SCFcDNA中的原核稀有密码子定点突变为同义的高频密码子 ;酶切连接构建多种原核表达载体并比较它们在宿主菌中诱导表达结果。表达产物经SDS PAGE、WesternBlot和MTT活性鉴定。结果　扩增SCF膜外区序列测定正确。构建的原核表达载体 pET32a(+) SCF在大肠杆菌中获得高效融合表达 ,表达产量达 30 %以上 ,WesternBlot鉴定正确 ,初步纯化产物的活性与标准品相似。结论　根据使用的载体 /宿主系统 ,翻译起始序列 (TIS)的RNA结构和自由能优化、稀有密码子突变对表达无影响 ;SCF基因在 5′端融合要比Objective To explore and establish a high level prokaryotic expression system of human recombinant stem cell factor (SCF).Methods The out membranous fragment of SCF was obtained by method of RT PCR with special primers from human fetal liver tissue, and cloned into pGEM T vector. The structure and free energy of translational initiating sequence (TIS) were optimized by the Goldkey software. The prokaryotic rare codons in SCF cDNA sequence were mutated into high frequency synonymous codons by method of PCR. The various expression vectors of SCF were constructed and then expressed in host bacterium. The expression products were identified by SDS PAGE, Western Blot, and MTT assay. Results The out membranous SCF sequence was correct. The expression results of different expression vectors were different. The expression vector pET32a(+) SCF was constituted and expressed in origami TM (DE3), and the expression efficiency was more than 30 percent of total protein. The activity of purified SCF fusion protein was similar to the standard SCF. Conclusion The expression yield of SCF is independent of rare codons,structure and free energy of TIS. But the expression efficiency is much higher when SCF gene is fused into 5' terminal than when it is fused into 3' terminal.

关 键 词：干细胞因子 重组 大肠杆菌 高效表达 

分 类 号：Q813.1[生物学—生物工程] Q591.2