可溶型人干细胞因子cDNA的克隆表达、复性与纯化

目的 :构建可溶型人干细胞因子 (hSCF)基因的表达载体。优化培养条件 ,实现hSCF基因在大肠杆菌中的高表达。方法 :利用PCR技术 ,以人淋巴结cDNA为模板扩增并克隆hSCF基因。用简并PCR对hSCF基因 5′端的序列进行修饰 ,以实现在大肠杆菌中的高效表达 ;用MTT比色法测定初步复性和纯化的hSCF蛋白的生物学活性。结果 :扩增并克隆了hSCF成熟肽cDNA。将其插入表达载体pBV2 2 0构建了hSCF基因的表达质粒 ,并在大肠菌中初步表达。表达量占总菌体的 2 0 %以上 ,优化培养条件后表达量可达到 4 0 %以上 ,表达产物为包涵体。将包涵体溶解在 8mol/L脲或 7mol/L盐酸胍中 ,用疏水色谱法进行复性并同时纯化 ,得到具有天然生物学活性的rhSCF蛋白。结论 :成功地克隆hSCF基因 ,并实现在原核系统中的高表达 ,所获表达菌株可用于生产以获得具有生物学活性的rhSCF蛋白产品。

Cloning,expression,renaturation and purification of soluble hSCF

AIM: To construct an expression vector for soluble recombinant human stem cell factor (rhSCF) gene, and optimize culture conditions for high-level expression of rhSCF in E.coli. METHODS: hSCF cDNA was amplified by PCR with total cDNA from human lymph node as a template, followed by cloning into pMD18-T vector. The 5' terminal of the hSCF cDNA was modified by degenerative PCR to obtain high-level expression in E.coli. The biological activity of the refolded rhSCF, purified with high performance hydrophobic interaction chromatography, was examined by MTT colorimetry. RESULTS: hSCF cDNA was amplified and cloned, and was inserted into an E.coli expression vector pBV220. The expressed rhSCF accounted for about 20% of total bacterial proteins and reached 40% of total bacterial proteins under optimal culture conditions. The expressed rhSCF appeared in bacterial lysates in the form inclusion body. The rhSCF with biological activity was obtained after solubilization of the inclusion body with 8 mol/L urea or 7 mol/L guanidine chloride, followed by preliminary refolding and purification. CONCLUSION: hSCF was cloned and expressed in E.coli successfully. The E.coli strain expressing rhSCF can be used to produce rhSCF with biological activity on large-scale production.