重组人白血病抑制因子的表达、包涵体复性、纯化及其活性

Expression, inclusion body renaturation, purification, and activity of recombinant human leukemia inhibitory factor

白血病抑制因子（LIF）是一种在生物体内具有独特活性和重要调节作用因而广泛应用的多功能细胞因子.利用基因工程技术表达重组人白血病抑制因子（Recombinant human leukemia inhibitory factor,rhLIF）并进行包涵体的复性及纯化,将rhLIF基因克隆到原核表达载体pET32a中,成功构建重组hLIF基因工程菌,并研究该菌株发酵条件和纯化工艺.先将种子液进行发酵培养,诱导表达,收集诱导后的细胞;对细胞破碎,收集包涵体沉淀,洗涤包涵体;用6 mol/L盐酸胍变性重溶包涵体,镍离子亲和层析柱上直接复性及纯化,并用阴离子交换进一步纯化,复性率为75%,纯度达97%.结果显示基因序列与理论序列一致,SDS-PAGE分析结果表明其表达的外源蛋白质分子量与预期的目的蛋白质相对分子量大小相一致,均为35×10~3;Western blotting、MTT活性检测结果表明,此重组hLIF融合蛋白具有良好的抗原性和免疫原性.本研究成功建立了rhLIF原核表达、复性和纯化体系,可为进一步研究LIF生物学活性及产品开发提供试验基础.

Leukemia inhibitory factor（LIF） is one of the multifunctional cytokines that plays an important regulatory role in organisms. Because of its unique biological properties, LIF has been widely used. In the present study, recombinant human LIF（rhLIF） was expressed by gene engineering technique. The gene rh LIF was cloned into a prokaryotic expression vector pET32 a to construct a recombinant h LIF strain. Furthermore, the fermentation conditions and purification process of the strain were studied. Initially, the seed liquid was fermented in liquid culture; the fermentation products were collected and crushed. The inclusion bodies were collected and washed. The mixture was redissolved with 6 mol/L guanidine hydrochloride, the column was directly refolded and treated, and then further purified by ion exchange chromatography. The purity reached 97% with a refolding rate of 75%. The resulting DNA sequence obtained from hLIF was identical to the published h LIF sequence. The sodium dodecyl sulfate-polyacrylamide gel electrophoresis（SDS-PAGE） revealed that the molecular weight（Mr） of rhLIF was 35 × 10~3. The western blotting and（3-（4,5-dimethylthiazol-2-yl）-2,5-diphenyltetrazolium bromide） tetrazolium assay（MTT） showed that the recombinant hLIF fusion protein has good antigenicity and immunogenicity. A reproducible and efficient prokaryotic expression system for rhLIF, and highly effective purification and refolding procedures were established in the present study, which would provide reliable data for further studies on the biological activities of LIF and for its exploitation by manufactures.