重组人粒细胞集落刺激因子在大肠杆菌中的表达(英文)

Expression of recombinant human granulocyte-macrophage colony-stimulating factor gene in Escherichia coli

背景:粒细胞巨噬细胞集落刺激因子(human granulococyte-macrophage colony stimulating factor,hGM-CSF)是刺激造血前体细胞分化及增殖的细胞因子,同时在机体免疫调节过程中具有重要作用。大肠杆菌表达系统所具有的低成本、高产量等优点则是其他体系无法比拟的,因此仍是目前最常用的外源基因表达系统。目的:观察重组人粒细胞集落刺激因子在大肠杆菌中的表达。设计、时间及地点:观察性实验,于2007-02/06在三峡大学生物技术研究中心实验室完成。材料:质粒pBR322hGM-CSF购自美国ATCC菌种保藏中心。hGM-CSF抗体购自Sigma公司,IPTG、X-gal和载体pMGT-18,购自上海生物工程技术公司。方法:根据hGM-CSF基因序列设计出引物,以克隆载体质粒pBR322-hGM-CSF为模板,得到hGM-CSF基因并重组入原核表达载体pGEX-4T-1中,将经酶切和测序鉴定正确的重组质粒转化大肠杆菌DH5α,用IPTG诱导表达,表达产物经SDS-PAGE鉴定并用Western bloting检测。主要观察指标:hGM-CSF在大肠杆菌中的表达。结果:SDS-PAGE显示,得到相对分子量为40500的目的蛋白,表达量可达菌体总蛋白的17.9%。Western bloting检测表明,抗hGM-CSF单抗可与相对分子量为40500大小的电泳条带发生特异性反应。结论:实验构建了原核表达载体pGEX-hGM-CSF,并在大肠杆菌中诱导表达,得到hGM-CSF融合蛋白。

BACKGROUND： Human granulocyte-macrophage colony stimulating factor （hGM-CSF） is a kind of cytokine which can stimulate the differentiation and proliferation of haematopoietic precursor cells and plays an important role in the process of immunoloregulation. Escherichia coli （E.coli） expression system has advantages, such as low cost and high output, over other systems. Therefore, E.coli is still the most commonly used exogenous gene expression system. OBJECTIVE： To observe the expression of hGM-CSF in the E.coli strain DH5α. DESIGN, TIME AND SETTING： The present observational experiment was performed at the Central Laboratory of Biotechnology Research, China Three Gorges University between February and June 2007. MATERIALS： Plasmid pBR322hGM-CSF was purchased from American Type Culture Collection, USA. hGM-CSF antibody was sourced from Sigma Company, USA. 1PTG, X-gal, and vector pMGT-18 were purchased from Shanghai Bioengineering Technology Company, China. METHODS： Primer was designed according to hGM-CSF gene. Taking cloning vector plasmid pBR322-hGM-CSF as template, hGM-CSF gene was acquired and recombined into prokaryotic expression vector pGEX-4T-1. Recombinant plasmid confirmed by enzyme digestion and sequencing was transformed into E.coli strain DH5α and induced by isopropy-β-D-thiogalactoside （IPTG）. Expression product was identified by sodium dodecyl sulfate polyacrylamide gel electropheresis （SDS-PAGE） and detected by Western blotting assay. MAIN OUTCOME MEASURES： hGM-CSF expression in the E.coli strain DH5α. RESULTS： SDS-PAGE results revealed that the recombinant hGM-CSF protein with relative molecular weight of 40 500 was produced up to approximately 17.9% of total protein. Western blotting detection results indicated that there was a 40 500 bp brand. CONCLUSION： The present study reconstructed prokaryotic expression vector pGEX-hGM-CSF, induced its expression in the E.coli strain DH5α, and obtained hGM-CSF fusion protein.