不同IL-8亚型的原核表达

Prokaryotic Expression of Different Subtype IL-8

目的构建一种精确表达小分子蛋白的技术平台,原核表达IL-8的3种不同亚型,使产物仅为研究对象本身,以避免大分子载体片段对下游研究的干扰。方法采用PCR方法扩增仅仅编码IL-8(1-77aa)I、L-8(6-77aa)和IL-8(9-77aa)成熟肽片段的基因,分别TA克隆到载体PET SUMO。测序验证后的重组质粒转化至表达菌BL21(DE3)中诱导表达,融合蛋白用基质辅助解吸电离飞行时间质谱仪进行串联质谱鉴定(MALDI-T0F-MS/MS),通过特异性的SUMO蛋白酶酶解载体SUMO蛋白,利用SUMO蛋白和其蛋白酶带6×His标签的性质,经镍螯合柱亲和层析将二者去除,以MALDI-T0F-MS/MS验证不同IL-8亚型,即IL-8(1-77aa)I、L-8(6-77aa)和IL-8(9-77aa)成熟肽片段的获得。结果产物经肽指纹图谱(PMF)分析,结果分别在NCBI和Swissport蛋白数据库搜索,证实蛋白片段属于IL-8且分子量大小与IL-8(1-77aa)I、L-8(6-77aa)和IL-8(9-77aa)成熟肽片段相符。结论本研究成功地构建了一种精确表达小分子蛋白的技术平台,并获得目的产物,为基因工程精确表达小分子蛋白提供了有效的方案,为深入研究不同IL-8亚型在卵巢恶性肿瘤中的作用机制打下实验基础。

Objective To inhibit the impact of macromolecular vector fragment in the expressed products,a special prokaryotic expression technology was applied to express three subtypes of IL-8,which only included the expectant research fragments.Methods The different subtype IL-8 domain genes amplified by polymerase chain reaction（PCR） were cloned into the prokaryotic expression vector PET SUMO.The recombinant expression plasmids were transformed into E.coli BL21（DE3） and expressed.The fusion products were analyzed by MALDI-T0F-MS/MS.The SUMO protein protease was used to removed the SUMO protein.The mat peptides of different subtype IL-8 were testified by the MALDI-T0F-MS/MS.Results Through PMF analysis,the end products were comfirm to be the exact IL-8（1-77aa）,IL-8（6-77aa） and IL-8（9-77aa） with the correct PMF characters and molecular weights.Conclusion The results suggested that above research successfully structured a technical platform for accurately expressing the micromolecular protein,and finally got the mat peptides of IL-8（1-77aa）,IL-8（6-77aa） and IL-8（9-77aa）,which also built the solid experimental foundation for the research of different subtype IL-8.