花生致敏原Ara h 1的重组表达与纯化  

Expression and Purification of Recombinant Peanut Allergen Ara h 1

作　　者：田阳[1] 饶欢[1] 薛文通[1] Tian Yang;Rao Huan;Xue Wentong(College of Food Science and Nutritional Engineering,China Agricultural University,Beijing 100083)

机构地区：[1]中国农业大学食品科学与营养工程学院,北京100083

出　　处：《中国食品学报》2020年第8期20-28,共9页Journal of Chinese Institute Of Food Science and Technology

基　　金：国家重点研发计划食品安全关键技术研发专项(2019YFC1605000);国家自然科学基金项目(31872904)。

摘　　要：Ara h 1是花生中含量最高的过敏原,也是致敏性较高的蛋白之一。目前提纯Ara h 1的方法大多涉及2至3步柱层析,步骤繁琐且成本较高。本文中,将带有6×his标签的Ara h 1基因与pET-32a表达载体融合,构建重组质粒并转化大肠杆菌BL21(DE3)pLysS,诱导使其表达目标蛋白。菌体裂解后使用Ni-NTA吸附、梯度洗脱对Ara h 1进行纯化。采用质谱及Western-blot鉴定纯化蛋白的种类及免疫活性。结果显示,质粒中目标基因的序列与NCBI数据库中Ara h 1的基因数据相符;300 mmol/L异丙基-β-D-硫代吡喃半乳糖苷22℃诱导菌液22 h时蛋白表达量最高;添加15 mmol/L十二烷基磺酸钠可将蛋白释放到上清液中,使用含50,100 mmol/L咪唑的洗脱液分别洗脱2次和1次后得到纯度较高且免疫原性良好的重组Ara h 1。As the dominant allergen in peanut,Ara h 1 has a relatively high allergenicity among peanut allergens.According to the previous researches,methods used for Ara h 1 purification usually involve with two or three steps of chromatography,which is tedious and costly.In this study,pET-32a-Ara h 1 plasmid was constructed and Escherichia coli BL21(DE3)pLysS was utilized for recombinant Ara h 1 expression.Ni-NTA affinity column and gradient elution were used for purification.MALDI-TOF MS and western-blot were used to identify recombinant Ara h 1 and analysis its sensitivity respectively.According to the results,the sequence of Ara h 1 in the plasmid was consistent with the gene data of Ara h 1 in the NCBI database.When 300 mmol/L IPTG(isopropyl-β-D-thiogalactopyranoside)was added and cells were cultured at 22°C for 22 h,the yield of recombinant protein is at a relatively high level.After adding 15 mmol/L sodium laurylsulfonate to the lysis buffer,the recombinant protein could be effectively released to the supernatant.The elution buffer containing 50 mmol/L and 100 mmol/L imidazole were used in the elution process.Recombinant Ara h 1 with high purity and immunoreactivity could be obtained by using the method above.

关 键 词：花生过敏原 Ara h 1 重组蛋白 蛋白纯化 

分 类 号：TS20[轻工技术与工程—食品科学] S565.2[轻工技术与工程—食品科学与工程]