文章摘要
田阳;饶欢;薛文通.花生致敏原Ara h 1的重组表达与纯化[J].中国食品学报,2020,20(8):20-28
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
  
DOI:
中文关键词: 花生过敏原  Arah1  重组蛋白  蛋白纯化
英文关键词: peanutallergen  Arah1  recombinantprotein  proteinpurification
基金项目: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)
作者单位
田阳;饶欢;薛文通 中国农业大学食品科学与营养工程学院 
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中文摘要:
      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。
英文摘要:
      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.
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