生物技术通报 ›› 2020, Vol. 36 ›› Issue (3): 193-198.doi: 10.13560/j.cnki.biotech.bull.1985.2019-0661

• 研究报告 • 上一篇    下一篇

牛奶过敏原β- 乳球蛋白的重组制备及磁微粒化学发光检测法的建立

杭鑫茹1, 2, 耿荣庆1, 2, 钱林3   

  1. 1. 南京工业大学,南京 210000;
    2. 盐城师范学院,盐城 224051;
    3. 江苏省免疫诊断工程技术研究中心,苏州 215028
  • 收稿日期:2019-07-24 出版日期:2020-03-26 发布日期:2020-03-17
  • 作者简介:杭鑫茹,女,硕士研究生,研究方向:过敏原检测;E-mail:xinruhang@163.com
  • 基金资助:

    江苏省产学研合作前瞻性联合研究项目(BY2016066-01)

Preparation of Recombinant Milk Allergen β-Lactoglobulin and Establishment of Magnetic Particle Chemiluminescence Detection Method

HANG Xin-ru1, 2, GENG Rong-qing1, 2, QIAN Lin3   

  1. 1. Nanjing Tech University,Nanjing 210000;
    2. Yancheng Teachers University,Yancheng 224051;
    3. Jiangsu Immuno-diagnostic Engineering and Technology Research Center,Suzhou 215028
  • Received:2019-07-24 Published:2020-03-26 Online:2020-03-17

摘要:

运用原核系统表达牛奶β-乳球蛋白(Bos d5)蛋白,建立一种纳米磁微粒化学发光方法用于检测牛奶组分过敏原β-乳球蛋白特异性Ig E抗体的含量。通过优化合成牛奶β-乳球蛋白基因,与质粒重组导入Rosetta原核表达菌株中表达目标蛋白,纯化的重组蛋白采用生物素标记后,在化学发光平台上进行过敏原项目检测。获得纯度﹥85%的22.5 kD重组牛β-乳球蛋白,将生物素化的重组蛋白用于牛奶β-乳球蛋白纳米磁微粒检测试剂盒,检测临床110例血清样本,和Phadia进行方法学比对阳性符合率为88.9%,阴性符合率97.3%,总符合率94.5%,P<0.001,χ2=84.238,Kappa=0.874,其与Phadia参照试剂盒同样具有良好的检测能力。原核表达系统中获得的重组牛奶过敏组分β-乳球蛋白具有较好的免疫活性,开发的免疫诊断试剂盒具备良好的性能,可用于临床辅助诊断。

关键词: 牛奶, β-乳球蛋白, 过敏原, 重组蛋白, 纳米磁微粒化学发光免疫检测

Abstract:

Using prokaryotic system to express milk β-lactoglobulin(Bos d5)protein,a magnetic nanoparticle chemiluminescence immunoassay method was established to detect specific Ig E antibody content of allergen milk β-lactoglobulin. The recombinant bovine β-lactoglobulin gene was artificially optimized and synthesized with the plasmid,which then was introduced into the prokaryotic expression strain Rosetta to express the target protein. The purified recombinant Bos d5 protein was biotinylated and used on chemiluminescence platform,and the allergen items were detected on the chemiluminescence platform. The 22.5 kD recombinant bovine β-lactoglobulin with >85% purity was obtained and the biotinylated recombinant protein was used to develop the bovine β-lactoglobulin magnetic nanoparticle detection kit. The positive coincidence rate of serum samples was 88.9%,the negative coincidence rate was 97.3%,the total coincidence rate was 94/5% P<0.001,χ2=84.238,Kappa=0.874,while aligned with Phadia reference kit in 110 serum samples. The recombinant milk allergy component β-lactoglobulin obtained from the prokaryotic expression system has high immunoactivity,and the developed immunoassay kit performs well and can be used in medical clinical diagnosis.

Key words: milk, β-Lactoglobulin, allergens, recombinant protein, magnetic nanoparticle chemiluminescence immunoassay