生物学杂志

生物学杂志 ›› 2023, Vol. 40 ›› Issue (5): 111-.doi: 10.3969/j.issn.2095-1736.2023.05.111

• 技术方法 • 上一篇    下一篇

高效表达人源可溶性转铁蛋白受体sTfR抗原及多克隆抗体制备和应用

卢振华1,2， 吴振兴1， 袁新松3， 王　剑1， 吴坚平2   

  1. 1. 宁波普瑞柏生物技术股份有限公司， 宁波 315033； 2. 浙江大学 化学工程与生物工程学院，
    杭州 310027； 3. 合肥师范学院 化学与化学工程学院， 合肥 230601
  • 出版日期:2023-10-18 发布日期:2023-10-17
  • 通讯作者: 吴坚平，博士，教授，研究方向为生物催化与转化，E-mail:wjp@zju.edu.cn
  • 作者简介:卢振华，博士，研究方向为IVD原料开发，E-mail:11728005@zje.edu.cn
  • 基金资助:
    安徽高校自然科学研究项目（KJ2021A0932）

Efficient expression of human soluble transferrin receptor sTfR antigen and the preparation and application of its polyclonal antibody

LU Zhenhua1,2, WU Zhenxing1, YUAN Xinsong3, WANG jian1, WU Jianping2   

  1. 1. Ningbo Purebio Biotechnology Co., Ltd., Ningbo 315033, China;
    2. College of Chemical and Biological Engineering, Zhejiang University, Hangzhou 310027, China;
    3. College of Chemistry and Chemical Engineering, Hefei Normal University, Hefei 230601, China
  • Online:2023-10-18 Published:2023-10-17

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摘要： 通过多种策略构建人源可溶性转铁蛋白受体（sTfR）抗原的高效表达体系获得足量的sTfR抗原，并免疫得到稳定的兔多克隆抗体，以此制备高灵敏度的体外检测试剂盒。首先通过筛选表达体系，最终确定以人胚胎肾细胞293（HEK293）表达体系进行sTfR抗原的表达，表达量可达0.52 mg/mL。为进一步增强sTfR表达量，优选CMV启动子及白蛋白信号肽，使抗原的表达量进一步增强至0.86 mg/mL。随后优选0.3 mg的sTfR抗原为免疫剂量，持续5次免疫新西兰兔后可获得高效价的sTfR兔多克隆抗体，该抗体耦联胶乳微球制成试剂盒后进行样本检测，其检测效果准确稳定。sTfR试剂盒及原料的开发显著降低生产成本，也为其他检测项目的试剂盒开发提供参考意义。

关键词: 可溶性转铁蛋白受体, 重组表达, 多克隆抗体, 体外诊断, 免疫

Abstract: An efficient expression system of human soluble transferrin sTfR antigen was constructed by various strategies and the stable rabbit polyclonal antibody was obtained by immunization. High sensitivity in vitro detection kit was developed using sTfR antigen and antibody. A suitable expression system was chosen to express sTfR in human embryonic kidney cells 293 (HEK293), and the expression amount of sTfR was 0.52 mg/mL. To further improve the expression level of sTfR, the CMV promoter and the albumin signal peptide were added, leading to a significant increase in the amount of sTfR (0.86 mg/mL). High titer of rabbit sTfR polyclonal antibody was obtained using 0.3 mg sTfR antigen for the fifth immunization. When using sTfR kit to analysis human serum samples, the kit developed in this work was credible and stable. The development of sTfR kit and raw materials had significantly reduced the production cost. This work also offered an efficient approach for the development of other in vitro diagnostic reagents.

Key words: serum transferrin receptor, recombinant expression, polyclonal antibody, in vitro diagnosis, immunity

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