生物学杂志

生物学杂志 ›› 2021, Vol. 38 ›› Issue (3): 108-.doi: 10.3969/j.issn.2095-1736.2021.03.108

• 技术方法 • 上一篇    下一篇

可溶性B淋巴细胞刺激因子原核表达及其单克隆抗体制备

  

  1. 空军军医大学 第二附属医院 检验科, 西安 710038
  • 出版日期:2021-06-18 发布日期:2021-06-21
  • 通讯作者: 董　轲，副主任医师，副教授，研究方向为肿瘤分子生物学研究及临床检验诊断学相关工作，E-mail:tdjyk3@fmmu.edu.cn
  • 作者简介:刘　冲，助理实验师，主要研究方向为恶性肿瘤发生、发展机制研究，E-mail：lc83832578@163.com
  • 基金资助:
    国家自然科学基金项目(81702732)

Prokaryotic expression of soluble B lymphocyte stimulating factor and preparation of anti-BLys monoclonal antibody aginst sBLyS

  1. Department of Clinical Laboratory, The Second Affiliated Hospital, Air Force Medical University, Xi′an 710038, China
  • Online:2021-06-18 Published:2021-06-21

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摘要： 为开发可溶性B淋巴细胞刺激因子(sBLyS)检测试剂，研究克隆sBLyS编码基因，并在大肠杆菌BL21(DE3)表达系统中表达sBLyS重组蛋白。sBLyS重组蛋白经镍柱亲和层析纯化后，免疫BALB/c小鼠，通过细胞融合技术构建可分泌抗sBLyS单克隆抗体的杂交瘤细胞株。采用正辛酸-硫酸铵沉淀方法纯化单克隆抗体，并利用Western Blot和ELISA方法检测抗体活性和效价。以未标记抗体作为捕获抗体，以辣根过氧化物酶(HRP)标记抗体作为检测抗体，进行双抗体夹心ELISA试验，筛选最佳组合。结果显示：成功表达纯化了sBLyS重组蛋白，获得的4株分泌抗sBLyS单克隆抗体的杂交瘤细胞株(2E4、2H8、4C5和4C12)，抗体效价分别为9.6、10.8、20.4和26.6ng/mL。Western Blot实验结果显示所获抗体具有sBLyS蛋白结合活性。双抗体夹心ELISA实验显示：2H8和HRP-4C5组合可用于检测sBLyS，检测线性度范围为0.4~1.6ng/mL(R2=0.995)。

关键词: 自身免疫病, B淋巴细胞刺激因子, 原核表达, 单克隆抗体

Abstract: To develop a soluble B lymphocyte stimulating factor (sBLyS) detection assay, the sBLyS encoding gene was cloned in this study, and sBLyS recombinant protein was expressed by using Escherichia coli BL21(DE3). The recombinant protein was purified by nickel column affinity chromatography. BALB/c mice were immunized with purified sBLyS recombinant protein, and cell lines secreting anti-BLyS monoclonal antibody (mAb) were obtained by using cell fusion technology. The mAb was purified by using n-Caprylic acid combined ammonium sulfate precipitation assay, and Western Blot and ELISA were used to analyze the activity and titer of the mAb. The unlabelled mAb and the HRP labeled mAb was used as the capture antibody, and the detection antibody, respectively, and then the double antibody sandwich ELISA test were performed to screen the optimal combination. The results showed that the sBLyS recombinant protein was expressed and purified. Four cell lines secreting the sBLyS monoclonal antibody hybridoma were obtained (named 2E4, 2H8, 4C5 and 4C12) with the titer of 9.6， 10.8, 20.4 and 26.6ng/mL, respectively. Western Blot results showed that the purified monoclonal antibodies had sBLyS protein binding activity. Double antibody sandwich ELISA showed that the combination of 2H8 and HRP-4C5 could be used to detect sBLyS, and the testing linearity ranged from 0.4ng/mL to 1.6ng/mL (R2=0.995).

Key words: autoimmunity disease, B lymphocyte stimulating factor, prokaryotic expression, monoclonal antibody

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