天津医药

天津医药 ›› 2017, Vol. 45 ›› Issue (9): 897-901.doi: 10.11958/20170284

• 细胞与分子生物学 •    下一篇

抗 IL-4R 单链抗体原核表达载体的构建与表达

杨光勇，刘茜明，刘莉莉，王文佳，何光志   

  1. 贵阳中医学院基础医学院（邮编 550002）
  • 收稿日期:2017-03-07 修回日期:2017-05-12 出版日期:2017-09-15 发布日期:2017-09-25
  • 通讯作者: 何光志 E-mail:392724549@qq.com
  • 基金资助:
    2011 年贵州省社会发展科技攻关项目[黔科合 SY 字( 2011) 3020];贵州省委组织部高层次人才科研条件特助经费( TZJF - 2011年28 号);2012年贵州省科学技术基金（黔科合[2012]2075号）联合资助;贵州省高等学校工程研究中心项目(黔教合KY字20153377)

Construction and expression of anti IL-4R single antibody of prokaryotic expression vector

YANG Guang-yong, LIU Qian-ming, LIU Li-li, WANG Wen-jia, HE Guang-zhi   

  • Received:2017-03-07 Revised:2017-05-12 Published:2017-09-15 Online:2017-09-25

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摘要： 摘要：目的 通过 BL21（DE3）原核表达系统制备抗白细胞介素-4 受体（IL-4R）鼠抗人单链抗体（scFv）。方法 在前期研究结果的基础上优化抗 IL-4R scFv 序列，优化后分析 scFv 序列，构建重组质粒 pET-32a-scFv，将该重组质粒酶切鉴定，将其转入 BL21（DE3）原核表达菌进行诱导表达，并进行 SDS-PAGE 分析检测，对表达蛋白进行纯化和复性，通过 SDS-PAGE 分析抗 IL-4R 单链抗体的分子质量，运用 Western blot 检测融合蛋白的特异性。结果 插入pET-32a 载体的 scFv 序列长度 761 bp，抗 IL-4R 单链抗体的分子质量在 45 ku 左右，重组蛋白具有较高的特异性。结论 本实验成功构建 pET32a-scFv 原核表达系统，重组蛋白具有较高的免疫反应性，为进一步研究抗 IL-4R 单链抗体作为药物靶点提供了工作基础。

关键词: 受体, 白细胞介素 4, 重组蛋白质类, 电泳, 聚丙烯酰氨凝胶, 单链抗体, 原核表达

Abstract: Abstract: Objective To construct anti-IL-4R murine anti-human single-chain variable fragment (scFvs) antibodies through BL21 (DE3) prokaryotic expression system. Methods The anti-IL-4R scFv sequence was optimizated on the basis of previous findings. The optimized scFv sequence was analyzed. The recombinant plasmid pET-32a-scFv was constructed. The recombinant plasmid was detected through enzyme identification, and was turned into BL21 (DE3) prokaryotic expression bacteria to express the pET- 32a- scFv recombinant protein in E.coli BL21 (DE3). The purification and renaturation were researched, and SDS- PAGE analysis was studied. The molecular weight of ScFv against IL- 4R was analyzed by SDS-PAGE. The expression of the fusion protein was detected by Western-blot assay. Results The length of fusion gene scFv-MLT sequence was 761 bp. The molecular weight of the recombinant expression of proteins of anti-IL-4R single antibody was approximately 45 ku. The recombinant proteins showed high specificity with anti-6×His-tag antibody. Conclusion This experiment successfully constructs pET-32a-scFv prokaryotic expression system of recombinant protein with high immune reactivity, which provides the basis for further study of anti-IL-4R single chain antibody as drug target.

Key words: receptors, interleukin- 4, recombinant proteins, electrophoresis, polyacrylamide gel, scFv, prokaryotic expression