重庆理工大学学报（自然科学）

重庆理工大学学报（自然科学） ›› 2023, Vol. 37 ›› Issue (2): 350-356.doi: 10.3969/j.issn.1674-8425(z).2023.02.039

• 药学·生物工程 • 上一篇    下一篇

东亚飞蝗DSCAM2多克隆抗体制备

邱佳, 余瑛   

  1. 重庆理工大学 药学与生物工程学院，重庆 ４０００５４
  • 出版日期:2023-03-21 发布日期:2023-03-21
  • 作者简介:邱佳，女，硕士研究生，主要从事药理学研究，Ｅｍａｉｌ：７４００２９３６５＠ｑｑ．ｃｏｍ；通讯作者：余瑛，女，博士，副教授，主 要从事生化与分子生物学研究，Ｅｍａｉｌ：ｘｘｙｙ＠ｃｑｕｔ．ｅｄｕ．ｃｎ

Preparation of polyclonal antibodies against Locustamigratoria DSCAM2

  • Online:2023-03-21 Published:2023-03-21

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摘要： 制备唐氏综合征细胞粘附分子 ２（ＤＳＣＡＭ２）多克隆抗体，拟为 ＤＳＣＡＭ２功能探索 提供免疫学工具。采用生物信息学方法对 ＤＳＣＡＭ２抗原结构进行分析；利用 ＤＮＡ重组技术构 建 ＤＳＣＡＭ２８２８－９５７重组表达菌株；采用 Ｎｉ亲和层析技术纯化重组 ＤＳＣＡＭ２８２８－９５７蛋白，免疫小鼠 制备 ＡｎｔｉＤＳＣＡＭ２多克隆抗体；采用 ＥＬＩＳＡ法检测抗体效价、ＷｅｓｔｅｒｎＢｌｏｔ法检测抗体特异性、 瑞氏 －吉姆萨染色和免疫荧光检测 ＤＳＣＡＭ２在东亚飞蝗血淋巴细胞定位；采用 ｓｉＲＮＡ敲低 ＤＳＣＡＭ２，进行血淋巴细胞计数。结果表明，所构建的 ｐＥＴ３０ａ（＋）ＤＳＣＡＭ２８２８－９５７／ＢＬ２１（ＤＥ３） 原核菌株成功表达重组 ＤＳＣＡＭ２８２８－９５７蛋白；用 Ｎｉ螯合亲和层析分离获得的 ＤＳＣＡＭ２８２８－９５７重组 抗原免疫小鼠，获得了效价高且特异性好的 ＤＳＣＡＭ２多克隆抗体；细胞定位分析显示 ＤＳＣＡＭ２ 在东亚飞蝗嗜碱性粒细胞高表达；敲低 ＤＳＣＡＭ２表达水平，引起东亚飞蝗总血淋巴细胞数量 增多。

关键词: 东亚飞蝗, 唐氏综合征细胞粘附分子 ２, 结构域, ＤＮＡ重组, 蛋白质纯化, 抗体制备

Abstract: In this study,the prepared Down syndrome cell adhesion molecule 2 (DSCAM2) polyclonal antibodiesare intended to provide an immunological tool for functional exploration of DSCAM2. This project employs bioinformatics approaches to analyze the DSCAM2 antigenic structure, and constructs DSCAM2828-957recombinant expression strain by DNA recombination technology.The recombinant DSCAM2828-957 proteinis purified by Ni affinity chromatography and Anti-DSCAM2 polyclonal antibodiesare prepared by immunized mice. Antibody titers are detected by ELISA, antibody specificity is detected by Western Blot,and DSCAM2 localization in Locustamigratoria hemocytes is detected by Wright-Giemsa staining and immunofluorescence.DSCAM2 is knocked down by siRNA,and hemocytes are counted. The results show that the constructed pET30a(+)-DSCAM2828-957/BL21(DE3) prokaryotic strain can successfully express the recombinant DSCAM2828-957 protein.The immuned mice with DSCAM2828-957 recombinant antigens obtained through Ni chelating affinity chromatography gain polyclonal antibodies with high titer and good specificity.Cell localization analysis shows a high expression of DSCAM2 in basophils of Locustamigratoria.A knockdown of DSCAM2 expression level increases the number of total hemocytes in Locustamigratoria.

中图分类号: 

  • Q784