东亚飞蝗DSCAM2多克隆抗体制备  

Preparation of polyclonal antibodies against Locustamigratoria DSCAM2

作　　者：邱佳 余瑛 QIU Jia;YU Ying(School of Pharmacyand Bioengineering,Chongqing University of Technology,Chongqing 400054,China)

机构地区：[1]重庆理工大学药学与生物工程学院,重庆400054

出　　处：《重庆理工大学学报（自然科学）》2023年第2期350-356,共7页Journal of Chongqing University of Technology：Natural Science

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、Western Blot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。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 DSCAM2828-957 recombinant 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.

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 

分 类 号：Q784[生物学—分子生物学]