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作 者:刘佳[1,2,3] 宋洁 李帅 王尚辉 李江南 王春凤[1] 翁长江 曾艳[1] LIU Jia;SONG Je;LI Shuai;WANG Shanghui;LI Jjiangnan;WANG Chunfeng;WENG Changiang;ZENG Yan(College of Veterinary Medicine,Jilin Agricultural University,Changchun 130118,China;State Key Laboratory for Animal Disease Control and Prevention,Harbin Veterinary Research Institute,Chinese Academy of Agricultural Sciences,Harbin 150069,China;Heilongjiang Provincial Key laboratory of Veterinary Immunology,Harbin 150069,China)
机构地区:[1]吉林农业大学动物医学学院,吉林长春130118 [2]中国农业科学院哈尔滨兽医研究所动物疫病防控全国重点实验室,黑龙江哈尔滨150069 [3]黑龙江省兽医免疫重点实验室,黑龙江哈尔滨150069
出 处:《中国兽医科学》2024年第7期968-973,共6页Chinese Veterinary Science
基 金:“十四五”国家重点研发计划项目(2022YFD1800300);黑龙江省自然科学基金面上项目(C2016061)。
摘 要:为制备猪源GSDMD多克隆抗体,构建了重组表达质粒pET-21a-nGSDMD,成功表达并纯化了重组nGSDMD蛋白。将纯化的重组蛋白免疫新西兰大白兔制备nGSDMD多克隆抗体(pAb),经ELISA检测,抗体效价为1∶64000。Western-blot和IFA显示该抗体能特异性识别HEK293T细胞中瞬时表达的Flag-GSDMD蛋白和PRRSV感染肺泡巨噬细胞(PAMs)后表达的GSDMD蛋白。该多克隆抗体特异性良好,无交叉反应,能识别检测病毒感染条件下GSDMD切割片段,可用于PRV和PRRSV诱导的细胞焦亡检测。综上,本研究为探究GSDMD蛋白提供了良好的工具,为深入探究GSDMD蛋白的生物学功能奠定了基础。In order to prepare porcine GSDMD polyclonal antibody,the recombinant expression plasmid p ET-21a-n GSDMD was constructed,and the recombinant n GSDMD protein was successfully expressed and purified.n GSDMD polyclonal antibody(p Ab)was produced by immunizing New Zealand white rabbits with purified recombinant protein.The antibody titre was 1∶64000 by ELISA.Western-blot and IFA showed that the antibody could specifically recognize Flag-GSDMD protein expressed in HEK293T cells and GSDMD protein expressed in alveolar macrophages(PAMs)after PRRSV infection.The polyclonal antibody has good specificity and no cross-reactivity.It can identify and detect GSDMD fragments under the condition of viral infection and can be used to detect PRV and PRRSV-induced pyroptosis.In conclusion,this study provides a good tool for exploring GSDMD protein and lays a foundation for further exploring the biological function of GSDMD protein.
分 类 号:S852.659.6[农业科学—基础兽医学]
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