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.
Abstract
In order to prepare porcine GSDMD polyclonal antibody,the recombinant expression plasmid pET-21a-nGSDMD was constructed,and the recombinant nGSDMD protein was successfully expressed and purified.nGSDMD polyclonal antibody(pAb)was produced by immunizing New Zealand white rabbits with purified recombinant protein.The antibody titre was 1∶64 000 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.
基金项目
"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)
国家科技期刊平台
NSTL
万方数据