猪源GSDMA蛋白多克隆抗体的制备与初步应用  

Preparation and application of polyclonal antibody against porcine GSDMA protein

作　　者：李帅 宋洁 周仕君 刘佳 李江南[1,2] 翁长江 LI Shuai;SONG Jie;ZHOU Shi-jun;LIU Jia;LI Jiang-nan;WENG Chang-jiang(Division of Fundamental Immunology,Harbin Veterinary Research Institute,Chinese Academy of Agricultural Sciences,State Key Laboratory of Veterinary Biotechnology,Harbin 150069,China;Heilongjiang provincial Key Laboratory of Veterinary Biotechnology,Harbin 150069,China)

机构地区：[1]中国农业科学院哈尔滨兽医研究所兽医生物技术国家重点实验室/基础免疫创新团队,黑龙江哈尔滨150069 [2]黑龙江省免疫学重点实验室,黑龙江哈尔滨150069

出　　处：《中国预防兽医学报》2022年第12期1311-1316,1331,共7页Chinese Journal of Preventive Veterinary Medicine

基　　金：国家自然科学基金面上项目(31872448);黑龙江省自然科学基金优秀青年项目(YQ2019C033)。

摘　　要：Gasdermin A(GSDMA)作为Gasdermin(GSDM)蛋白家族的一员,参与细胞的炎症反应,其经切割产生的片段可以诱导细胞焦亡。为制备猪GSDMA(pGSDMA)多克隆抗体,本研究构建了重组表达质粒pET-21apGSDMA,转化BL21(DE3)感受态细胞后经IPTG诱导,表达并纯化了重组pGSDMA蛋白。将纯化的该重组蛋白免疫小鼠制备pGSDMA多克隆抗体(pAb),并进行western blot和间接免疫荧光试验鉴定。结果显示,pGSDMA pAb能够特异性地识别外源表达的pGSDMA蛋白和猪肺泡巨噬细胞(PAMs)中内源表达的pGSDMA蛋白。利用该pAb检测猪主要组织器官中pGSDMA蛋白的表达,结果显示其能识别猪不同组织器官中的pGSDMA蛋白,且pGSDMA在猪心脏中的表达量最低,在肝、脾、肺和肾中的表达量较高。利用该pAb检测不同剂量高致病性猪繁殖与呼吸综合征病毒(HP-PRRSV)感染PAMs中p GSDMA的表达,结果显示pGSDMA蛋白的表达量随着病毒感染剂量的增加而降低,表明pGSDMA在HP-PRRSV感染过程中发挥相关生物学作用。本研究首次制备了pGSDMA pAb,为深入探究pGSDMA生物学功能奠定了基础。Gasdermin A(GSDMA), a member of Gasdermin(GSDM) family, is involved in antiviral inflammatory responses.GSDMA could be cleaved to produce fragment that leads to pyroptosis. In order to prepare polyclonal antibodies against porcine GSDMA(pGSDMA), the recombinant expression plasmid p ET-21a-pGSDMA was constructed and then transformed into BL21(DE3) competent cells and induced by IPTG. The recombinant pGSDMA was purified, and five BALB/c mice were immunized to prepare pGSDMA polyclonal antibodies(pAb). Western blot and indirect immunofluorescence assay results demonstrated that pGSDMA pAb could specifically recognize the exogenous Flag-pGSDMA in HEK293 cells transfected with p CAGGS-FlagGSDMA and the endogenous p GSDMA in porcine alveolar macrophage(PAMs). The pGSDMA pAb was used to detect pGSDMA in pig tissues, and the results showed that pGSDMA protein in different tissues and organs of pigs could be recognized by pGSDMA p Ab. The expression level of p GSDMA in the heart was the lowest but higher in the liver, spleen, lung, and kidney. In addition, we found that the expression of p GSDMA protein in PAMs decreased with the increase of the infectious dose of highly pathogenic-porcine reproductive and respiratory syndrome virus(HP-PRRSV), demonstrating that p GSDMA plays a biological role in the infection period of HP-PRRSV. In this study, the p GSDMA p Ab were prepared for the first time. This study lays a foundation for further study of p GSDMA and its biological function during PRRSV infection.

关 键 词：GSDMA 原核表达 多克隆抗体 高致病性猪繁殖与呼吸综合征病毒 

分 类 号：S852.4[农业科学—基础兽医学]