GSDMD单域抗体的筛选及功能探究  

creening and functional investigation of anti-GSDMD single-domain antibodies

作　　者：高秋雲 孙亚楠 马振毅 GAO Qiu-yun;SUN Ya-nan;MA Zhen-yi(Department of Biochemistry and Molecular Biology,School of Basic Medical Sciences,Tianjin Medical University,Tianjin 300070,China)

机构地区：[1]天津医科大学基础医学院生物化学与分子生物学系,天津300070

出　　处：《天津医科大学学报》2023年第6期622-627,共6页Journal of Tianjin Medical University

基　　金：天津市教委科研计划(2021KJ251)。

摘　　要：目的:筛选并验证gasdermin D(GSDMD)单域抗体(single-domain antibody,sdAb)及其生物学功能。方法:利用原位邻近连接分析(in situ proximity ligation assay,is PLA)结合高通量测序筛选抗GSDMD sdAbs候选序列;利用is PLA、Co-IP、GST pull down、等温滴定量热法(isothermal titration calorimetry,ITC)验证这些sdAbs与GSDMD的特异性结合;在脂多糖(LPS)和nigericin处理的细胞焦亡模型中,观察细胞表型变化;检测细胞上清液中白细胞介素1-β(IL-1β)水平变化以及乳酸脱氢酶(LDH)的释放;通过Western印迹检测经上述处理后的细胞中GSDMD以及GSDMD N端结构域(GSDMD N terminus,GSDMD-NT)量的变化。结果:通过is PLA结合高通量测序方法筛选出GSDMD sdAb的候选序列,其中sdAb#26与GSDMD C端结构域(GSDMD C terminus,GSDMD-CT)相互作用;与sdAb Con对照组相比,sdAb#26处理高表达GSDMD细胞产生焦亡表型的细胞显著增多;细胞上清中释放的IL-1β以及LDH显著提高(t=68.54,P<0.001;t=5.909,P<0.01);GSDMD-NT产生量显著增加。结论:GSDMD sdAb具备操控GSDMD介导焦亡的潜力,为焦亡相关疾病的治疗提供了新思路。Objective:To screen and investigate single-domain antibodies(sdAbs)against gasdermin D(GSDMD)and their biological functions.Methods:Using in situ proximity ligation assay(isPLA)followed by high-throughput sequencing,the candidate sequences of anti-GSDMD sdAbs were obtained.The specific binding of these sdAbs against GSDMD was verified by isPLA,Co-IP,GST pull down,and isothermal titration calorimetry(ITC),respectively.In the pyroptosis model of lipopolysaccharide(LPS)and nigericin treated THP-1 cells,the cellular morphology,the level of interleukin-1β(IL-1β)and the release of lactate dehydrogenase(LDH)in cell supernatants were detected.Western blotting was also used to detect the expression of GSDMD and GSDMD N-terminal domain(GSDMD-NT)in the above treated cells.Results:The candidate sequences of anti-GSDMD sdAbs were screened by isPLA followed by high-throughput sequencing.One of them,sdAb#26 was verified to interact with the C-terminus of GSDMD(GSDMD-CT).Compared with the sdAb control group,the number of cells producing pyroptosis morphology in high expression GSDMD cells treated with sdAb#26 was significantly increased.The release of IL-1βand LDH in cell supernatants significantly increased(t=68.54,P<0.001;t=5.909,P<0.01).The production of GSDMD-NT significantly increased.Conclusion:Anti-GSDMD sdAb has the potential to manipulate GSDMD-mediated pyroptosis,which may provide a novel manipulation for the treatment of pyroptosis-related diseases.

关 键 词：细胞死亡 焦亡 gasdermin D 单域抗体 筛选 

分 类 号：R34[医药卫生—基础医学]