|本期目录/Table of Contents|

[1]高秋雲,孙亚楠,马振毅.GSDMD单域抗体的筛选及功能探究[J].天津医科大学学报,2023,29(06):622-627.
　GAO Qiu-yun,SUN Ya-nan,MA Zhen-yi.Screening and functional investigation of anti-GSDMD single-domain antibodies[J].Journal of Tianjin Medical University,2023,29(06):622-627.
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GSDMD单域抗体的筛选及功能探究(PDF)
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《天津医科大学学报》[ISSN:1006-8147/CN:12-1259/R]

卷:
29卷
期数:
2023年06期
页码:
622-627
栏目:
基础医学
出版日期:
2023-11-20

文章信息/Info

Title:
Screening and functional investigation of anti-GSDMD single-domain antibodies
文章编号:
1006-8147(2023) 06-0622-06
作者:
高秋雲; 孙亚楠; 马振毅
（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）
Author(s):
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）
关键词:
细胞死亡; 焦亡; gasdermin D; 单域抗体; 筛选
Keywords:
cell death;  pyroptosis;  gasdermin D;  single-domain antibody;  screening
分类号:
R34
DOI:
-
文献标志码:
A
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，isPLA）结合高通量测序筛选抗GSDMD sdAbs候选序列；利用isPLA、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isPLA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。
Abstract:
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-1β and 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.

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备注/Memo

备注/Memo:
基金项目 天津市教委科研计划（2021KJ251）
作者简介 高秋雲（1995-），女，硕士在读，研究方向：生物化学与分子生物学；通信作者：孙亚楠，E-mail：yanansun@tmu.edu.cn；马振毅，E-mail:zhyma@tmu.edu.cn。
更新日期/Last Update: 2023-12-01