摘要
目的构建并鉴定蛋白磷酸酶2A(protein phosphatase 2A,PP2A)调节蛋白磷酸酶2调节亚基B"alpha(protein phosphatase 2 regulatory subunit B"-α,PPP2R3A)的相互作用蛋白图谱,以探索PPP2R3A在心肌细胞中的作用机制。方法首先构建人类原代心肌细胞的cDNA文库,并通过质粒转化技术进行验证。然后,采用酵母双杂交技术,以PPP2R3A为诱饵,筛选出与之相互作用的蛋白。最后,对筛选出的相互作用蛋白进行功能分类与分析。结果研究成功构建人类原代心肌细胞的cDNA文库,重组效率达到100%,滴度为1.2×10^(7)CFU/mL,cDNA片段长度大部分超过1200 bp,保证了心肌细胞基因表达的广泛覆盖。研究成功鉴定出19种与PPP2R3A相互作用的蛋白,包括GIPC1、AXIN1、RGS19、COL1A2、MYO15B、Vimentin、RBM10、EWSR1、BCL6、PBXIP1、HTRA1和KAT5等。其中COL1A2的筛选频率最高,为12.5%。回转验证实验表明,这些相互作用蛋白能够激活His、Ade和LacZ报告基因,进一步确认了它们与PPP2R3A的相互作用。结论PPP2R3A通过与多种蛋白的相互作用,参与了心肌细胞的多条生物学途径,揭示了其在心脏生理与病理过程中复杂的调控网络,为心血管疾病的靶向治疗提供了新的研究思路和潜在的治疗靶点。
Objective To construct and identify the interaction protein map of protein phosphatase 2A(PP2A)regulat-ing protein phosphatase 2 regulatory subunit B"-α(PPP2R3A),and to explore the mechanism of PPP2R3A in cardio-myocytes.Methods Firstly,a cDNA library of human primary cardiomyocytes was constructed and verified by plasmid transformation technology.Then,the proteins interacting with PPP2R3A were screened by yeast two-hybrid tech-nique.Finally,the selected interacting proteins were functionally classified and analyzed.Results The cDNA library of human primary cardiomyocytes was successfully constructed.The recombination efficiency was 100%,the titer was 1.2×10^(7)CFU/mL,and the length of most cDNA fragments was more than 1200 bp,which ensured the wide coverage of gene expression in cardiomyocytes.The study successfully identified 19 proteins that interact with PPP2R3A,in-cluding GIPC1,AXIN1,RGS19,COL1A2,MYO15B,Vimentin,RBM10,EWSR1,BCL6,PBXIP1,HTRA1 and KAT5.Among them,the screening frequency of COL1A2 was the highest,which was 12.5%.Rotating validation ex-periments showed that these interacting proteins could activate His,Ade and LacZ reporter genes,further confirming their interaction with PPP2R3A.Conclusion PPP2R3A participates in multiple biological pathways of cardiomyocytes by interacting with various proteins,revealing its complex regulatory network in cardiac physiology and pathology,and providing new research ideas and potential therapeutic targets for targeted therapy of cardiovascular diseases.
作者
高晶
吴春燕
宋贵波
GAO Jing;WU Chunyan;SONG Guibo(Department of Medical Laboratory,The First Affiliated Hospital of Kunming Medical University,Kunming 650032,Yunnan,China;Department of Blood Transfusion,The Second People′s Hospital of Qujing,Qujing 655000,Yunnan,China)
出处
《系统医学》
2025年第12期1-5,共5页
Systems Medicine
基金
国家自然科学基金(81460064)
云南省基础研究计划项目(202301AT070236)
云南省“高层次人才培养支持计划”青年拔尖人才(YNWR-QNBJ-2020-261)
云南省应用基础研究-昆医联合专项(2014FB034)。
作者简介
高晶(1982—),女,本科,主管技师,研究方向为心肌病的分子诊断及致病机制研究;通信作者:宋贵波(1986—),男,博士,副主任技师,研究方向为心肌病的分子诊断及致病机制研究,E-mail:songgb1229@163.com。
