人蛋白质组芯片筛选宿主中与结核分枝杆菌Rv1705c相互作用的蛋白质  

Screening for host proteins interacting with Mycobacterium tuberculosis Rv1705c by human proteome microarray

作　　者：刘涛[1] 洪文旭 邹小飞[1] 桂静[1] 傅钰[1] 洪湘雅 叶春立 Liu Tao;Hong Wenxu;Zou Xiaofei;Gui Jing;Fu Yu;Hong Xiangya;Ye Chunli(Department of Clinical Laboratory,Shenzhen Center for Chronic Disease Prevention,Shenzhen 518020,China)

机构地区：[1]深圳市慢性病防治中心临床检验科,深圳518020

出　　处：《中华微生物学和免疫学杂志》2022年第2期128-133,共6页Chinese Journal of Microbiology and Immunology

摘　　要：目的探究人体中与结核分枝杆菌(Mycobacterium tuberculosis,Mtb)Rv1705c蛋白相互作用的蛋白质。方法原核表达Rv1705c蛋白基因,收集包涵体,裂解后透析纯化Rv1705c并测定其浓度,ELISA法检测细菌Rv1705c刺激巨噬细胞后细胞因子IFN-γ的分泌量,使用纯化并带有生物素标记的Rv1705c孵育人蛋白质组芯片HuProt^(TM),筛选与Rv1705c相互作用的人类蛋白质,使用GenePix Pro 6.0软件对蛋白质芯片的信号图像进行数据提取,使用GO、KEGG等多个数据库进行生物信息学分析,GST pulldown验证Rv1705c与PSMA3、RSAD2的相互作用。结果纯化结果显示,Rv1705c在包涵体中表达,Rv1705c刺激巨噬细胞后IFN-γ分泌量显著上升。芯片结果显示共筛选出了29个与Rv1705c相互作用的潜在阳性蛋白质,其中PSMA3、NLN、THOP1、UPF3A、RSAD2、OMG、PNKD、STEAP3、MED8共9个蛋白质的信噪比(signal-to-noise ratio,SNR)>1.6。进一步的生物信息学分析发现候选蛋白PSMA3、RSAD2、C1QBP参与固有免疫应答激活信号转导,并且PSMA3、RSAD2与干扰素存在交互作用,GST pulldown验证PSMA3、RSAD2与Rv1705c确有相互作用。结论发现并验证PSMA3、RSAD2与Rv1705c存在相互作用,为Mtb感染机制的研究提供参考。Objective To investigate the proteins interacting with Mycobacterium tuberculosis Rv1705c in human body.Methods Rv1705c was prokaryotically expressed and inclusion bodies were collected for further lysis and the purification of Rv1705c.ELISA assay was used to detect the secretion of IFN-γafter stimulating macrophages with Rv1705c protein.Purified and biotin-labeled Rv1705c sample was incubated on the HuProt^(TM) human proteome microarray to screen the interacting proteins.GenePix Pro 6.0 software was used to extract all features of the data obtained from the scanned images and further analysis was performed based on bioinformatics databases such as GO and KEGG.GST pull-down was performed to verify the interaction of Rv1705c with PSMA3 and RSAD2.Results The purification results showed that Rv1705c was expressed in endosomes.The secretion of IFN-γincreased significantly after stimulating macrophages with Rv1705c.A total of 29 potential Rv1705c-interacting proteins were screened,and nine of them showed signal-to-noise ratio(SNR)>1.6,namely PSMA3,NLN,THOP1,UPF3A,RSAD2,OMG,PNKD,STEAP3 and MED8.Further bioinformatics analysis revealed that PSMA3,RSAD2 and C1QBP were involved in innate immune signaling pathway,and there were interactions of PSMA3 and RSAD2 with IFN.GST pull-down assay validated that PSMA3 and RSAD2 interacted with Rv1705c.Conclusions This study showed that PSMA3 and RSAD2 interacted with Rv1705c,providing reference for further investigation on the mechanism of Mycobacterium tuberculosis infection.

关 键 词：人蛋白质组芯片 Rv1705c 生物信息学 结核病 

分 类 号：R52[医药卫生—内科学]