ABSTRACT
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-γ after stimulating macrophages with Rv1705c protein. Purified and biotin-labeled Rv1705c sample was incubated on the HuProt? 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-γ increased 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.