The Experimental Study Of Androgen Receptor Interacting Protein In Male Mononuclear Cells

Objective:The incidence of atherosclerosis?AS?is gradually increasing,and the pathogenesis,prevention and treatment of AS and its related diseases have become hot spots.Dihydrotestosterone?DHT?is the sterol formed by the 5?-reductase reducing testosterone double bond and is the most active androgen.Previous studies have found that physiological levels of DHT can significantly reduce m RNA expression of lectin-like oxidized low-density lipoprotein receptor 1?LOX-1?in macrophages,and at least In part,it acts through the androgen receptor?AR?.To further explore the role of androgen and its receptors in the development of atherosclerosis,our group previously screened proteins interacting with AR in male peripheral blood mononuclear cells through yeast two-hybrid screening to obtain ribosomal protein L10?RPL10?,pre-B cell leukemia homeobox protein 1?PBXIP1?,but yeast two-hybridization does not fully confirm the role of protein and protein,and there may be false positives.Therefore,this experiment aims to use pull down technology to further verify the interaction between AR and the above proteins in vitro,and then to explore the signal transduction pathway of the interaction between protein and AR in male monocytes,possibly as an auxiliary regulatory factor.The interaction with AR provides a theoretical basis and target for atherosclerosis pathogenesis and treatment.Research methods:This study uses gene cloning technology,pGADT₇-RPL10,pGADT₇-PBXIP1 as templates,to design specificprimers for amplification ofRPL10and PBXIP1 gene fragments,polymerase chain reaction?PCR?method was used to amplify the destination segment?RPL10,PBXIP1?.The RPL10 and PBXIP1 gene fragments were cloned into the pGEX₄T₂ vector containing GST tags after restriction enzyme digestion with Eco R I and Bam H I and the recombinant plasmids pGEX₄T₂-RPL10 and pGEX4T2-PBXIP1 were constructed,sequenced and analyzed.The inducer IPTG was used to induce E.coli expressed protein RPL10,PBXIP1.The protein was separated by polyacrylamide gel electrophoresis and was stained with Coomassie Blue.Protein expression was observed after decolorization.Using pull down system,pGBKT₇-AR recombinant plasmid as a capture protein for in vitro transcription;the RPL10,PBXIP1protein expressed in E.coli was extracted,and immobilized as a bait protein on MagneGST^?Particles.Then the two proteins were co-incubated,eluted and purified.Western blot was used to analysis the interaction of protein complexes,chemiluminescence method was used to identify the results and analysis.Result:The target gene fragment was obtained by PCR and the recombinant plasmids GST-RPL10 and GST-PBXIP1 were successfully constructed.After IPTG-induced RPL10 and PBXIP1 fusion proteins were successfully expressed,they were confirmed by pull down and Western blot techniques.The size of the GST-RPL10fusion protein was approximately 50 KD,the size of the GST-PBXIP1 fusion protein is about 52KD,the blank control GST protein size is about 26KD,and the AR-LBD fusion protein size is about 37KD;the two proteins are incubated together and detected with different antibodies to detect GST-RPL10/The PBXIP1 fusion protein can specifically pull down the c-myc-AR fusion protein in vitro.The blank control GST protein cannot pull down the c-myc-AR fusion protein.Conclusion:It was further confirmed AR protein can interact with RPL10,PBXIP1respectively in vitro by the pull down technology.