Molecular Cloning,protein Expression And Preliminary Functional Study Of C4orf22 Gene

In the past ten years,the infertility rate of couples of childbearing age is about 10-15%,among which male infertility accounts for 33.15%.One of the important reasons is male spermatogenesis disorder,which is mainly manifested by spermatozoa or severe oligospermia.Spermatogenesis is a complex process,which is regulated by genes,hormones and environment,among which spermatogenic genes play a key role.At present,many genes related to spermatogenesis or apoptosis of spermatogenic cells have been discovered through experiments,but many genes involved in regulating sperm development have not yet been discovered.Therefore,cloning and identification of novel genes related to spermatogenesis will help to elucidate the physiological or pathological mechanisms involved in spermatogenesis.In this study,we cloned a new gene C4orf22 in testicular tissues by digital differential display(DDD),and analyzed its spatial-temporal profile systematically,as well as its function was preliminarily studied.Bioinformatics analysis showed that the cDNA of mouse C4orf22 gene was 891 bp in length,located in E3-E4 region of mouse chromosome 5,containing 6 exons and 5 introns,encoding a protein with a molecular weight of 26.9 KD and 233 amino acids.C4orf22 mRNA was predominantly expressed in the testicular of sexually mature mice and weakly expressed in lung,while no expression was detected in other tissues.Immunohistochemical analysis showed that C4orf22 protein was mainly expressed in spermatogonial cells and spermatogonial cells of testicular tissue.The prokaryotic expression vector pET28b/C4orf22 was constructed and transferred into Ecoli BL21,the recombinant protein was inducd by IPTG and isolated by affinity chromatography.The results of subcellular localization showed that after transfection of pEGFP-C3/C4orf22 plasmid in GC-1 cells and GC-2 cells respectively,green fluorescence was observed mainly in the cytoplasm,indicating that C4orf22 protein was mainly expressed in the cytoplasm of these two kinds of cells.Over-expression of C4orf22 gene in GC-1 cells and flow analysis of cell cycle and apoptosis showed that over-expression of C4orf22 gene in GC-1 cells could significantly inhibit apoptosis.The results of qRT-PCR analysis of the apoptosis-related genes Bax,bcl-2,caspase-3 and caspase-8 showed that the mRNA expression of C4orf22 gene was significantly increased in the bcl-2 gene,but decreased in the Bax,caspase-3 and caspase-8 genes.On the other hand,interference with the expression of this gene in GC-1 cells and flow analysis of cell cycle and cellapoptosis showed that interference with the expression of this gene promoted cell apoptosis.The above experimental results showed that the new C4orf22 gene was related to the occurrence and apoptosis of spermatogenic cells,and it may affect the apoptosis of spermatogenic cells in GC-1 cells through the bcl-2 regulatory pathway.