Cloning, Expression And Initial Characterization Of An Unknown Function Gene Induced By HSV-1

HSV is a group of do uble-stranded DNA virus, which contains envelope components including glycoprotein that can attach to the cell surface to initiate infection. Previous studies on the interaction between H SV-1 and human fibroblast( KMB-17 strain) demonstrated that HSV-1 binding to KMB-17 strain cells could induce a specific gene response. In this study, HSV-1 stimulation related gene 1 (hsrg1) — a new unknown function gene of cellular response induced by the specific stimulation of HSV-1 was cloned from cDNA library established from mRNA of early gene response of KMB-17 cells, This gene (named as hsrg1) encoded an ORF of 1641 bp. To analyze the function of the coding protein, an expression vector pGEX-HSRG1 was constructed, and the purified protein was obtained as well. By immunizing the mice, the anti-sera of hsrg1 were obtained, and the speciality of the anti-sera was detected by western blotting. The anti-HSRG1 antibody was used to detect the expression of HSRG1 protein in KMB-17 cells stimulated by HSV-1 in immunoprecipitation assay, result showed that HSRG1 protein was actually induced in fibroblast bound by HSV-1 in high level.Northern blot of poly A~+ RNA sample extracted from 8 human tissues, were performed with the labeled probe of 572bp fragment of hsrgl and the results indicated that no expression of hsrgl gene in the pancreas and kidney was noticed and a higher expression was observed in skeletal muscle, brain, heart and skin.By using bioinformatic analysis, a conserved domain was found within the hsrgl, which showed a significant homology in six eukaryotic species. The protein encoded with this gene was identified in its structure to be a member of the SAND protein family. A comparison between the hsrgl cDNA sequence and Public Database of Human Genome Sequence revealed that the hsrgl gene was localized in chromosomel6q23.1, and was composed of 5 exons and encompassing genomic DNA of approximately 7kb.The location of HSRG1 protein in cell was determined by the expression of the pEGFP-HSRG1 plasmid in KMB-17 cells and PSORT Ⅱ software, results from two methods confirmed HSRG1 is predominantly located in cytoplasm. KMB-17 cells were transfected with pcDNA-HSRG1, transfected cells and control cells were infected with HSV-1, virus titer of two groups did not show significant differences. dsRNA were transfected into KMB-17 cells, the cell cycles and apoptosis were detected by...