Expression Analysis Of Carp Ubc9 And Its Effect On CGSIV DNA Replication

The Ubc9 gene encodes the only E2 binding enzyme(UBE2I)in the SUMOylation modification system.The E2 binding enzyme can directly bind to the SUMO molecule and bind it to the substrate protein.It is a key node in the SUMO modification and plays an important role in the SUMO modification.Studies have shown that Ubc9-mediated SUMOylation modification affects viral replication.In this paper,the cloning,expression,analysis and subcellular localization of squid epithelial cell(EPC)Ubc9 c DNA were used to up-regulate and down-regulate the expression of EPC Ubc9 and its effect on the proliferation and DNA replication of CGSIV virus,and to screen and verify the interaction with Ubc9.The role of viral proteins,exploring the relationship between cellular Ubc9 gene and viral DNA replication.The main results are as follows:1.Cloning,expression,subcellular localization and bioinformatics analysis of the EPC Ubc9 gene.Using EPC cell c DNA as a template,the c DNA fragment of EPC Ubc9 was amplified and prokaryotically expressed as a material for protein purification and preparation of antibodies.At the same time,EPC Ubc9 was expressed in eukaryotic cells,and the fusion protein Ubc9 was localized in the cytoplasmic region of the circular nucleus.The sequence structure analysis and phylogenetic tree construction of EPC Ubc9 showed that EPC Ubc9 has a characteristic conserved domain with ubiquitinated ligase family and SUMO ligase,ubiquitinated ligase active site,secreted signal peptide Site,E3 interacting residue,cysteine??active site,with SUMO transferase activity.The phylogenetic tree of Ubc9 sequence reconstruction showed that carp Ubc9 and other scorpionidae belonged to one branch,and were more similar to the genus genus.The predicted result of the CGSIV ORF SUMO modification site revealed the presence of a SUMOylation modification site on the CGSIV ORF.2.Up-regulation and down-regulation of EPC Ubc9 expression have an effect on the proliferation and DNA replication of CGSIV virus,and screening for viral proteins that interact with Ubc9.Overexpression of Ubc9 promotes CGSIV virus proliferation and DNA replication,and construction and screening of cell lines stably expressing Ubc9.Ubc9 was overexpressed in EPC cells,and viral DNA copy number and virus titer were detected by real-time PCR and virus titer experiments,respectively.The results showed that the over-expression group was significantly up-regulated compared to the control group with average virus titer and viral DNA copy number.Studies have shown that the Ubc9 gene plays a key role in the replication of the CGSIV virus.Then,the corresponding vector was transfected into EPC cells,and the cell line stably expressing Ubc9 was screened by G418 to prepare materials for the later Ubc9 proteinfunction study.Three small interfering RNAs were designed by RNA interference technology,and RNA interference experiments were performed according to the best efficiency selection.The results showed that there was no significant difference in virus copy number between the si R-343 RNA interference group and the negative control group and the blank control group after virus infestation for 3 h;after the virus infection for 48 h,the si R-343 RNA interference group was compared with the negative control group.Both the copy number and the virus titer of the blank control group were significantly decreased,and the difference was statistically significant.The interaction of Ubc9 with CGSIV-PCNA was verified by CO-IP.Ubc9 and PCNA genes were co-expressed in Hela cells,and proteins were collected 48 h after transfection.The protein concentration was determined by BCA,and IP experiments were performed.Western results were obtained.The results indicate that Ubc9 interacts with the viral protein PCNA.