Bioinformatics Analysis Of HCMV UL124 Gene And Preparation And Detection Of Polyclonal Antibody

Human cytomegalovirus(HCMV)is a widespread pathogen that infects majority of people all over the world.Like other herpes viruses,HCMV can cause productive and latent infections.For normal hosts,HCMV latent infections are usually asymptomatic,but for those immunodeficient hosts,it can cause diverse diseases or even death.The HCMV latency has been studied for decades,but mechanisms for its latent infection is still poorly understood.The HCMV UL124 gene was found in cells that were latently infected by the virus,which is one of the antisense latent transcripts,presumably playing an important role in establishment and maintenance of virus latency.In order to further understand and study the UL124 gene,this study carried out bioinformatics analysis of UL124 and preparation and detection of polyclonal antibody for UL124,This work will provide the foundation for the futuer subsequent research.The detailed studies are shown below:1.By using bioinformatics related softwares and websites,we predicted the basic properties,structure and function of UL124 gene encoding protein;We analyzed the immunological characteristics and gene ontology of UL124 gene and studyed the homology and polymorphism of UL124 gene.The results showed that the UL124 protein has a molecular weight of about 15kDa and is a hydrophobic protein.It contains a N-terminus signal peptide,a transmembrane domain that located around 80-100 amino acid region,a helical structure,and a non-transmembrane domain contained multiple glycosylation sites and phosphoylation sites.Immunological characterization analysis screened two antigenic epitope peptides,which can be used for subsequent antibody preparation and other research.The gene ontology prediction illustrated that the protein was associated with membrane structure and functions primarily related to protein-protein interactions.The homology and polymorphism analysis of UL124 gene showed that the gene was highly conserved among different HCMV strains,but was not highly conserved in CMV and?herpesvirus with some similarity in structure.2.The full length of the UL124 gene was successfully cloned from the HCMV TB40/E library,and expressed as a his-tagged recombinant protein,which was exist mainly in form of inclusion body.The expression conditions were optimized and we determined that the optimal expression temperature was 30?,the optimal IPTG concentration was 0.8mM,and the optimal induction time was 6h.Using the optimized conditions,we obtained enough recombinant protein.The recombinant protein was purified by nickel column affinity chromatography and then desalted by dialysis and concentrated by ultrafiltration to immunize New Zealand white rabbits.Finally we collected antiserum to check the titer by ELISA.The titer reached 10~5.Then the whole blood of the rabbit was collected and purified to produce a higher purity polyclonal antibody.Using the prepared polyclonal antibody,our experiment showed that the UL124 gene had the overexpression in 293T cells,and had the transcription and expression in the HCMV latency and the lysis.We also determined that UL124 was localized around the nucleus.