Studies On Some Characteristics Of Duck Enteritis Virus UL18Gene And Applications Of The Recombinant UL18Protein

1. Analysis of duck enteritis virus(DEV) UL18gene for its sequence features and codon bias. The DEV UL18gene sequence (GeneBnak accession EU195093) and its protein sequence were analyzed with bioinformatics software. The results showed that DEV UL18gene was969bp,its encoded protein was a polypeptide which comprised of322amino acids residue, the relative molecular weight was35.250KD, the theoretical value of its isoelectric point was8.37,which had no N-terminal signal peptide, but contained15potential phosphorylation sites,2glycosylation sites,and15potential B-cell epitopes. The phylogenetic tree analysis showed that DEV UL18belonged to one of the Alphaherpesvirinae, which was evolutionarily closest to the MeHV-1.It is proper to choose eukaryotic expression system to express this gene based on the codon analysis.So it might be necessary to screen different host bacteria and optimize the condition if using a prokaryotic expression system to expressing this protein.2. Prokaryotic expression and antibody preparation of DEV UL18gene and its application. The primers were designed based on the DEV UL18gene sequence using software Primer Premier6.0. The UL18gene fragment was amplified from DEV genomic DNA by PCR and was cloned into pMD18-T vector. The recombinant construct, named pMD18-T-UL18, was verified by PCR and double digestion (Hind Ⅲ and Xho Ⅰ) and DNA sequencing. Then the UL18gene fragment was subcloned into the prokaryotic expression plasmid, pET32a(+).The recombinant plasmid pET32-UL18was transformed into the host bacteria BL2](DE3), the optimal concentration was0.6mmol/L IPTG as inductor, the optimal time was4h at37℃, and the recombinant expressed protein is about55KD, which was mostly existed in inclusion body. The recombinant protein was purified, rabbits were immunized four times after immunization, then collected the hyperimmune serum of the rabbit anti pET32a/DEV-UL18. Based on the purified recombinant protein pET32a/DEV-UL18, DEV-UL18-ELISA was developed for detecting the DEV serum antibodies in an indirect ELISA. Indirect immunoperoxidase assay (IPA) and indirect immunofluorescence assay (IFA) with the rabbit IgG raised against UL18recombinant protein was used to study the distribution in the tissues of infected ducks.3. Transcription and translation characteristics of DEV UL18gene in infected host cells. UL18mRNA and protein were detected through qRT-PCR and western blotting respectively. The result showed that the transcription of DEV UL18were started at2h post infection (PI),and the detection of its expression products were at12h PI,the transcripts and expression products were surging at24h PI,afterwards came to a peak at36h and48h PI,then both of them were reduced.The expression products was nearly35KD. It can be inferred that this gene was in the last period of the virus by analyzed synthetically the characteristics of DEV UL18’s transcripts and expression.