Prokaryotic Expression, Antibody Preparation Of The UL45 Des-transmembrance Domain From Duck Enteritis Virus And The Study Of Transcription Phase, Protein Characteristics

The bioinformatics feature of the the new-identified UL45 gene (Genbank accession no. EU195107) from duck enteritis virus (DEV) was studied in this essay. We expressed the des-transmembrane domain of UL45 gene(295-675bp of UL45, named UL45Δgene) successfully and produced the polyclonal antibody. Some biological characteristics of UL45 were studied using the prepared antibodies. The results were summarized as follows:1. Molecular characterization of UL45 gene from Duck enteritis virus The DEV CHv UL45 gene was composed of 675 basic groups, encoding a polypeptide of 224 amino acid residues, and its isoelectric point was 6.51. The UL45 protein had no signal peptide but a transmembrane helix. The sequence contained 13 possible sites for phosphorylation, 8 on serine,4 on threonine, and 1 on tyrosine residues, when the threshold was defined as 0.5. The possible B-cell surface antigens were located in 13-17,20-28,40-47,51-56, 157-161 and 202-206 of amino acids.The possible T-cell surface antigens were located in 68-73,130-133 and 157-160 of amino acids. Sequence comparison of UL45 among DEV CHv and other reference herpesvirus strains showed that UL45 gene was more homologous to Avian herpetoviruses. Codon usage analysis showed that the tropism of UL45 gene was low and with a low heterologous expression level. The E.coli expression system was the most suitable system for UL45 gene.2. Prokaryotic expression and polyclonal antibody preparation of des-transmembrane domain of UL45 gene Prokaryotic expressional plasmids of UL45 and des-transmembrane domain of UL45 (pET-32-UL45 and pET-32-UL45Δ) were constructed successfully and transformed to the E.coli BL21(DE3)PlysS. The results of induction by IPTG showed that the UL45 gene couldn't express while des-transmembrane domain of UL45 was highly expressed. The soluble polypeptide was about 33kDa with an expected size. The optimized condition of expression was inducing 4h at 30℃after adding 0.2 mmol/L IPTG. Purified protein was used to immunize rabbits for the UL45Δanti-serum preparation. Agar diffusion reaction showed that the antibody titer was up to 1:32. Western blot analysis showed that the recombinant protein was recognized by the polyclonal antibody and the UL45 gene was a member of DEV genome.3. Transcription characteristics analysis of UL45 from DEV Real-time quantitative PCR was performed to study the transcription kinetics of the DEV UL45 gene during the viral infection. The results showed that the the expression of UL45 mRNA was at a low level from 0 to 18h post-infection (pi), then accumulated quickly at 24h pi and peaked at 42 h pi. The UL45 mRNA can be detected till 72 h pi. The results indicated that DEV UL45 gene might be a late viral gene.4. Characteristics analysis of UL45 protein The purified DEV was got from ultra-high speed centrifugation. Using the detergent NP-40 to extract the virus and got the supernatant (envelop protein and very few tegument protein) and sediment (majority tegument protein and capsid protein).Western blot analysis showed that the UL45 protein resided in the purified virus and the supernatant and so we inferred that UL45 protein was a envelop protein of DEV.