Transcriptional Studies Of Duck Plague Virus UL47 Gene And Prokaryotic Expression, Antibody Preparation Of Major Antigen Determinant Sequence

This article concentrates on the UL47 gene (Genebank No. EU195109) of Duck Plague Virus. The bioinformatics analysis of DPV UL47 gene was studied first. Then, we designed two pairs of primers for the full-length UL47 gene and UL47 gene major antigen determinant sequence, amplified them by PCR and did the prokaryotic expression, respectively. We expressed protein of UL47 gene major antigen determinant sequence successfully and utilized the protein to prepare polyclonal antibodies.The biological characteristics of DPV UL47 gene were also carried out later. The results are as follows:The UL47 gene of DPV was composed of 2367 base pair, encoding for a polypeptide of 788 amimo acid, with a molecular mass 87.95kDa. There were 106 acidic amino acids (13.5%),121 basic amino acids (15.3%),242 hydrophobic amino acids (30.7%) and 227 hydrophilic amino acids (28.8%) in the polypeptide. The predicted isoelectric point was 6.23. The polypeptide had 73 potential phosphorylation sites and three potential N-linked glycosylation sites. In DPV UL47 protein, the main hydrophobic region were between aa 369-370,393-406,490-493,510-516,526-528,622-630 and 641-648. In addition, UL47 protein had no transmembrane domain and signal peptide. Subcellelar localization preduction showed that the UL47 protein mainly concentrated in nuclear (69.6%), the rest were existed in plasma membrane (13%), cytoplasm, mitochondrial and vesicles of secretory system. The secondary structure prediction of DPV UL47 protein indicates the DPV UL47 protein consists of 40.36% alpha helix,11.93% Beta turn and 11.93% random coil.The EMBOSS CHIPS and CUSP programs were used to deduce the CAI, ENC value and GC3S content of DPV UL47 gene, and the results were 0.71,52.40 and 51.58%, respectively. A high level of codon usage bias existed for encoding the selection of Leu, Arg, Gly, Val, Ser, His, Ala, Pro. And an extremely low level of codon usage bias existed for encoding the selection of Leu, Ser, Ala and Pro. Rare codons analysis showed that there were 72 rare codons in DPV UL47 gene. Twenty-one AGA codons, thirteen AGG codons and six CGA codons, which are the rare Arg codons. Twelve CTA codons are the rare Leu codons. Sixteen ATA codons are the rare Ile codons. Four CCC codons are the rare Pro codons. Multiple sequence alignment of the amino acid sequences and phylogenetic tree analysis demonstrated that the DPV UL47 and some fowl herpesviruses such as GaHV-2, GaHV-3, MDV-2 and MeHV-1 were clustered within a monophyletic clade and as a result it has a close evolutionary relationship with alphaherpesviruses.We used PCR primers to amplify the UL47 gene and its major antigen determinant sequence, respectively.Then the products was directionally inserted into pMD18-T and pET-32a(+) vector to construct prokaryotic expression system. The recombinant plasmids of T-cloned and sub-cloned were identified by PCR, restriction analysis and sequencing. Then the sub-cloned plasmid pET-32a (+)/UL47 was transformed into BL21 and expressed by IPTG introduction. SDS-PAGE showed that a distinct band of approximately 55kDa molecular weight, corresponding to the expected size, but we didn't observe a distinct band of approximately 108kDa molecular weight appearing in the corresponding position. These results may confirm that the full-length UL47 gene couldn't be expressed.Purified protein was used to immunize rabbit for the UL47 anti-serum preparation, and the antibody titer was up to 1:16 by agar diffusion reaction. Western blot analysis using the resultant sera showed that the recombinant protein was recognized by the polyclonal antibody. Thus, the polyclonal antibody prepared here may serve a good tool for study of the functional involvement of UL47 in the DPV life cycle. For dot blot analysis, one digoxigenin-labeled DNA probe of UL47 gene was designed. To test species specificity, the recombinant plasmid pMD18-T/UL47, DPV, GPV, PRV, DHV, ILTV, MDPV and MDV were extracted and analyzed by the dot blot assay. Non-infected DEF served as the blank control. The results showed that the DPV DNA and pMD18-T/UL47 gave a very distinct color signal, and others showed no signals. It implied that the UL47 gene existed only in DPV.The phase analysis of DPV UL47 gene transcription was detected by FQ-PCR method. The results showed that the transcripts of UL47 gene slowly rised up at the first 8 hours post-infection (hpi), then increased rapidly with the extension of the infection time and reached a peak at 36 hpi, lowed down slowly but still detected the transcripts of UL47 gene until 72 hours after infection.