The Prokaryotic Expression, Polyclonal Antibody Preparation,and Function Study Of DPV UL16Gene

This paper has carried out a series of researches of the identified UL16gene (GeneBank accession no.EU195095) of the duck plague virus (DPV) in our laboratory, containing characteristics prediction and analysis of DPV UL16gene, cloning and prokaryotic expression, preparation of polyclonal antibody, time courses of transcription and expression analysis, subcellular localization of DPV UL16gene in infected DEFs, establishment an indirect ELISA for detection of antibodies against UL16protein of DPV. The results were as follows:1. Characteristics prediction of DPV UL16geneDPV UL16gene contained an open reading frame (ORF), which was composed of1089nucleotides. Multiple sequence alignment of the DPV UL16amino acid sequence with the other16reference alphaherpesviruses of UL16amino acid sequences suggested that the UL16gene was higher similarity with MeHV-1, GaHV-2and GaHV-3. At the same time, a conserved domain of Herpes_UL16superfamily was detected in the deduced362-aa protein and encoded a tegument protein. There were2putative N-linked glycosylation sites,16possible sites for phosphorylation, but no signal peptide and transmembrane region along the amino acid sequence. Moreover, DPV UL16amino acids sequence contained12epitopes in the hydrophilic region of random coil and turn. DPV UL16protein was located mostly in mitochondrial and cytoplasm by prediction. The codon usage bias of DPV UL16gene revealed that UL16gene do not have a strong codon bias and the synonymous codons with A and T at the third codon positon have widely usage. Compared with those of E.coli, Yeast and Human codon bias, suggest that codon usage of DPV UL16is similar with prokaryote and eukaryote, which are suitable for expressing UL16gene.2. Cloning, prokaryotic expression and preparation of anti-UL16protein antiserumA predicted1098bp product containing the entire ORF of DPV UL16was amplified using PCR, then inserted into pMD18-T vector. The constructed pMD18-T-UL16was cut with HindⅢ and XhoⅠ, and the insert was ligated into prokaryotic expression vector pET-32b(+) precut with the same enzymes. The resulting pET-32b(+)-UL16was verified and transformed into E.coli Rossetta(DE3). The E.coli Rossetta(DE3) was successfully expressed in the IPTG induced cells. A60kDa fusion protein UL16was expressed. The fusion protein was purified, dialyzed renaturation, and immunized the rabbits to raise antisera against UL16. The collected antisera titer was up to1:8by agar diffusion assay. The SDS-PAGE analysis showed that the UL16antiserum reacted specifically with the recombinant protein His6-taggedUL16.3. Transcription and expression analysis of UL16gene in DPV infected DEFsThe transcription of UL16gene in DPV infected DEFs was analyzed using qRT-PCR. The results showed UL16mRNA was transcripted at a low level from0-18h post-infection (p.i). Then increased over time and peaked at36h, reduced after48h p.i. To determine the UL16transcription was highly dependent on viral DNA synthesis, infected DEFs were maintained for36h after a1-2h adsorption in the presence of ACV. The result showed that the UL16couldn’ t be detected in the presence of ACV, indicating that the UL16transcription was highly dependent on viral DNA synthesis. Moreover, the expression kinetics of DPV UL16gene was confirmed using Western-blot assay. At early phase of infection, UL16gene expressed at a low level, and rose to peak level at48h p.i. All these suggesting that DPV UL16gene belonged to late gene and might encode a structural protein to take part in virion assembly and maturation.4. Subcellular localization of DPV UL16in infected DEFsThe inntracellular distribution of DPV UL16protein was examined by indirect immunofluorescence assays utilizing rabbit anti-UL16antisera as primary antibody. The results showed that UL16protein appeared in the perinuclear cytoplasm area at18h p.i. As the infection time passed, a strong fluorescence was detected as mass discrete granules in the cytoplasm at later times of infection. As over time the cells dissolved apoptosis, this specific fluorescence gently reduced. These indicated UL16protein may distribute in organelles as Golgi apparatus to take part in virion assembly and maturation.5. Establishment of an indirect ELISA for detection of antibodies against UL16protein of DPVRecombinant DPV UL16protein was used as a coating antigen to estabish an indirect ELSIA for specifically detecting anti-DPV antibodies in serum samples from ducks. In the optiminzed UL16-ELISA, the fusion protein was coated at1.25μg/ml and duck serum samples were diluted at1:160. The endpoint cut-off value of this assay was0.598. The inter-assay and intra-assay coefficients of variation(CV) were both<10%. There was no cross reaction with duck positive serum of DHBV, DHV, RA, E.coli, S.anatum, H5N1and DSHDV. The assay was successfully applied to the examination of the suspected duck seras and showed95.5%(76+10/90) agreement with the SNT. The results showed that the developed UL16-ELISA was rapid, specific, sensitive and repetitive for diagnosis of DPV and epidemiological surveys.