The Time Courses Of Transcriptional And Expressional Analysis Of DPV UL46 Gene And The Study Of Prokaryotic Expression, Preparation Of Polyclonal Antibody, And The Double Sandwich Antibody ELISA To Detect DPV Based On Its Main Antigen Region

Abstract:This article has carried out series researches on the identified UL46 gene (assigned accession No. EU195108) of the duck plague virus (DPV) in our laboratory by the means of cloning, molecular characteristics analysis by the analysis of bioinformatics analysis, prokaryotic expression, preparation of polyclonal antibody, time courses of transcriptional and expressional analysis of UL46 gene, the analysis of attribute of UL46 protein and the establishment of the double antibody sandwich ELISA basing on the anti-DPV UL46M polyclonal antibody to detect DPV antigen. Results were reported as follows:1. Molecular characteristics of the DPV UL46 geneThe DPV UL46 gene included 2220 nucleotides encoding 739 amino acid residues. Its encoding protein (VP11/12), which had a functional domains among 20-500aa, and a large of consistent sequences among 17-470aa, especially highly conserved at the domains of 356-413aa, belonged to highly conserved the Herpes_UL46 proteins. Gln372,Asn376,Tyr382 and Trp385 were completely conserved at the single amino acid sites.It shared low homology with 24 strains other herpesvirus UL46 gene and the phylogenetic analysis showed that the DPV had a closest evolutionary relationship with poultry alphaherpesvirus such as GaHV-3 and MDV-2. The DPV UL46 had 10 transcription promoter sequences and 35 epitopes situating in the N-terminal regions of Ser423-Thr449, Va1451-Ser500, Phe502-Tyr526, Asn540-Thr596, Glu614-Ala651, Ser655-Ser671 and Gly681-Cys700. The UL46 protein located around the cell nucleus domain in cytoplasmic with better hydrophilicity, flexible regions and higher antigen indexs, and no transmembrance domain and signal peptide. It had 72 phosphorylation sites,2 N-Glycosylation potential sites and many flexible regions,α-helices andβ-strands, but only a few of random coils. Codon bias analysis showed that the UL46 gene was biased in some codons, especially those codons encoding amino acids Trp, Met, His, Lys and Glu. The tertiary structure had not been gained by the way of homology modelling of molecular confirmation. These results provided elementary data to research the biological function of this gene. We definited the gene sequence encoding the main antigen domains locating 699-2220bp of the UL46 gene, which combinated the antigenicity, hydrophilia and the need of the following work, and designated it DPV UL46M.2. Cloning, prokaryotic expression, preparation of polyclonal antibody of DPV UL46 and UL46M geneThe DPV UL46 gene sequence (GenBank accession No. EU195108) was analyzed and two pairs of primers were desigened. The amplified fragment of DPV UL46 (2558bp) and UL46M gene (1522bp) were inserted into pMD18-T vector, then the pMD18-T/UL46, pMD18-T/UL46M and prokaryotic vector pET-32a(+) were digested with BamH I and Xho I, and the UL46 and UL46M gene were subcloned into pET-32a(+). After identification of the recombinant plasmids pET-32a(+)/UL46 and pET-32a(+)/UL46M by PCR and restriction digestion of BamH I and Xho I, the fragments were transformed into E.coli Rosetta competent cell. By IPTG-inducible expression, a polypeptide with a size corresponding approximately to that expected for the recombinant protein (about 79kD) was expressed with the concentration of the IPTG of 0.2 mmol/L and the induced for 4h at 37℃in a form of inclusion body. Unfortunately, the complete UL46 gene was fail to expression. The purified expression protein was used to immunize the rabbit, and the antibody titer was up to 1:8 by the agar diffusion reaction. Western blot results showed that the anti-DPV UL46M serum can react specially with the recombinant protein. The result of the Dot-ELISA established using the polyclonal antibody to apply on detecting DPV disclosed that the polyclonal antibody was specificity for DPV. The results provided the scientific materials for the study on function of UL46 gene and the diagnostic of DPV.3. Time courses of transcriptional and expressional analysis of DPV UL46 geneWe desigened two pairs of primers U3/U4 (121bp) and U1/U2 (178bp) according to the DPV UL46 and duck P-actin gene sequences, respectively. mRNA from DEF cells at postinfection times including 2h,4h,6h,8h,12h,18h,24h,30h,36h,42h,48h, 54h,60h,66h,72h and 80h was extracted and performed reverse transcription into cDNA. The transcriptional analysis result of UL46 gene products in infected duck embryo fibroblasts (DEF) detected by FQ-PCR showed the transcriptional products of DPV UL46 gene began to increase at 12h, and significant increased at 24h, appearing a peak at 54h, and then lowed down slowly, which owes the typical characterization of herpervirus late gene. A time course of expression of DPV infected DEF was analyzed by western blot using the purified rabbit anti-DPV UL46M IgG by freezed thawing three times to extract the virus protein. A specific immunoreactive band migrating was observed at the expected position for protein DPV UL46 protein (about 81.8kD). The UL46 protein could be detected at 12h, then it gradually increased and maximal amount at 60h. The results proved that the transcription and expression of the DPV UL46 gene were accord with the genes life circle regulation from transcription to translation, moreover it satisfied the model that transcription is the former one and expression is later after it. We could summarize that the DPV UL46 gene is a late gene from our research.4. The attribute analysis of the DPV UL46 proteinThe DEF was harvested after infecting 25h and centrifugalized with low speed, then washed by PBS. The NP-40 lysate was used to schizolysis cells and performed the Western blot test using the supernatant and the sediment after centrifugalization. The results disclosed that the sediment which could not dissolve in the NP-40 lysate appeared the specific immunoreactive band with the anti-DPV UL46M IgG of the expect molecular weight, and it confirmed that the DPV VP11/12 was the structural protein of DPV, as well as one of the ingredient of the tegument.5. The establishment and application of the double antibody sandwich ELISABy using purified rabbit anti-DPV UL46M IgG as the primary antibody and purified mouse anti-DPV IgG as the second antibody, a double antibody sandwich ELISA to dectect DPV was developed and optimized. The results showed that the optimal concentration of the rabbit anti-DPV UL46M IgG was dilution of 1:320, mouse anti-DPV IgG was dilution of 1:20 and HRP-goat anti mouse IgG dilution of 1:5000. The minimum DPV content detection is 52ng purified DPV. The established double antibody sandwich ELISA was used to detected doubtful samples and compared with the PCR. The results showed that the assay was specific and of high sensibility, which could used to detect DPV quickly, largely and specificly.