DPV US4 Gene Prokaryotic Expression And The Study Of Its Cellular Localization And Expression Time Course In Virus-infected Cells

Molecular characteristics analysis, cloning and expression of the US4 gene of duck plague virus (DPV) had been carried out in this article. And then polyclonal antibody was prepared. Furthermore, we established indirect ELISA method of detecting DPV antibody using the purified US4M protein as antigen, and identify the cellular localization and time course of DPV US4 gene products in infected host cells through the method of immunofluorescence. The results were as follows:1. Molecular characteristics analysis of the identified DPV US4 gene DPV US4 gene open reading frame (ORF) was 1380 bp in length and its primary translation product was a polypeptide of 459 amino acids long. The amino acid sequence of DPV US4 protein (gG) had higher homology with gG homologous protein of Alphaherpesvirus than others. Analysis of evolutionary relationship by construction of a phylogenetic tree showed that DPV was clustered within a monophyletic clade and independent to others, despite its close evolutionary relationship with GaHV-1 and PsHV-1, indicating that the DPV should be placed into a single cluster within the subfamily Alphaherpesvirinae. Sequence analysis indicated that gG possessed typical characteristics of membrane glycoproteins, containing a N-terminal hydrophobic signal peptide sequence, two transmembrane domains and 9 potential N-glycosylation sites. Further analysis showed that it was a type I transmembrane protein. The analysis of subcellular localization indicated that the protein located at golgi, plasma membrane and endoplasmic reticulum. The result of secondary structure presumption and estimating potential antigenic epitopes indicated that DPV-gG had good immunogenicity.2. Prokaryotic expression, preparation of anti-gG polyclonal sera After US4 gene subcloned into prokaryotic vector pET-32a(+), Escherichia coli strain BL21 (DE3) was transformed with plasmid pET-32a(+)/Us4. After induction overnight with 1.0 mM isopropyl-(3-D-thiogalactopyranoside (IPTG) at 25℃, these cells expressed large quantities of the 6-His-tagged gG fusion protein with an apparent molecular mass of 70 kDa, corresponding to the expected size. The proteins were purified using Ni2+affinity resins under denaturing conditions. Then the rabbit was immunized with the purified protein for raising antibody. Western blotting experiments showed that the fusion protein was an antigen recognized by the DEV-immunized duck antiserum and the gG antiserum had a high level of specificity and we could use this antiserum for further experiments to characterize the gG protein of DPV.3. Establish indirect ELISA method of detecting DPV antibody using the purified US4M protein as antigen Based on the bioinformatics analysis of DPV US4 gene, we expressed efficiently the truncated-DPV US4 gene which contained main antigenic domains (68aa-377aa). Western blot showed this recombination protein had a high level of antigenicity. Then we used the purified US4M protein as antigen and established indirect ELISA method. The results showed that with the coating concentration of US4M antigen of 2.5ug/100μl, favourable sensitivity can be achieved. Experiments also indicated that the US4M-indirect ELISA method had favourable repeatability and specificity, thus could be used to identify the serum of DPV infection.4. Cellular localization and expression time course of DPV gG in virus infected DEF Localization Expression of gG was not affected by ACV, indicating that its expression occurred before viral DNA synthesis and thus indicated that DEV gG is an early gene product. Specific fluorescence was detectable in the cytoplasm of infected cells which were fixed and permeabilized as early as 4 h post-infection (h.p.i) and with the extension of the infection time, more specific fluorescence appeared. Specific fluorescence was detectable at the surface of infected cells which were only fixed at 8 h.p.i.