Development Of Indirect ELISA Method Based On 3A Protein Of Duck Hepatitis A Virus And Subcellular Localization Of 3A Protein

Duck viral hepatitis(DVH),which causes A highly lethal virus infectious diseases mainly for goose of cheeper Duck.by Duck hepatitis virus(DHAV).DHAV-1 virus is the most widely distributed and most harmful gentype of DHAV.In this study,its 3 A Was expressed in pmkaryotic system,and purified to act as the immunogen to prepare rat antiserums.The subcellular localization of 3A in recombinant plasmid expressing and virus infection was determined by the indirect immunofluorescence test.Moreover,a 3A based indirect ELISA method for DHAV antibodies detection was developed.1.Expression,purification and identification of 3A of DHAV-1 andpreparationofanti-3A3A truncated gene fragment which is removing the 3A gene across the membrane area was obtained form PCR.Purpose fragment cloning to pET-32 a(+),to construct recombinant plasmid pET-32-3A-198.Turn recombinant plasmid into the express bacterium BL21(DE3)induction of express is about 31 kD after 3A truncated protein,named 3A-198.Its expression quantity reached top when induced by 0.1mmol/L IPTG for 6h at 37℃,and it was in the form of inclusion bodies anyway.Take the Ni affinity chromatography column method to purify 3A-198,Western blot analysis indicated the recombinant protein was able to react with anti 3A-198 polyclonal antibody and showed good reactionogenicity.Rats were immuned by purified 3A-198 protein emulsion and they all reached 1:8 by agar gel immunodiffusion after the fourth immunity.23 A protein subcellular localizationatDEFConstructed based on 3 a full-length recombinant plasmid pCMV-myc with myc label-3A,Western blot results show that the eukaryotic expression plasmid expression product in duck embryo fibroblasts can be duck serum anti DHAV-1 identification,and strip size and 3A-myc recombinant protein size,recombinant plasmid to express success 3A protein.In virus infection and transfection recombinant plasmid pCMV-myc-3A in duck embryo fibroblast,in order to obtain the serum as a resistance,in the treatment of 48h after the duck embryo fibroblast to fly on a chip,it respectively by using the method of indirect immunofluorescence with the golgi apparatus and endoplasmic reticulum in cells.The results showed that 3A protein mainly locate in the endoplasmic reticulum,and golgi apparatus without apparent positioning signal3.Development of a indirect ELISA method based on 3A protein of DHAV-1The detemfined optimal condition of 3A protein based indirect ELISA method was that coating antigen at 6.185μg/ml at 4℃ overnight,using 5%BSA as blocking buffer and incubating at 37℃ for 90min,then diluting the serum samples to 1:20 and incubating at 37℃ for 30min,diluting the HRP-labeled rabbit anti-duck IgG to 1:2000 and incubating at 37℃ for 30min.React with TMB at room temperature for 10min.The cut off value determined was 0.274.The method proved good sensitivity,repeatability and specific and was capable of detecting antiserums to DHAV-3.The coincidence rate between the novel method and the DHAV-1 based indirect ELISA was 92.7%.