Duck Hepatitis Virus Type 1 VP1 Truncated Protein Expression And Application

Duck Viral Hepatitis(DVH)is mainly caused by Duck Hepatitis A Virus 1(DHAV-1),which is an infectious disease with high infectiousness and high lethal rate.Until now the clinical detections of DHAV-1 are include neutralization test?indirect hemagglutination test and enzyme linked immunosorbent assay etc.This research constructed five recombinant protein of DHAV-1 VP1 gene superior antigen region,and choose VP1-a(1-60aa)as envelope antigen for prepared a polyclonal antibody of rabbit anti DHAV-1,on the other hand the VP1-a protein establish an indirect ELISA method.Providing a method for the rapid diagnosis and detection of DHAV-1 and DHAV-1 serum in clinical practice.1.VP1 protein truncated segment prokaryotic expressionBy bioinformatics software DNAStar and online protein analysis website analyzes antigenicity of DHAV-1 VP1.Combine those analysis results select five superior antigen region to truncate VP1 protein.The truncated protein sequences cloning to pET-32a(+)expression vector,choose Rosetta(DE3)as expression bacterium,the express condition temperature is 37 ? and the concentration of IPTG is 0.4 mmol/L inducing expression for 6 h,get consistent with the expected size of the recombinant protein VP1-a,the VP1-b,the VP1-c,VP1-d and VP1-e.Western blot analysis showed that,with the exception of truncated recombinant protein VP1-d cannot react with duck DHAV-1 resistance positive serum,the remaining four short length of recombinant protein could react with duck DHAV-1resistance positive serum and the reaction of the VP1-a most strongly.This study built four good response of the original short length of recombinant protein and antibody detection for DHAV-1 provides the basic material.2.Preparation and application of rabbit anti-DHAV-1 VP1-a hyperimmune serumSelection of rubber cutting purified recombinant protein VP1-a and immune rabbit,obtain the VP1-a polyclonal antibody,use indirect ELISA method to detect the antibody titer for 1:512000.The preparation of rabbit anti VP1-a hyperimmune serum for indirect immunofluorescence,and immunohistochemical testing in vitro DHAV-1 DEF infected cells and the distribution of virus infected duck liver tissues.According to the results in vitro DHAV-1 virus in the infected DEF cells mainly distributed in the cytoplasm,a small amount of distribution in the nucleus;and the duck liver tissues virus mainly distributed in the cytoplasm of Liver cells.The experiment shows that the rabbit anti VP1-a hyperimmune serum,can well identify DHAV-1 indicates that the constructed recombinant protein VP1-a good immunogenicity and response to the original,the preparation of VP1-a antibody detection can be used for DHAV-1.3.Establish an indirect ELISA method based on VP1-a proteinWith VP1-a protein as envelope antigen to establish an indirect ELISA detection antibody DHAV-1.Package is best antigen concentration is 2.875 ng/?L,best for serum dilution degrees for 1:160,the optimal dilution degree of resistance to 1:400;The best sealing fluid is 5% BSA,Positive critical value is 0.461,negative critical value is 0.394,between them were suspicious.Plate within the average coefficient of variation is 2.43%,plate between the average coefficient of variation is 3.15% with duck pestilence.This research doesn't with the duck plague virus,duck origin influenza virus,duck Tembusu virus etc immune serum cross reaction.There are two methods of indirect ELISA that VP1-a and DHAV-1 as envelope antigen to detected clinical specimens,the result shows that those two methods coincidence rate is 84%.Therefore the application of recombinant protein VP1-a indirect ELISA method for building strong specificity and good repeatability can be used in the DHAV-1 detection of clinical serum antibody.