Indirect Enzyme-linked Immunosorbent Assay For Simultaneous Detection Of Antibodies Against Duck Hepatitis A Type 1 And 3 Viruses

Duck hepatitis A(DHAV) is an acute, highly contagious and fatal disease in young ducklings, which mainly infects duckings under 3 weeks. The increasing morbidity and mortality in ducklings result in severe loss.DHAV has been a severe disease threaten to duck industry.In recent years, while the duck industry is developing in the direction of industrial,intensive and large-scale,reports of serotypes unlike DHAV-1 increase gradually. DHAV-3 has been widely popular in many areas of the country.Failure of the traditional active or passive immunization of duck hepatitis A occur frequently, which put forward a new challenge to the prevention and control of duck hepatitis A virus.VP1 and VP3 genes are the primary antigen genes of DHAV. This experiment ma inly studied the expression of the VP3 gene of DHAV-1 and the VP1 gene of DHAV-3 in E.coil.We proposed to develop an indirect enzyme-linked immunosobent assay(ELIS A) with bacterially expressed recombinant viral protein as antigen that can simultaneou sly detect antibodies against Duck hepatitis A type 1(DHAV-1) and type 3(DHAV-3)viruses.We cloned the VP3(DHAV-1) and the VP1(DHAV-3) into p ET-32 a expressio n vector in the same open reading frame to obtain a prokaryotic expression vector, desig nated p ET-1VP3- 3VP1. The recombinant antigen produced in Escherichia coil expresse d an in-frame fusion protein between viral protein 3(VP3) of DHAV-1 and viral prot ein 1(VP1) of DHAV-3,which was used to develop an indirect ELISA assay. This re search mainly includes the following three contents:1. The construction of recombinant expression vector of pET-1VP3-3VP1Two pairs of primers were designed and synthesized according to duck hepatitis A virus genome sequences in Gen Bank. We amplified the full-length VP3 gene of DHAV-1 and the full-length VP1 gene of DHAV-3 through reverse transcription-polymerase chain reaction (RT-PCR) and then cloned them into p MD18-T, respectively. Following sequencing confirmation, we cloned enzyme-digested products the VP3(DHAV-1) and the VP1(DHAV-1) into p ET-32 a expression vector to obtain a prokaryotic expression vector,designated p ET-1VP3- 3VP1.Screenings were done to ensure that the recombinant expression vector with authentic sequence and orientations.2. Prokaryotic expression of p ET-1VP3-3VP1The p ET-1VP3-3VP1 recombinant expression vector was transformed into BL21(DE3).DHAV-1VP3- 3VP1 fusion protein expressed steadily and in a high yield following induction by Isopropyl-beta-D-1-thiogalactopyranoside(IPTG).SDS-PAGE analysis of extracts of p ET-1VP3-3VP1 transformed E.coil revealed that the recombinant protein with a mass of74.1 KDa approximatly, which was consistent with expected weight.Western blot showed that the expressed protein was very antigenic and reactive to both DHAV-1 and DHAV-3 positive sera, showing good immunogenicity.3. Establishment of an indirect enzyme-linked immunosobent assay(ELISA)for simultaneous detection of antibodies against duck hepatitis A type 1 and 3 virusesUsing the purified DHAV-1VP3-3VP1 recombinant protein as envelope antigen, and DHAV-1 or DHAV-3 positive sera as primary antibodies, as well as HRP labeled Goat Anti-Duck Ig G as second antibody,an indirect enzyme-linked immunosobent assay for simultaneous detection of antibodies against duck hepatitis A type 1 and 3 viruses was established.The optimal working concentration for coating antigen was 1.0 μg per well and the working concentration of serum samples was 1:200 dilution and the cut-off value that distinguished the positive from negative serum samples was OD650≥0.38. The correlation coefficient for positive sera of DHAV-1 and DHAV-3 was higher than 0.9, which indicated that the established standard line equation to count the ET value of single dilution based on serum samples possessed a preferable reference. The correlation coefficient between ELISA and virus neutralization test(VN) was 96.3% and 96.77% respectively for detecting DHAV-1 or DHAV-3 antibodies present in serum samples. This result was also supported by further correlation analysis involving the detection of viral antibodies in serum samples from ducklings in poultry farms as well as from experimentally infected animals.This ELISA method was highly specific and sensitive in detection of antibodies to both DHAV-1 and DHAV-3 viruses. The experimental results show that the ELISA method based on the prokaryotic expression of VP3(DHAV-1) and VP1 proteins(DHAV-3) can be used effectively for the clinical detection antibodies against DHAV-1 and DHAV-3.