Development Of Indirect ELISA Method Based On VP3 Protein Of Duck Hepatitis A Virus And Identification Of B-cell Epitope For VP3 Protein

Duck hepatitis A virus (DHAV) is a single-stranded, non-enveloped, positive-sense RNA virus. It encodes structural proteins and nonstructural proteins, of which the structural proteins act essential roles in assembly of the viral capsid and its function of pathogenicity. VP3 is the main conservative structural protein in the outer capsid and acting as the main determinant of DHAV’s antigenicity. Numerous antigenic epitopes exist on VP3 protein and can induce immune response. DHAV-1 is the most widely spread and harmful gentype of DHAV, in this study, its VP3 was expressed in prokaryotic system, and purified to act as the immunogen to prepare rabbit antiserums. Chicken embryo neutralization test was conducted to researching neutralizing activities of VP3 antiserums; the probable B-cell epitopes of VP3 was identified; moreover, a VP3 based indirect ELISA method for DHAV antibodies detection was developed.1. Expression, purification and identification of VP3 of DHAV-1.Expected sized VP3 fragment was obtained from RT-PCR, it was successively cloned to pMD19-T (simple) to construct pMD19-VP3 plasmid and pGEX-4T-1 to construct pGEX-VP3. pGEX-VP3 was expressed with a molecular weight about 50kD after transformed to BL21 (DE3). Its expression quantity reached top when induced by 0.2mmol/L IPTG for 8h at 37℃, and it was in the form of inclusion bodies anyway. The purification by using gel extraction proved high pure. Western blot analysis indicated the recombinant protein was able to react with DHAV-1 and showed good reactionogenicity.2. Neutralizing activity analysis of VP3 antiserums and B-cell epitopes identification of VP3 from DHAV-1.Rabbits were immuned by purified VP3 protein emulsion and they all reached 1:16 by agar gel immunodiffusion after the fifth immunity. Western blot analysis proved that the prepared rabbit antiserums react specifically with VP3 protein. Chicken embryo neutralization test proved the neutralization titer of VP3 antiserums was 2-5.29. By using bioinformatics softwares to analyse the flexibility, surface accessibility, hydrophilicity and antigenicity of VP3 sequence comprehensively, four probable B-cell epitopes were obtained and submitted to artificial synthesis. The prepared polyclonal antibodies were served as the primary antibodies, and VP3 protein was served as antigen and determined the optimal dilution of peptides was 1:1280. Peptides were subsequently diluted to optimum for indirect ELISA to identify the B cell epitopes. As was proved by the result that 1GKRKPCRRPIHKPKNPPQEP20,131FNTGRYQMSWYPIADGEQSL150 and 200VNSSAPSNID209 reacted specifically with VP3 polyclonal antibodies and were the B-cell epitopes of VP3 protein. Further evaluation for antibody detection ability of the identified B-cell epitopes by detecting a panel of DHAV-1 clinical duck serum samples revealed the epitopes were capable of detecting DHAV-1 antibodies.3. Development of a indirect ELISA method based on VP3 protein of DHAV-1The determined optimal condition of VP3 protein based indirect ELISA method was that coating antigen at 9.375 ng/μL at 4℃ overnight, using 1% gelatin as blocking buffer and incubating at 37℃ for 90min, then diluting the serum samples to 1:160 and incubating at 37℃ for 90min, diluting the HRP-labeled rabbit anti-duck IgG to 1:2000 and incubating at 37℃ for 45min. The cutoff value determined was 0.332. The method proved good sensitivity, repeatability and specific and was capable of detecting antiserums to both DHAV-1 and DHAV-3 at one time. The coincidence rate between the novel method and the DHAV-1 based indirect ELISA was 96%.