| In recent years, duck hepatitis A virus type 1(DHAV-1) has already shown new pathogenic characteristics, the main gross lesion was pancreatic hemorrhage and the color turned yellow, unlike the characteristic lesions of classic liver bleeding. The morbidity and mortality of duck hepatitis A virus type 1 with pancreatitis were 42.8% and 77.8%, respectively.Clinical samples were collected from Zhejiang province in 2011, pathogen were isolated and purified. After that virus genome sequencing and analysis, pathogenicity tests were conducted, the pathogen turn out to be duck hepatitis A virus type 1. Duck embryo serum cross neutralization test was conducted.Then analyzed the correlation between pancreatic type DHAV-1 and classical DHAV-1,the result showed that the R value was 0.62.These experiments proved that pancreatitis type DHAV-1 belongs to duck hepatitis A virus type 1 subtype(DHAV-1a).One pair of primer set was designed based on sequences available in GenBank to amplify the VP3 gene of DHAV-1a. VP3 gene was sucessfully amplified by RT-PCR using DHAV-1a MPZJ1206 strain served as template. Then we cloned, sequenced and analyzed the VP3 gene. The sequencing of VP3 gene of DHAV-1a was analyzed and compared with ZJ strain(EF382778.1) and CE3 strain(EU687756.1) of DHAV-1. The identity of nucleotide between ZJ strain and CE3 strain were 94.8% and 94.7%, the amino acids similarity was 99.2% and 97.9%,respectively.Construct the positive recombinant pEASY-VP3 cloning plasmids. Recycling VP3 gene fragment after double enzyme digestion. The recombinant expression plasmid pET-32a-VP3 was constructed by inserting the target gene fragment into prokaryotic expression vector pET-32a(+) and transformed into Escherichia coli BL21(DE3) competent cell. SDS-PAGE analysis showed that the recombinant VP3 protein approximately 47 kDa in molecular weight was highly expressed in E.coli after pET-32a-VP3 induced with 1.0 mmol/L IPTG at 37 ?for 4 h. And we knew that VP3 fusion protein was expressed as inclusion bodies. Purified VP3 protein by using His Trap HP purification column that produced by GE health company. The result of western blot analysis showed that purified VP3 fusion protein can be identified by DHAV-1a positive hyperimmune serum which showing a good immunogenicity.Purified VP3 protein was used as antigen to establish the indirect Enzyme-linked immunosorbent assay(ELISA) for detection of anti-DHAV-1a antibody. The optimal reaction conditions were determined as following: The antigen coating concetration of VP3 fusion protein was 1.5ug/mL(the antigen was diluted at 1:800) and placed at 4 for more ?than 12 h, duck serum sample was diluted at the ratio of 1:160 and incubated at 37?for 90 min, HRP labeled Goat anti-Duck IgG was diluted 4000 times and incubated at 37?for 90 min, the optimum time of colour reaction was 20 min after TMB was attached to the ELISA plate. Specificity test and repeatability test were conducted at optimal reaction condition, and showing excellent specificity and good reproducibility. The positive rate of 84 duck sera tested by an established indirect ELISA method was 32.14%. The indirect ELISA based on VP3 fusion protein has an consistant rate of 87.1% with that bases on purified DHAV-1a virus particles.Six balb/c strain female mice were chosen to immunized with purified VP3 protein of DHAV-1a for cell fusion. Indirect ELISA method was used to screen positive hybridoma cells. After four times of subclone, two positive hybridoma cells, designated 1C9 and 2C9 were obtained. Monoclonal Ig types and subtypes were identified by antibody isotype Kit, the result showed that the antibody belonged to IgG1 subtype and kappa type. Ascites titer of 1C9 and 2C9 were identified by ELISA, the ascites titers were 1:1024000 and 1:512000, respectively. |
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