Prokaryotic Expression And Indirect ELISA Method Development Of Duck Hepatitis A Virus Type 1 VP2 And VP4 Protein

Duck hepatitis A virus (DHAV) can cause duckling breaking out duck virus hepatitis, which has acute onset, short duration, spread rapidly, and high mortality, major clinical manifestations of loss of appetite, neurological symptoms sudden death and liver enlargement bleeding, also known as Waibo disease. Currently it is existing in almost all the countries and regions around the world,bringing duck industry great economic losses. DHAV can be divided into DHAV-1, DHAV-2 and DHAV-3 three genotypes.In this study,we focused on DHAV-1 VP2, VP4 gene which were carried out Molecular Biology character analysis, Tcloned and subcloned prokaryotic expression, protein purification, the establishment of indirect ELISA detection methods based on two prokaryotic expression proteins, the main results are as follows:1. Bioinformatics analysis and prokaryotic expression of DHAV-1 VP2 and VP4 genesOn the basis of our laboratory submitted sequence duck hepatitis A virus type 1 strain H (accession No. JQ301467.1) in GenBank sequence and professional protein cleavage site prediction software,NetPicoRna V1.0,we got the gene sequences of DHAV-1 VP2 and VP4,which are 543bp and 225bp, respectively. Using bioinformatic analysis tools on the genes VP2, VP4 and encoding proteins VP2, VP4 to do the analysis,separately.The predicted results show that VP2, VP4 encoding 181 and 75 amino acids each,with the molecular weight of 20.6 kDa and7.7 KDa, respectively. Andcontaining 4 and 1N- myristoylation sites each,3 and 1 N-glycosylation sites each, both containing 3 casein kinase II phosphorylation site,And having many antigenic sites both.According to the bioinformatic prediction.then prokaryotic expression plasmid pProEx-HTb-VP2^ pET32c(+)-VP4 successfully constructed after RT-PCR amplification,T cloning subcloning and sequencing.The plasmid pProEx-HTb-VP2 was transformed into the express host bacterium BL21 and induced expression at 37℃,0.2 mmol/L IPTG and 8h conditions.The plasmid pET32c(+)-VP4 was transformed into the express host bacterium BL21and induced expression at 37℃,0.6mmol/L IPTG and 4h conditions. The protein VP2 was mainly present in the inclusion bodies. It was purified by the means of cutting gel with high purity.And the protein VP4 was mainly present in soluble bodies. It was purified by the means of hanging Ni2+NTA column with high purity. Then, in westernblotting experiments, two recombinant proteins were reacting with an anti-rabbit serum of anti-DHAV-1,which all have had an good reactogenicity confirming that VP2 and VP4 genes of duck hepatitis A virus have had expressing successfully in prokaryotic expression system.2.To establish the methods of ELISA based on the duck hepatitis A virus-1 proteins VP2 and VP4The indirect-ELISA methods were developed with purified recombinant VP2 and VP4 protein as an antigen coating ELISA plate respectively. And the optimal reaction conditions were determined. As following:The best detection condition of VP2 antibody level was coating with 2.713μg/mL (100μl) of VP2 protein overnight at 4 degree,and blocking 1 h at 37 degree with 5% skimmed milk powder, then incubate 2 h at 37 degree with 1:40 diluent of serum, following with 1:2000 diluent of HRP-labeled goat-anti-rabit IgG at 37 degree celsius for 2 h and using TMB coloration for 10 min avoid light. The value of OD450-OD630 were read to be the specified absorbance. The positive threshold value was determined as 0.359 with detecting 60 negative serum samples. Meanwhile, the conditions based on VP4 were coating with 3.375 μg/ml(100μl) at 37 degree celsius for 3 h and then at 4 centigrade degree overnight, and blocked with 5% skimmed milk powder at 37 degree celsius for 1 h, incubating with 1:80 diluent serum for 2 h and then 1:1600 dilute HRP-labeled rabbit-anti-duck IgG for 0.5 h, coloration for 30 min at 37centigrade degree. And the positive threoshold was determined as 0.203 with detecting 60 negative serum samples. The results of repeatability showed that the coefficient of variation is both less than 10%,indicating the two methods have good repeatability. In both two methods, there was no cross-reactivity with positive-serums of five kinds duck common dieases, indicating that they were highly specific.The both ELISA detection methods all have good sensitivities.Contrasting with having reported established ELISA method based on the whole DHAV-1 virus in detecting 48 serum samples The coincidence rates of two methods were 91.1% and 70.5%,respectively.