Establishment Of Indirect Immunofluorescence Assay (Ifa)with Epitope Of Vp2of Infectious Pancreatic Necrosis Virus

Infectious pancreatic necrosis virus (IPNV) is a pathogen of infectious pancreatic necrosis (IPN) causes haemorrhage and putrescence of hepatopancreas and other interal organs. This disease is especially destructive in artificial breeding fishing, primarily infect larvae and young fish of salmonid. Highly virulent strains may cause greater than90%mortality in hatchery stocks over a period of two months. The fish of surving after infection carry virus lifelong, become potential pathogen infection. The disease is wide range prevalence and high rates of morbidity and death, is China and other countries’one of the important disease to salmon trout death, bringing significant economic loss in the aquaculeure farming.We amplified a616bp epitope of the VP2gene from the RNA extracted directly from IPNV-ZYX infected EPC cells, called IPNV VP2COE. Comparing nucleotide sequence with a strain of VP2gene published on GenBank (AF342728), the homology was99.8%, mutated A�'T at710. The VP2COE gene was cloned into the prokaryotic expression vector pET30a、pET28a、 pProEx HTb、pGEX-6p-1、pCold TF and expressed in E. coli Rosetta(DE3) to get recombinant protein respectively. The former four expressed fusion proteins existed in the form of inclusion body by SDS-PAGE, which the expressed pProEx HTb-VP2COE accounted for23%of the total cellular proteins are the most. The analysis results of Western-blot showed that the pProEx HTb-VP2COE recombinant strain was correct expressed, a30kDa protein was detected according with the theoretic molecule weight and the recombinant protein could be recognized by6×His monoclonal antibody. So, we choose pProEx HTb-VP2COE recombinant protein to study further. In addition, the expressed pCold TF-VP2COE recombinant protein was soluble by SDS-PAGE. The analysis results of Western-blot showed that the pCold TF-VP2COE recombinant strain was correct expressed, a78kDa protein was detected according with the theoretic molecule weight and the recombinant protein could be recognized by6×His monoclonal antibody.In order to obtain good immunogenicity of recombinant protein, we purified the fusion protein pProEx HTb-VP2COE with ProBondTM resin from the suspension centrifuged (degeneration method) and cutting gel, the purity of the expressed protein was98%and99%. Then, we used glutathione redox system dislysis renaturation the protein which were purified by cutting gel; we used urea gradient dislysis renaturation the protein which were purified with ProBondTM resin from the suspension centrifuged and chose the optimized condition, the best conditon of protein renaturation is:PBS buffer,4℃, pH8.0, urea gradient change is6M,4M,2M,1.5M,1.0M,0.5M,0M, recovery rate was30%. Gel purification of pProEx HTb-VP2COE recombinant protein、 affinity purification of pProEx HTb-VP2COE recombinant protein、urea gradient dislysis renaturation of pProEx HTb-VP2COE recombinant protein、glutathione redox system dislysis renaturation of pProEx HTb-VP2COE recombinant protein all were diluted to0.3mg4nl and were used to immunize6～8weeks BALB/c mice.. The prepared antisera all reacted specifically with IPNV (ATCC VR-1318) antigen by indirect ELISA. The antisera against pCold TF-VP2COE fusion protein、the antisera against affinity purification of pProEx HTb-VP2COE recombinant protein、the antisera against urea gradient dislysis renaturation of pProEx HTb-VP2COE recombinant protein、the antisera against glutathione redox system dislysis renaturation of pProEx HTb-VP2COE recombinant protein and the antisera against gel purification of pProEx HTb-VP2COE recombinant protein was respectively at a dilution of1:12800、1:1600、:6400、1:3200、1:1600. Neutralization test showed the five antiseras had ability of neutralizing IPNV (ATCC VR-1318), the neutralization ability of serum antibody of the antisera against pCold TF-VP2COE fusion protein、the antisera against affinity purification of pProEx HTb-VP2COE recombinant protein, the antisera against urea gradient dislysis renaturation of pProEx HTb-VP2COE recombinant protein、the antisera against glutathione redox system dislysis renaturation of pProEx HTb-VP2COE recombinant protein and the antisera against gel purification of pProEx HTb-VP2COE recombinant protein were1:51.29、1:44.67、1:25.70、1:12.88、1:3.02. The result show the expressed pCold TF-VP2COE fusion protein has good immunogenicity and immune reactivity.An indirect immunof luor escence assay (IFA) test was developed to diagnosis IPNV using the antisera against pCold TF-VP2COE fusion protein as the primary antibody and the f luorescein isothiocy anated (FITC) labeled goat anti-mouse IgG as the second antibody. The optimum conditions of IFA were as follows:the optimum vaccinate concent ration and culture condition of IPNV were that the IPNV was diluted by10"5to vaccinate EPC cells and were cultivated fo24h at temperator (18℃). Cell fixed agent was4%polyoxymethylene. The optimum working concentration of the antisera against pCold TF-VP2COE fusion protein and FITC-labeled goat anti-mouse IgG were1:80and1:200.Through the above condition exploration, establishment of indirect immunofluorescence assay for detecting IPNV. To further evaluate the specificity of the test, the result of IFA confirmed that the method, with the best stability by repetitions, had no reactive capability with IHNV-ZYX isolated、IBDV; With the same method for the detection of the IPNV (ATCC VR-1318), when pCold TF-VP2COE fusion protein of serum is diluted to1:320, EPC cells still visible obvious specific fluorescence, indicating the sensitivity of the detection method. It is effective in studying the antigen locations of IPNV. IFA can be applied in diagnosing and study ing the distribution of the IPNV in infected EPC cells. The results showed the results of the RT-PCR and IFA were consistent, indicates the establishment of the indirect immunofluorescence assay with epitope of VP2gene of isolated IPNV-ZYX is accurate and reliable, can be used for IPNV detection.