Virus Detection Of Shrimp Virus Disease And The Establishment Of Sandwich ELISA For IHHNV

In 1980s,the global shrimp aquaculture was expanded into a great scale.Shrimp culture transformed into intensive culture mode.The high density and the poor prevention technology was leading to outbreak of shrimp viral diseases.After that,the shrimp aquaculture industry suffered serious losses.In recent years,the shrimp aquaculture in China still suffered White Spot Disease(WSD),Infectious Hypodermal and Hematopoietic Necrosis(IHHN),Taura Syndrome(TS)and other traditional viral diseases.WSD,IHHN and TS were both spread fast,caused a high incidence and a high mortality.The yield and the quality were significantly decreased.At present,the effective drugs are still the bottleneck of shrimp viral diseases.The detection and control are the main means of healthy shrimp farming.We collected 348 shrimps from Jiangsu,Anhui and Guangdong during September 2012 to September 2014.White Spot Syndrome Yirus(WSSV),Infectious Hypodermal and Hematopoietic Necrosis Virus(IHHNV)and Taura Syndrome Virus(TSV)were detected by nested PCR,step PCR and reverse transcription PCR respectively.Compared ORF14/15 of WSSV-TH,WSSV-CN,WSSV-TW with 9 WSSV strains separated in lab,then established the phylogenetic tree and analyzed the homology.Using bioinformatics software analyse hydrophobicity,transmembrane,signal peptide,secondary structure,antigen epitope,glycosylation and phosphorylation sites of IHHNV-NJ.Compared ORF3 of IHHNV-HI,IHHNV-CA,IHHNV-FJ,IHHNV-TH,IHHNV-TW,IHHNV-EC with IHHNY-NJ,then established the phylogenetic tree and analyzed the homology.The recombinant plasmid pET32a-ORF3 was transformed to E.coli BL21(DE3).Expressed and purified the pET32a-ORF3 fusion protein.Immunized ICR mice and New Zealand rabbits.Collected serum of mice and rabbits and purified the serum respectively.Explored a sandwich ELISA method for detection of IHHNV.The positive rate of WSSV,IHHNV,TSV was 15.31%,4.31%,0%.Analysis of ORF 14/15 shows,9 strains of WSSV were divided into 2 branches:the first one was including BA,BC,BE,BK,BI;the second one was including AG,SW,PH,JX.The divergence rate between two branches was 60%-80%.Bioinformatics data of IHHNV shows that the characteristics of ORF3 protein was in accordance with the capsid protein.The length of ORF3 was 990 bp,encoding 329 amino acids.The protein revealed a good hydrophile.The secondary structure of the protein contained 55.9%of a-helix,52.0%of ?-fold and 13.4%of ?-corner.Epitopes located in multiple sequence intervals,and the protein showed a strong antigenicity.There were 13 potential O-glycosylation sites and 17 potential phosphorylation sites but with no potential N-glycosylation sites.The homology between ORF3 of IHHNV-NJ strain and the other strains of IHHNV was higher than 96%,indicating that IHHNV ORF3 was strong conserved.The recombinant plasmid pET32a-ORF3 was successfully constructed.The pET32a-ORF3 fusion protein and mouse polyclonal antibody,rabbit polyclonal antibody were successfully purified.Explored a sandwich ELISA method for detection of IHHNV.The optimal conditions were 0.02 mol/L coating liquid with mouse antibody(1:6400).The coating time and the temperature was 4? 12 h+37? 1 h.Using 10%calf serum to block at 37 ? for 1 h.The optimal time of antigen was 1 h.Using rabbit antibody(1:3200)to detect the antigen at 37? for 1 h.The sandwich ELISA method provided a new method and basis for the preparation of IHHNV detection kit.