To research the characteristics and structural features of ORF3 gene from infections hypodermal and hematopoietic necrosis virus NJ strain(IHHNV-NJ)isolated in the laboratory, the primers were designed upon ORF3 gene sequence.Then we used PCR method to clone the ORF3 gene sequence and inserted it into the prokaryotic expression vector pET-32a(+).The successfully constructed vector pET32a-ORF3 was transformed into BL21(DE3)competent cells for prokaryotic expression.ORF3 gene sequence was studied by means of bioinformatics software.As the results showed, the length of ORF3 gene sequence was 990 bp, encoding 329 amino acids.The theoretical molecular mass of the protein was 37 385.2 u and the isoelectric point was 7.22.The grand average hydrophobicity was -0.619 and the hydrophilic zone(score<0)was evenly and widely distributed, which revealed a good hydrophile.The ORF3 sequence did not have transmembrane regions and signal peptide.The secondary structure of the protein contained 55.9% of α-helix, 52.0% of β-fold and 13.4% of β-corner.Epitopes were 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.Phylogenetic analysis showed that the highest homology was between NJ strain and Ecuador strain.Comparing NJ strain with Ecuador strain, Hawaii strain, Taiwan strain, Fujian strain, California strain and Thai strain, the homology was 99.7%, 99.1%, 99.1%, 98.8%, 98.2% and 96.6%.The recombinant expression vector pET32a-ORF3 successfully expressed a 49 ku fusion protein, which conformed to the expected size.Research shows, ORF3 gene may encode IHHNV capsid protein.O-glycosylation sites can participate in the assembly of capsid proteins and cellular infection process.Phosphorylation sites can participate in the reproduction process of the virus inside the shrimp's cells.ORF3 gene sequence has a strong conservative property that won't impact the viral toxicity and infection ability among individuals.The study provided the guidance and laid the foundation for those researches such as the IHHNV ORF3 gene's genetic information, gene functions and establishment of IHHNV molecular epidemiology methods.