Abstract:In recent years, Vibrio harveyi has become a main pathogen for mariculture animals. It caused serious diseases and mortalities of many aquaculture animals and caused huge economic loss worldwide. The isolation and identification of virulence factors of V. harveyi become research hotspot recently. The V. harveyi strain ML01 (CGMCC No. 11720) was isolated from diseased juvenile hybrid grouper Epinephelus fuscoguttatus (♀) × Epinephelus lanceolatus (♂) suffering from severe skin ulcer. In this research, the virulence factors, including extracellular products (ECPs) and secretory proteins of strain ML01 were extracted and purified by cellophane plate and gel filtration techniques. Furthermore, extracellular products (ECPs) and secretory proteins were characterized and identified through toxicity tests, mass spectrometry MALDI-TOF/TOF, and molecular cloning methods. The results showed that ECPs of strain ML01 had the activity of gelatinase, amylase, lipase and caseinase, but no the activity of urease. The hemolysis to sheep red blood cells of ECPs was negative. The toxicity tests showed that ECPs of strain ML01 were lethal to zebrafish and the LD50 value was 19.55 μg/g body weight. Three major secretory proteins (P42, P36, and P31) corresponding to the molecular weight of 42 kDa, 36 kDa and 31 kDa on SDS-PAGE were purified from strain ML01. These proteins were identified as membrane proteins of OmpU, OmpN and a hypothetical protein of V. harveyi by mass spectrometry MALDI- TOF/TOF. Based on homology cloning techniques, the genes of proteins P42, P36 and P31 were amplified from the genome of strain ML01 and sequenced. The sequence alignment results showed that similarities of the open reading frames (ORFs) between strain ML01 and V. harveyi ATCC 33843 were 97.08% (p42), 100% (p36) and 99.67% (p31), while the similarities of peptide sequences were 99.71% (p46), 100% (p36) and 99.93% (p31), respectively. This research is of significance to further analysis of the pathogenic mechanism and the development of subunit vaccines of V. harveyi strain ML01.
