Development of recombinant protein adjuvants based on Vibrio harveyi flagellin gene in Epinephelus coioides
Andre Giovanni(a), Shun Maekawa(a,f*) , Pei-Chi Wang(b,c,e) , Shih-Chu Chen(a,b,c,d,e*)

a)International Degree Program of Ornamental Fish Technology and Aquatic Animal Health, International College, National Pingtung University of Science and Technology, Pingtung, Taiwan
b)Southern Taiwan Fish Diseases Research Center, College of Veterinary Medicine, National Pingtung University of Science and Technology, Pingtung 91201, Taiwan
c)Department of Veterinary Medicine, College of Veterinary Medicine, National Pingtung University of Science and Technology, Pingtung, Taiwan.
d)Research Center of Animal Biologics, National Pingtung University of Science and Technology, Pingtung 91201, Taiwan
e)Research Center for Fish Vaccine and Diseases, College of Veterinary Medicine, National Pingtung University of Science and Technology, Pingtung 91201, Taiwan
f)General Research Service Centre, National Pingtung University of Science and Technology, Pingtung, Taiwan

Abstract

Vibrio harveyi is a gram-negative bacterium that has flagella which are useful for locomotion. This bacterium is an important pathogen causing vibriosis in shrimp and some marine fish, including the orange-spot grouper (Epinephelus coioides) can become infected with these pathogens. In V. harveyi virulent genes such as chitinase, exotoxin, metalloprotease, serine protease, hemolysin and flagellin genes have been identified. The toxin produced by Vibrio harveyi flagella can be a source of infections in fish. The purpose of this research is to look at how gene responses in orange-spot grouper (Epinephelus coioides). Binding of VhFliA to TLR5 could activate the innate and adaptative immune system to stimulate production of cytokines. This study used VhFliA and mutants gene and analyzed cytokine (IFN&#947-, IL-1&#946-, IL-6, IL-8, and IL-10) and also IFN&#947- downstream gene (MHC class I, MHC class II, CD4, CD8) immune response gene. Determine the responsiveness of Vibrio harveyi flagellin (VhFliA) gene expression in the spleen and head kidney of grouper. Total RNA was extracted from the spleen and head kidney, and qRT-PCR was used to analyze it. The post-injection observation period is repeated several times. There are four different treatments: EGFP as a control, wild-type VhFliA, and two mutagenesis of wild-type VhFliA (&#8710-MH-VhFliA and &#8710-D0MH-VhFliA). We^ve found an upregulation in cytokines and downstream genes. In this study, we identified and characterized V. harveyi flagellin A (VhFliA) gene. We investigated the in vivo activities of recombinant VhFliA protein and its mutant forms (&#8710-MH-VhFliA and &#8710-D0MH-VhFliA) in grouper. This study will provide the development of epidemic prevention strategies in aquaculture species using recombinant VhFliA and its mutants protein

Keywords: Vibrio harveyi- flagellin- toll-like receptor 5- Immune response- Epinephelus coioides

Topic: Marine and Fisheries Biotechnology (International)