Molecular Cloning And Characterization Of Grouper(epinephelus Coioides)phagocyte Nadph Oxidase And Myeloperoxidase

Stimulated neutrophils undergoes the respiratory burst and produce large amounts of reactive oxygen species(ROS)that act as powerful microbicidal agents.NADPH oxidase is a major source of ROS in cells,and it can convert O2 into O-2,eventually producing H2O2 by the disproportionation.As a highly motile phagocytic cell,neutrophils strong engulf and degrade microbes,which constitute the first line of defense of the innate immune system.Myeloperoxidase(MPO)mainly exists in the cytoplasmic granules of the neutrophil,and the microbicidal action of MPO was attributed to its unique enzymatic capacity to produce hypohalous acid using H2O2 and halide.Cryptocaryon irritans is a ciliated obligate parasite and has severely limited mariculture development and results in mass economic losses.Previous studies have demonstrated that C.irritans can induce the immune response of fish although the specific mechanism is unclear.To study whether neutrophils were involved in grouperanti-C.irritans infection,we cloned 5 subunit genes of the grouper NADPH oxidase complex(gp91phox,p22 phox,p67phox,p47 phox and p40phox)and the grouper myeloperoxidase gene c DN A based on the transcriptome data of grouper skin,gill,spleen and head kidney,and analysed the expression pattern of these genes in different tissues at the different time-points post C.irritans infection.We also expressed the partial MPO protein and prepared the polyclonal antibody against recombinant protein by immunizing rabbits.A comparison between the different time-points post C.irritans infection was aim at investigating the involvement of MPO in grouper anti-C.irritans combining q RT-PCR and immunohistochemistry.The main results were as follows:we cloned 6 genes of the grouper NADPH oxidase and MPO in this thesis.The open reading frame of the gp91 phox,p22phox,p67 phox,p47phox,p40 phoxand MPO gene is 1698 bp,564 bp,1497 bp,1290 bp,1050 bpand 2313 bp,and encode a putative protein of 565,187,498,429,349 and 770 amino acid residues,respectively.Phylogenetic analysis reflected their phylogenetic origin between mammalian and teleost MPO amino acid sequences,which suggested that they may share a common ancestor.These genes m RNA is expressed in all thirteen tissues detected,with higher expression in the lympho-haematopoietic organs(head kidney and spleen)than in the non-immune organs(muscle,heart,brain and stomach).Interestingly,these genes also have higher expression in the skin,gill and trunk kidney.The relative expression level of these genes m RNA in 4 organs(skin,gill,head kidney and spleen)after C.irritans infection shows significant increase at almost all time-points,whichdemonstrate these genes may be involved in grouper anti-C.irritans.We successfully expressed the partial MPO protein and prepared the polyclonal antibody against recombinant protein by immunizing rabbits,and this antibody can recognize the native protein of grouper head kidney well.It provides a good basis for the labeling of neutrophils.A comparison immunohistochemistry between the gill tissues at the different time-points post C.irritans infection shows the number of the positive cells are increasing firstly and then decreasing as time goes on,and large numbers of positive cells gathered round the trophont.It suggests neutrophils are involved in grouper anti-C.irritans.Deeper mechanism about the function of neutrophils needs to be further researc hed.