cDNA Cloning and Expression Characterization of Three Regulatory Subunits of NADPH Oxidase Within Cytoplasm from Mandarin Fish, Siniperca chuatsi

The phagocyte NADPH oxidase plays a crucial role in host defense against invading microorganisms by catalyzing the formation of reactive oxygen species, which is the precursor of a variety of microbicidal oxidants such as hydrogen peroxide (H₂O₂). In the present study, full-length cDNAs of three regulatory subunits of NADPH oxidase, including p40phox, p47phox, p67phox were cloned from head kidney of mandarin fish utilizing the reverse transcription polymerase chain reaction and rapid amplification of cDNA ends. Sequence analysis showed that the full length cDNA of p40phox is 1 406 nt, containing a 1 050 nt open reading frames that encodes a 349 amino acid protein, the full length cDNA of p47phox is 1 686 nt, containing a 1 209 nt open reading frames that encodes a 402 amino acid protein, the full length cDNA of p40phox is 2 185 nt, containing a 1 488 nt open reading frames that encodes a 495 amino acid protein. Semi-quantitative RT-PCR analyses from various tissues indicated that mRNAs of the three subunits can be detected in the blood, brain, heart, spleen, kidney and thymus, but their expression intensity are different in tissues. Stimulating the mandarin fish with formalin killed Flavobacterium columnare G4 significantly up-regulated the expression of p40phox in blood and head kidney; and p47phox in head kidney and spleen; and p67phox in blood, head kidney and spleen. The results suggested that mandarin NADPH oxidase was involved in the immune responses against bacteria.