Characterizations And Prokaryotic Expression Of Myeloperoxidase And NADPH Oxidase Gene Of Mandarinfish (S. Chuatsi)

The mandarinfish (Siniperca chuatsi) is an important species for aquaculture industry in China. The understanding its immune system is essential for aquaculture. The present study is to clone NADPH oxidase and myeloperoxidase that related to respiration burst and to express part of heavy chain of myeloperoxidase with Prokaryotic expression.The MPO and the five subunit of NADPH oxidase cDNAs full sequence of mandarin fish have been cloned using RACE-PCR and PCR. The full cDNA of MPO is 3345 nucleotides. Open reading frame (ORF) is 2292 nucleotides which encode a 763 amino acid peptide. Comparison of MPO amino acid sequence with part of the known MPO protein sequence from other mammal demonstrated a lower level of similarity, but they have approximately molecular weight, MPO of the mandarin fish has similarity hemachrome group, a conserved banding calcium site, six intra-molecule disulfide bridges and six N-glycosylation sites as mammal. These results imply that mandarin fish MPO may have a similar structure and catalytic mechanism as mammal. The ORF of gp91phox subunit withl698 nucleotides may translate product of 565 amino acids. N-terminal amion acids can form six transmenbrance a-helices that contain three N-glycosylation sites and two heme groups. The ORF of p22phox contain 561 nucleotides for 186 amino acids. The hydrophobic analysis of N-termina p22phox predicts three transmenbrance a-helices. C-terminal has a proline-rich region. ORF of p67phox is 1488 nucleotides that encode a 495 amino acid peptide. The p67phox protein contains four TPR (tetratricopeptide repeat) motif, a PBldomain and two SH3 domains. ORF of p47phox contain 1209 nucleotides for 402 amino acids. The p47phox protein contains a PX profile and a SH3 profile. ORF of p40phox is 1050 nucleotides that encode a 349 amino acid peptide. The p40phox protein contains a PX domain and an SH3 domain. It is lower identity by comparing the deduced amino acid sequence of each subunit with respective counterpart of human's, but they have some similar functional domains and conservative sites. So it suggested that NADPH oxidase of mandarin fish might have similar function as human's.The expression primers were designed according to part of cDNA segment of mandarin fish...