The Studies On Cloning Expression And Bioinformatic Analysis Of Myeloperoxidase From Branchiostoma Belcheri Tsingtauens

Amphioxus is an animal under second-class state protection,which is an evolutionary developmental model on the evolution studying between vertebrates and invertebrates.In this paper,the peroxidase gene in Branchiostoma belcheri tsingtauense was cloned,We identified its myeloperoxidase(MPO)gene,which will provide a molecular basis for the evolutio and immune response of Branchiostoma belcheri tsingtauense.Myeloperoxidase is an important member of the peroxidase family,mainly founded in neutrophils and monocytes,which contains heme prosthetic group.It can serve as a specific indicator for the activation of neutrophils and macrophages.MPO exists in animals or their secretions,but not found in plants now.Most of papers about myeloperoxidase are focused mainly on human MPO research.MPO can catalyze the formation of a number of reactive oxidant species and chloramines(NH2Cl)based on oxidation reaction,which plays an important role in the host defense.However,excessive reactive oxygen species are harmful to the body beyond its removal ability,which could cause endothelial cells,lipid,DNA damage and lead to many diseases,such as atherosclerosis,leukemia,nephritis,multiple inflammation,cancer,etc.In this study,the full-length cDNA of MPO was cloned and characterized in amphioxus by using rapid amplification of cDNA ends(RACE).The full-length cDNA of MPO is 3403bp,including a 642bp 3’-untranslated region(UTR),a 379bp 5’-UTR,and its predicted open-reading frame codes whose full-length is 2379bp for a protein of 793 amino acids with a predicted molecular mass of approximately 87074.09Da and an isoelectric point of 4.78.It has a signal peptide and a transmembrane domain,and most of them locate outside the cell.Thus,the protein should belong to a secretory protein.It’s predicted that protein synthesis encoded by MPO gene begins in cytoplasm,and then MPO is transported out of cell through cell membrane if its process synthesis is finished.Phylogenetic analysis results indicate that the amphioxus MPO gene is situated between the genes of vertebrate and invertebrate,and has close relatives with the vertebrate.The results of in situ hybridization experiments and DAB dyeing experiments show that the MPO gene can express in the skin,hepatic caecum,spinal cord.Its expression levels of the skin and spinal cord are much higher than those of others,which may be related with that skin and hepatic caecum are the important parts of,amphioxus immune systems,and MPO is one of important enzymes that take part in immune response among these tissues or organs,but the reason why high expression of MPO gene in spinal cord remains unknown and is worthy of further investigation.