Molecular cloning and characterisation of a cell adhesion molecule, peroxinectin from the white shrimp Litopenaeus vannamei

摘要

Expression of peroxinectin cDNA was obtained from haemocytes of white shrimp Litopenaeus vannamei using oligonucleotide primers based on the peroxinectin sequence of tiger shrimp Penaeus monodon and freshwater crayfish Pacifastacus leniusculus. The peroxinectin of L. vannamei was constitutively expressed. The cloning of peroxinectin cDNA fragment was subjected to RT-PCR using degenerated primers. Both 3'- and 5'-regions were isolated by rapid amplification of cDNA end RACE method. Analysis of nucleotide sequence revealed that the cDNA clone has an open reading frame of 2415 bp encoding a protein of 805 amino acids including a 20 amino acid signal peptide. The calculated molecular mass of the mature protein (758 amino acids) is 89.1 kDa with an estimated pI of 7.5. Two putative integrin binding motifs, RGD (Arg-Gly-Asp) and KGD (Lys-Gly-Asp) were observed in L. vannamei peroxinectin. Sequence comparison showed that nucleotide homology has an overall similarity of 91% and 60% to that of P. monodon and P. leniusculus, respectively. Sequence comparison showed that peroxinectin deduced amino acid of L. vannamei has an overall similarity of 92% and 60% to that of P. monodon and P. leniusculus, respectively. The 432 bp fragment (partial cDNA) and 1016 bp fragment (genomic DNA) were obtained using specific primers PE2F and PE2R. There were three introns in the 1016 bp (genomic DNA) fragment. Peroxinectin transcript was significantly reduced when the shrimp was transferred to high temperature (34 degrees C) as compared to the shrimp transferred to 18 degrees C and the control shrimp (26 degrees C). However, peroxinectin transcript did not change in 24 h-post zymosan injection.