Cloning And Sequence Analysing Of Litopenaeus Vannamei Polyphend Oxidase Activated Factor And Peroxinectin Gene

Instead of real antibodies and acquired immune system,Invertebrates mainly rely on the innate immune system to resist against external pathogenic microorganisms. The prophenoloxidase activating system (Prophenoloxidase activating system, pro-PO) plays an important role in the innate immune mechanism of invertebrates. The prophenoloxidase-activating factor (prophenoloxidase activating factor PPAF) and the Cellular adhesive protein(Peroxinectin, PX) are two important factors in the prophenoloxidase activation system. How these immune-related factors resist exogenous biological invasion and participate immune activity has been attracted widespread concerned. We first cloned PPAF full-length cDNA(GenBank accession number JQ684529),and PX gene sequence by RACE.1.The sequence was amplified with the method of RACE,a full length cDNA of the PPAF from shrimp Litopenaeus vannamei was obtained. Afier being submitted to the GenBank database, the sequence accession number JQ684529was assigned. With the length of1986bp, which includes an open reading frame of1629bp(ORF),encoding a protein of amino542acids,the functional areas of the amino acid sequence in the269-517aa, an ALPHA-2macroglobulin thiolester region signature in the494-502position, Serine protease function site and histidine active site in the315-320aa.The full-length contained a3’-terminal untranslated region of336bp and a5’-terminal untranslated region of21bp Phylogenetic analysis showed the PPAF vannamei shared significant homology with other reported PPAFs,the highest is Penaeus monodon,whilefruit flies, mosquitoes are relatively low.Quantitative real-time PCR was used to assess the PPAF expression levels in the blood, which found that it decreased significantly the amount of expression in heart, pancreas, liver, intestines, stomach and muscle after infected the IHHNV comparede with the amount of normal tissues.2. A partial length cDNA of the PX is1533bp, which includes an open reading frame of1183bp(ORF), encoding a protein of amino380acidsthe,and it contained a3’-terminal untranslated region of318bp. The predicted molecular weight is43556.97and the isoelectric point was9.448. Analysis found that it has signal peptide in72-86aa, and it contained signal peptide cleavage site in82aa by signalP software. The deduced amino acid sequence was analyzed with the Motif Scan program, which found oxide activity in the336-378region.The deduced amino acid sequence of LvSPH shared with significant homology with others reported PPAF,sepecilly withblack tiger shrimp, while with the Drosophila is minimum.Quantitative real-time PCR was used to assess the PPAF expression levels in the blood, which found that it decreased significantly the amount of expression in heart, pancreas, liver, intestines, stomach and muscle after infected the. comparede with the amount of normal tissues.