The study of recombinant expression,preparation of antiserum and binding activity to microorganism of LGBP gene from Portunus trituberculatus

The lipopolysaccharide and beta-1,3-glucan-binding protein(LGBP)is a pattern recognition receptor which is fundamental for the innate immune response of crustaceans.A pair of primers were designed according to LGBP cDNA and were used for ORF amplification.Then the fragment was subcloned into pET-22b(+)and expressed in Escherichia coliBL21(DE3)plysE.We got a 41 ku clear visible band in the expected position by SDS-PAGE detection,which indicated that it was the same as the expected molecular weight,and the negative controls were blank.Expression of recombinant protein induced under different conditions indicated that lower concentration of IPTG(0.4 mmol/L)and lower temperature 30 ℃ could reduce the expression of background effectively by SDS-PAGE detection.The purified recombinant protein was used as antigen to prepare antiserum in mouse directly.Four weeks later,a polyclonal antiserum was obtained and it was specified by Western-blotting analysis.At last,three types of Gram-negative bacteria(Vibrio parahaemolyticus, V.alginolyticus, E.coli),four types of Gram-positive bacteria(Bacillus subtilis,B.megaterium,B.cereus,S.aureus)and one fungus(Sac charomyces cerevisiae)were utilized to analyze pET-LGBP binding activity.Our data suggested that pET-LGBP was able to bind S.cerevisiae,B.megaterium,V.Parahaemolyticus,V.alginolyticus and E.coli.