Cloning And Expression Analysis Of A Lipopolysaccharide -and Beta-1,3-glucan-binding Protein (LGBP) In 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. Based on the techniques of molecular cloning and recombination, LGBP gene was cloned from Portunus trituberculatus, the expression pattern, recombinant expression in vitor and the binding activity were also studied.Firstly, the RNA was extracted from Staphyloccocus aureus and Vibrio alginolyticus challenged haemocytes of P.trituberculatus. Then the amplification of conserved sequence, 3'and 5'cDNA ends were used by strategy of homology cloning and SMART RACE respectively. The full-length LGBP cDNA (1378bp) had a 1095bp open reading frame (ORF) encoding a protein of 365 amino acid residues including a 16 amino acid residues signal peptide, a 138bp 5'untranslated region (UTR) and a 144bp untranslated region in the 3'UTR with a 29bp polyA tail. The calculated molecular mass of the mature protein (349 amino acid residues) is 39825.24 Da with an estimated pI of 4.49. The gene sequence and secondary structure of LGBP were analyzed by bio-informatics. Additionally, a Glyco hydro 16 domain was identified.The expression of P.trituberculatus in various tissues were detected through RT-PCR methods. Results showed that LGBP gene expressed in all the detected tissues, including haemocytes, hepatopancreas, heart, gills and muscle. In response to the challenge of Staphyloccocus aureus and Vibrio alginolyticus, the LGBP gene expression in haemocytes of the group challenged with mixed bacteria were higher than the control group within 48 hours. So the LGBP gene was presumed to play an active role in immunologicprocess againsting bacterial infection.A pair of primers were designed from LGBP cDNA used for ORF amplification. Then the fragment was subcloned into pET-22b(+) and expressed in E.coli BL21(DE3)plysE. By SDS-PAGE electrophoresis, induced recombinant protein had a 41 kDa clear visible band, while the negative controls were blank. Expression of recombinant protein induced at different conditions analyzed by SDS-PAGE indicated that lower concentration of IPTG (0.4 mmol/L) and lower temperature 30°C could reduced the expression of background effectively. Then plenty recombinant protein was purified and a specific polyclonal antiserum was obtained from mouse after continuous injection of pET-LGBP.At last, three kinds of Gram-negative bacteria (Vibrio parahaemolyticus, V.alginolyticus, Escherichia coli), two kinds of Gram-positive bacteria (Bacillus subtilis, Bacillus megaterium, Bacillus cereus, S.aureus) and one fungus (Sac-charomyces cerevisiae) were utilized to analyze pET-LGBP binding activity. Our data suggested that pET-LGBP has ability to bind to S.cerevisiae, B.megaterium, V.Parahaemolyticus, V.alginolyticus and E.coli.This study is mainly about cloning and expression of LGBP gene in P.trituberculatus. It can provide a basic data of innate immune function and control mechanisms to P.trituberculatus and which is also useful to develop immunopotentiator and prevention disease of aquatic animals.