Gene Cloning And Prokaryotic Expression Of Liver-expressed Antimicrobial Peptide-2, In Larimichthys Crocea

Antimicrobial peptide is widely referred to peptides which revealed antimicrobial activity expressed inall kinds of cells, and has an important role in immune reaction. liver-expressed antimicrobial peptide-2（LEAP-2）is involved into blood circulation and shown antimicrobial activity.In this experiment，liver-expressed antimicrobial peptide-2（LEAP-2）gene from Larimichthys croceawas cloned and sequenced. The cloned sequence was2,236bp in length ranging from start codon to stopcodon, and included78bp exonⅠ,880bp intronⅠ,179bp exonⅡ,1044bp intronⅡ, and55bp exonⅢ. TheThe coding sequence was312bp in length, and encodes103amino acids. It is the first report ofLarimichthys crocea LEAP-2in China.The complete LEAP-2ORFs, which contains a potential signal sequence (residues1-29) and aLEAP-2superfamily domain (residues51-93), encodes103amino acid. There are4conserved cysteineresidues in C-terminal of LEAP-2superfamily domain which4conserved cysteine residues which wereproposed to form2disulphide bonds. The LEAP-2reveals a archetypal features as a LEAP-2superfamilymember. All of above features correspond to structure characteristic of all LEAP-2superfamily members.In the evolution, LEAP-2amino acid sequence was highly conservative, and the similarity amongParalichthys olivaceus, Pelteobagrus fulvidraco and Ictalurus furcatus was over95℅. Sequencecomparison and phylogenetic analysis showed that the LEAP-2in Larimichthys crocea was most similar toParalichthys olivaceus, and the relationships of the different LEAP-2coincided well with the evolutionaryrelationships of their organisms.Semi-quantitative expression of three different situations in9tissues (muscle, brain, eyes, heart, liver,kidney, intestines, spleen, gill) of LEAP-2in adult Larimichthys crocea were detected by conventionalRT-PCR. In healthy Larimichthys crocea, the results demonstrated that LEAP-2mRNA was highlyexpressed in liver and intestines, and that was not detected in eye, muscle, brain, gill, heart, spleen andkidney. After Pseudomonnas putida infection, the nine organization were taken at48h and7days. LEAP-2transcripts of every organizations significantly up-regulated after infection. And the LEAP-2transcriptswere most highly abundant in liver in three different situations. Compared with the normal group, challengeof Larimichthys crocea with Pseudomonnas putida significantly elevated LEAP-2mRNA levels in iver,kidney, spleen and intestines, and expressed faster than other organizations. LEAP-2has prevalentexpression in various tissues of Larimichthys crocea, but the LEAP-2expression levels are evidentlydifferent。These results suggest that LEAP-2may be involved in the immune response of Larimichthyscrocea.The aquired Larimichthys crocea LEAP-2cDNA carrying BamH Ⅰ and Hind III sites was amplifiedby PCR, and subsequently linked into pET32a plasmid with the same restriction sites to construct arecombinant expression vector pET-32a-LEAP-2. and the recombined plasmids were then transformed into E.Coli BL21. The inserted sequences are induced expression by1.0mmol/L IPTG. The complete LEAP-2gene coded protein is about27kDa.