Construction Of CDNA Library,EST Analysis And Preliminary Study On Antibacterial Peptide Gene Of Monopterus Albus Intestin Tract

The freshwater eel, Monopterus albus, is widely distributed throughout the world. In recent years it has become commercially important, resulting in the establishment of several facilities to culture this species in China. With the development of intensive aquaculture, several diseases, caused by bacteria, viruses and parasites, have emerged. We constructed an expressed sequence tag (EST) library (Uncut) to identify genes associated with nutrient metabolism and immunity in the intestine of M. albus, a commercially valuable species. Then preliminary study on M. albus hepcidin expression in prokaryotic expression vector.Our objective was to identify nutrient metabolism and immunity-related genes in M. albus. We constructed a cDNA library from the intestinal tissue of M. albus. The library capacity reaches 1.46×107, the percentage of recombination is as high as 96.88%. PCR results showed that the average size of inserts was 2.11Kb. We sequenced 1056 randomly selected clones from the cDNA library. We obtained 906 high quality ESTs (GenBank accession number:GW584265-GW585168) with an average length 812 bp after trimming the vector sequence and eliminating low quality sequences. The ESTs were assembled into 61 contigs with an average length of 1057 bp, leaving 701 singletons. In total, we identified 762 unique sequences.Several (523 of 762) of the unique sequences had shared significant homology (BLASTX, score> 80, e-values<10-10) with known genes. We identified 59 species putative nutrition-related genes based on their functional classification in the Gene Ontology database. A number (73) of the unique sequences (including 8 contigs and 65 singlets) were related to immune function. We identified immunity-related genes that coded for chitinase, ferritin, MHC II, Hsp 70, HSP90, lectin, complement components, lysozyme, transferrin, glutathione peroxidase, serine protease inhibitor, and immunoglobulin. Eosinophil chemotactic cytokine (ECC) is a member of the chitinase family of proteins. Ours is the first report of this cytokine in teleosts.This study provides a basis for the development of the innate and adaptive immune systems in M. albus.Application of PCR technology to screen out hepcidin (GenBank accession number: GU997139) antimicrobial peptide gene from the library. Bioinformatics analysis software, on the selection of the antimicrobial peptide hepcidin gene was sequenced, found that including a 273bp ORF region encoding 90 amino acid residues of the peptide. Further analysis of antimicrobial peptide protein sequence contains a signal peptide of 24 amino acids and pro-region part of 40 amino acids before the mature peptide of 26 amino acids, class of antimicrobial peptide hepcidin contains eight cysteine typical structure, the formation of four pairs of disulfide key; structure prediction shows that it belongs to P-sheet type peptide. Homologous than are found with a variety of animals, M. albus hepcidin sequences are highly homologous with the similarity of Larimichthys crocea croaker up to 81%.Using genetic engineering techniques, preliminary study on M. albus hepcidin expression in prokaryotic expression vector. We constructed a direct expression vector pET22b (+) found that M. albus hepcidin peptide can not directly expression in pET22b (+), prove that this peptide with bactericidal activity. Then pET43.1a (+) construction of hepcidin fusion expression vector was transformed into BL21 (DE3) pLysS, BL21Star (DE3) pLysS and BL21-CondonPlus (DE3)-RIPL strain of three expression was induced and expressed recombinant protein had no significant expression. In pET32a (+) vector were constructed encoding full sequence of hepcidin (pET32a (+)-hepcidin) and no signal peptide sequence (pET32a (+)-nhepcidin) recombinant plasmid was transformed into BL21 (DE3) pLysS, BL21Star (DE3) pLysS, BL21-CondonPlus (DE3)-RIPL expression of three different strains induced for expression, found to contain a signal peptide of pET32a (+) -hepcidin fusion protein in three kinds of strains are not normal expression, while no signal peptide of pET32a (+)-nhepcidin recombinant protein can be highly expressed in BL21-CondonPlus (DE3)-RIPL. Induced by optimizing the conditions to obtain pET32a (+)-nhepcidin recombinant protein in BL21-CondonPlus (DE3)-RIPL in the best conditions for the expression of bacterial concentration (OD600) 0.5～0.7, temperature 37℃, IPTG concentration of 0.5mM, the induction time 4～6h. Identification found pET32a (+)-nhepcidin recombinant protein expressed in BL21-CodonPlus (DE3)-RIPL strain for soluble expression.We successfully constructed cDNA library (uncut) from the intestinal tissue of M. albus and found a lot of nutrition metabolism and disease resistance-related EST sequences. Antimicrobial peptide hepcidin from the intestinal tissue was found not only functional gene of M. albus widening the road, but also the development of new disease for aquaculture provides an important candidate drugs and reference materials. Our study provides a basis for the identification of genetic variation among immune related genes and their relationship to disease resistance phenotypes.