Expressed sequence tags (ESTs) based identification of genes and expression analysis of leukocyte cell-derived chemotaxin-2 (LECT2) from Epinephelus bruneus Ramasamy Harikrishnan a, ⁎, Ju-Sang Kim a , Man-Chul Kim a , Chellam Balasundaram b , Moon-Soo Heo a, ⁎ a Marine Applied Microbes and Aquatic Organism Disease Control Lab, Department of Aquatic Biomedical Sciences, School of Marine Biomedical Sciences & Marine and Environmental Research Institute, Jeju National University, Jeju 690-756, South Korea b Department of Animal Science, Bharathidasan University, Tiruchirapalli-620 024, Tamilnadu, India abstract article info Article history: Accepted 29 August 2011 Available online 29 September 2011 Received by Dr. Jean-Marc Deragon Keywords: cDNA library Expressed sequence tags (ESTs) Epinephelus bruneus Leukocyte cell-derived chemotaxin-2 (LECT2) Recombinant expression The kelp grouper, Epinephelus bruneus, is an economically important intensively cultured species in Southeast Asia. Despite the insatiable demand its large-scale production has been hindered by problems associated with water quality, nutrition, and diseases especially due to increased rearing density. It is generally accepted that in fish both innate and adaptive immune system provide protection from diseases. In the present study a cDNA library of Streptococcus iniae-challenged kelp grouper was constructed to identify the genes that reveal molecular mecha- nism, physiological functions, and gene expression in different tissues using expressed sequence tags (ESTs) and RT-PCR strategy. Of a total of 2170 ESTs examined 279 (12.9%) were identified as contig and 860 (39.6%) as single- tons. A total of 190 important immune and enzyme related genes (16.7%) were identified in both contig and single- tons. The key immune molecules identified comprise complement factors, chemotaxin, chemokine, Fas ligand, ferritins, hepcidin, lysozyme c, MHC, and TLR which are involved in the innate or adaptive immune system. Among the genes a full-length cDNA of leukocyte cell-derived chemotaxin-2 (EbLECT2) with 540 base pair (bp) was identified; it consists of a 5′-untranslated region (UTR) of 17 bp, a 3′-UTR of 76 bp, and a stop codon TAA in 3′-UTR. The EbLECT2 is an important molecule in the innate immunity. It is a multifunctional protein involved in cell growth, differentiation, and autoimmunity. The open reading frame (ORF) of the EbLECT2 encodes with 155 amino acid (aa) residues with a predicted molecular weight and isoelectric point (pI) of 17 kDa and 9, respectively. The close phylogenetic relationship of EbLECT2 shares the highest similarity with the already reported LECT2 from Epinephelus coioides (96%) and Epinephelus akaara (94%). EbLECT2 mRNA was expressed predominantly in liver, spleen, and kidney while the expression was moderate in gills, heart, and muscle in E. bruneus after being chal- lenged with LPS from Escherichia coli and pathogenic bacterium Vibrio anguillarum both of which involve the im- mune defense system. Further, the recombinant mature EbLECT2 (rEbLECT2) was successfully expressed in E. coli BL21 (DE3), and the antiserum against EbLECT2 was obtained for further investigations. The significant number of ESTs genome results obtained constitutes a powerful resource for further investigation to establish the gene dis- covery, functional genomic research, molecular mechanisms, and development of microarrays for the gene expres- sion studies in kelp grouper. © 2011 Elsevier B.V. All rights reserved. 1. Introduction Kelp grouper, Epinephelus bruneus is one of the important marine aquaculture fish cultured in Korea and Southeast Asian countries be- cause of its sustained growth under crowded conditions, disease re- sistance, desired taste, and lucrative price. However, large-scale aquaculture production of the grouper has been hindered by many bacterial, parasitic, and viral diseases (Harikrishnan et al., 2001). Expressed sequence tags (ESTs) constitute the basis for functional ge- nomic research because they give a first glimpse of transcription pro- files in specific tissues (Ju et al., 2000) and provide detailed functional, structural, and evolutionary information (Quackenbush et al., 2000). The ESTs are important resources for discovery of mo- lecular markers which can be used for selection programs such as marker-assisted selection (MAS) by microsatellite tagging (Liu Gene 491 (2012) 88–101 Abbreviations: ESTs, expressed sequence tags; LECT2, leukocyte cell-derived chemotaxin-2; RT-PCR, reverse transcription polymerase chain reaction; MHC, major histocompatibility complex; TLR, toll like receptor; EbLECT2, Epinephelus bruneus leukocyte cell-derived chemotaxin-2; UTR, untranslated region; ORF, open reading frame; pI, isoelectric point; LPS, lipopolysaccharide; MAS, marker- assisted selection; SNP, single nucleotide polymorphism; QTL, quantitative trait locus; RAG, Recombinase-activating proteins; CFU, colony forming units; PBS, phosphate buffered saline; NCBI, National Center for Biotechnology Information; IPTG, isopropyl β-D-1-thiogalactopyranoside; SDS-PAGE, sodium dodecyl sulfate- polyacrylamide gel electrophoresis; ELISA, enzyme-linked immunosorbent assay; VWFC, von Willebrand factor type; CP, classical pathway; AP, alternative pathway; LP, lectin pathway; CRD, carbohydrate-recognition domain; PAMPs, pathogen-associated molecular patterns; TNF, tumor necrosis factor; NK, natural killer; IL-1, interleukin-1. ⁎ Corresponding authors. Tel.: +82 64 754 3473; fax: +82 64 756 3493. E-mail addresses: rhari123@yahoo.com (R. Harikrishnan), msheo@jejunu.ac.kr (M.-S. Heo). 0378-1119/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.gene.2011.08.029 Contents lists available at SciVerse ScienceDirect Gene journal homepage: www.elsevier.com/locate/gene