The http://www.selleckchem.com/CDK.html reaction was conducted at 42 °C for 60 min followed by an inactivation step at 70 °C for 15 min. PCR amplification of 1 μl cDNA was performed in a 25 μl reaction volume containing 1X Standard Taq buffer (50 mM Tris–HCl buffer (pH 9), containing 50 mM KCl, 1% Triton X-100 and 2.5 mM MgCl2) 5 U Taq polymerase and 0.25 mM dNTPs. Based on the previous publications, degenerate primers FIPE F1 (5′-ATG CGC CTC GTG GTC TG-3′), FIPE R1 (5′-TCC TTT TAC

TAA ITG ICA ICA-3′) were used for the partial amplification of F. indicus penaeidin [23]. Based on the partial sequence obtained, the complete sequence of Fein-Penaeidin cDNA was generated using RACE PCR. PCR reactions were performed as follows: 35 cycles of denaturation at 94 °C for 1 min, annealing at 50 °C for 1 min, and elongation at 72 °C for 2 min, followed by a 10 min extension at 72 °C. The PCR product was analyzed by electrophoresis in 1.5% agarose gels in TBE buffer, stained with 10 mg/ml ethidium bromide and visualized under UV transilluminator. Further amplified cDNA fragments were cloned into the pGEM-T Easy vector as per the instructions of the manufacturer (Promega Corporation, Madison, WI, USA). Recombinant bacteria were identified by

blue/white screening and confirmed by PCR. Plasmids containing the insert were purified (HiYieldTM Gel/PCR DNA Mini Kit-Real genomicsTM, GSI-IX chemical structure Taiwan) and used as a template for DNA sequencing. Nucleotide sequencing

Isotretinoin was performed using the dideoxynucleotide chain termination method on an ABI DNA sequencer (Applied Biosystems, Forster, CA, USA). The sequence homology and the translated amino acid sequences comparisons were carried out using the BLAST program at the National Center of Biotechnology Information (NCBI) (http://www.ncbi.nlm.nih.gov/blast). Gene translation and prediction of deduced protein were performed with EXPASY (http://www.au.expasy.org/). Elementary domain analysis was carried out on “InterPro Domain Scan” [24]. The signal peptide was predicted by the Signal P server (http://www.cbs.dtu.dk/services/SignalP/). The Multiple sequence alignments were performed on amino acid sequences of known penaeidins or penaeidins-like peptides from shrimps with MAFFT version 6 (http://mafft.cbrc.jp/alignment/server/) and the same were used to construct the phylogenetic tree by the Neighbor-Joining method and the UPGMA method with MEGA 3.0 (http://www.megasoftware.com). Bootstraps (1000) were performed for the UPGMA and NJ trees to confirm the repeatability of the results.