A Novel Shrimp DNA-Binding Protein LvDBP23 with the appropriate GeneRacerTM 59 Primer and a gene-specific primer. Nested PCR was performed by using GeneRacerTM 59 nested Primer and a nested gene-specific primer. ORF finder (NCBI) program was used for prediction of start codon position. The deduced amino acid sequence was analyzed with the NCBI Protein Sequence Analysis software. Predictions of protein function were initially analyzed by online software named Motif Scan (http://hits.isb-sib.ch/cgi-bin/PFSCAN). Search for conserved domains were analyzed on NCBI website (http://www. ncbi.nlm.nih.gov/Struc ture/cdd/wrp sb.cgi). DP-Bind, a web server for sequence-based prediction of DNA-binding residues, was used for searching DNA binding sequences (http://lcg.rit. albany. edu/dp-bind/). Expression of LvDBP23 recombinant protein in SF9 insect cell line The Insect cell expression (Novegen, Gibbstown, NJ) system was used to express recombinant LvDBP23 protein. Briefly, LvDBP23 and GFP gene were inserted to pIEx-5 plasmid between Nco I and Xho I restriction site. This plasmid has the protein secretion property. The plasmids pIEx-5- LvDBP23-His and pIEx-GFP-His were transformed into bacterial strain One ShotH TOP10. Plasmids were prepared by GeneJETTM Plasmid Miniprep Kit (Fermentas, Glen Burnie, MD), and inserts were confirmed by sequencing. The plasmids were transfected into Sf9 cells. Monolayer of Sf9 cells was grown in T-75 flasks at 28uC containing 9 ml of BacVector Insect cell medium supplement with 5% (v/v) fetal bovine serum by typical seeding density 1.06106 cell. Cell density was determined by hemacytometer counts and cell viability was evaluated by Trypan Blue exclusion dye at 0.4% (w/v) in 0.85% phosphate buffer saline (PBS). In T-75 flask , monolayers adherent Sf9 cells diluted with 10 ml serum free BacVector Insect cell medium with cell density 46105 cell/ml were transfected with 8 mg pIEx5-LvDBP23 and pIEx5-GFP which were diluted with 400 ml serum free BacVector Insect cell medium, using 40 ml GeneJuice Transfection Reagent (Novegen, Gibbstown, NJ), according to the manufacturer’s protocol. Transfectants were incubated at 28uC for 48 hrs. Transfected cells were lysed by 50 ml Insect PopCultureH Reagent per 1 ml culture volume, followed by 10 U BenzonaseH nuclease per 1 ml of the original culture volume [21]. The mixture was gently inverted several times and incubates 15 min at room temperature. Expression of LvDBP23-His was monitored by SDS-PAGE and western blot analysis with anti-His.tag primary antibody. RNA extraction Fresh different tissues of juvenile shrimp L. vannamei including heart (H), eyestalk (E), intestine (I), hepatopancreas (Hep), haemocyte (Cy), lymphoid (L), brain and nerve ganglion (BN), gill (G), pleopods (P), abdominal muscle (AM), swimming leg muscle (SM) and ovary (O) and muscle tissues of different PLoS ONE | www.plosone.org 6 May 2011 | Volume 6 | Issue 5 | e19959 Figure 6. LvDBP23 gene expression in different molting stages by RT-PCR. Six individual shrimp of each stages of postmolt (A), intermolt (B), premolt (C) were used for LvDBP23 gene expression analysis. Reverse transcription PCR of LvDBP23 partial mRNA sequence (451 bp) was carried out with mRNA isolated from shrimp abdominal muscle. EF-1 gene was used as a control gene. doi:10.1371/journal.pone.0019959.g006 RACE GeneRacer system (Invitrogen) with shrimp muscle mRNA template and LvDBP23 specific primer (GSP: 59 CCGCTT- CCGGCCCCGC CTGCCACGC 39) and nested specific primer (Nested GSP: 59CTGTATCCTGGC AGCTTCGCAGCGG 39). Abdominal muscle mRNA was dephosphorylated by calf intestinal phosphatase (CIP) to eliminate truncated mRNA and other RNA. Dephospholylated mRNA was treated with tobacco acid pyro- phosphatase (TAP) to remove 59cap structure from full-length mRNA. Treated mRNA was ligated with GeneRacerTM RNA oligo to 59end of mRNA using T4 RNA ligase. A cDNA template was generated by reverse transcription using SuperScriptH III and random primers. Firstly, the 59 end cDNA sequence was amplified Figure 7. DNA binding protein assay. LvDBP23 (A) and GFP (B) proteins binding with dsDNA cellulose and ssDNA cellulose bead in different total protein amount of 100, 200, 400 mg. P: only recombinant protein without DNA in the reaction. Both proteins were detected by anti-His antibody. doi:10.1371/journal.pone.0019959.g007