Characterization And Expression Of Flounder Recombination Activating Gene (rag)

Flounder (Paralichthys olivaceus) was extensively cultured under intensivesystems in the north coastal regions of China, and is becoming an important mariculturespecies. The frequent occurrences of diseases have hindered its production. Recentlyyears, it is necessary to conduct the ontogeny study of flounder. Because this will beuseful for better understanding the immunological prevention and therapy of fishdiseases, and will be valuable to the developmental and comparative immunologystudy.Rag (recombination activating gene) was cloned from flounder and rag expressionwas examined in flounder tissues and lymphocytes. The open reading frame of flounderrag1 had four exons and three introns. Two alternatively spliced first exons of rag1 5′untranslated coding region were found upstream of the coding region for rag1. Theflounder rag1 encoded a predicted protein of 1068 amino acids. The open readingframe of flounder rag2 was 1602 bp encoded a predicted protein of 533 amino acids. 3'UTR of rag2 shared the common overlapping polyadinylation region with rag1, whichwas 3128 bp in length.RT-PCR detection showed that the expression of rag1 and rag2 was mainly onpronephros (head kidney) and mesonephros (truck kidney). In situ hybridization onwhole-mount and paraffin sections was performed with rag1 and rag2 probe. At 8 dpf,rag1 and rag2 expression were detected in an area located anterior to the pectoral finand dorso-lateral to the gill, this were consisted with the location of the thymus at laterages. The hybridization of rag1 and rag2 on paraffin sections showed that both geneswere specifical to thymus with heavy stained markers in the cortex.Partial rag1 and full length of rag2 were recombined and fused on plasmidpPROEX^TM HTa, and was transferred into E. coli (BL-21) for RAG1 and RAG2expression. It is showed that the expression was induced by IPTG, and the opticalinducing concentration of IPTG and optical inducing time for RAG1 and RAG2 were0.1 mM, 0.2mM, 2 hrs and 3 hrs respectively. The recombined RAG2 protein waspurified by BD TALONTM Metal Affinity Resins.