Cloning And Expression Analysis Of Recombination Activating Genes In Red Snapper, Lutjanus Sanguineus

Recombination activating genes (rag1and rag2), involved in the V(D)Jrecombination of immunoglobulin and T-cell receptor genes play a crucial role in theadaptive immune response in vertebrate. Also they can be used as useful markersinvolved in the evolutionary analysis of the vertebrates. The expression of these geneswere required for the proper development and maturity of lymphocytes so that they canbe used to monitor the appearance and location of these cells throughout development ofthe immune system.Red snapper (Lutjanus sanguineus), which is one of the most important marinefishes in the south coastal regions of China, was studied in this paper. The full-lengthcDNA of red snapper rag1and rag2were cloned using homological cloning and rapidamplification of cDNA ends (RACE) methods. Results showed the full-length of rag1cDNA was3944bp, containing a5′untranslated region (5′-UTR) of200bp, a3′-UTR of561bp and an open reading frame of3183bp encoding1060amino acids with anestimated molecular weight of120.5kDa and an estimated isoelectric point of8.72. Thefull-length of rag2cDNA was2200bp, consisting of a141bp5′-UTR, a457bp3′-UTRand an open reading frame of1602bp encoding533amino acids with an estimatedmolecular weight of59.6kDa and an estimated isoelectric point of5.42. BLASTanalysis revealed that the RAG1and RAG2in red snapper shared a high homology of82%and87%with the corresponding proteins of other known species, respectively.Multiple sequence alignment confirmed that RAG1and RAG2were highly conserved.The phylogenetic trees constructed in this study showed that RAG1and RAG2of redsnapper shared closest relationship with the corresponding proteins of Hippoglossushippoglossus and Takifugu rubripes, respectively.The differential expression of rag1and rag2in head kidney, thymus, spleen, liver,heart, intestine, muscle and brain of healthy fish and the fish immunized by inactivatedVibrio alginolyticus were analyzed by fluorescent quantitative real-time PCR technologybased on the control genes of GAPDH and β-actin in this study. The results showed thatthe overall expression pattern of the two genes was quite similar. In healthy red snappers,the rag1and rag2transcripts were mainly detected in thymus, head kidney and spleen.After vaccinated with inactivated V. alginolyticus48h later, the rag1and rag2mRNA expression levels were significantly up-regulated in all studied tissues of red snapper. Aclear time-dependent expression pattern of rag1and rag2after immunization and theexpression reached the highest level at48h in thymus,60h in head kidney, spleen andbrain,36h in liver and96h in intestine, respectively.Partial rag1and full-length of rag2were cubcloned into pET-28a(+) vector toconstruct expression plasmids pET28-RAG1and pET28-RAG2. The recombinant RAG1and RAG2fusion proteins were overexpressed in E. coli BL21(DE3) cells in thepresence of isopropyl-β-thiogalactopyranoside (IPTG). The recombinant RAG1andRAG2fusion proteins were purified by His TrapTMHP column, and then identified bywestern blot using His-Tag Mouse mAb.Polyclonal antibody against RAG2fusion protein was raised in a New Zealandpedigree white rabbit immunized with the purified RAG2fusion protein, and theantibodytiter reached1︰40000. The result of the western blot revealed that specificantigen-antibody reaction was occurred between the antiserum and its correspondingrecombinant fusion protein. As a further means of studying the localization of RAG2inthe thymus, head kidney and spleen from red snapper, immunohistochemistry analysiswas carried out using the prepared polyclonal antibody. The results showed that RAG2protein mainly distributed in lymphocytes in this organs.