Molecular Cloning And Expression Analysis Of The PIgR Gene In Scophthalmus Maximus

Polymeric immunoglobulin receptor (pIgR) is a key immune factor, medicatingthe transport of polymeric immunoglobulins (pIg) through the epithelial cells into themucosal secretions to protect the organisms from pathogens. The efficient secretion ofpIgR is a prerequisite of polymeric Ig-mediated mucosal protection. With the furtherresearch on immunoglobulins (Igs) in fish, pIgR structure and function have becomeworldwide research focus. The pIgR is very conservative in evolution, and has alreadybeen cloned and studied in several kinds of teleosts.In this study, cDNA sequence of pIgR gene of Scophthalmus maximus wascloned, the expression level of the gene in different tissues was analyzed, and thedynamics changes of pIgR expression were evaluated following bath immunizationwith inactivated Vibrio anguillarum. Furthermore, the protein was recombined andpolyclonal antibody against pIgR was developed. In addition, the antibody wasapplied to detect pIgR in four mucosal tissues, which provided a certain value instudying the function of pIgR. The followings are the details:The pIgR of Scophthalmus maximus was first cloned and sequenced by rapidamplification of cDNA ends approaches (RACE). The pIgR contained1584nucleotides,3’UTR,5’UTR, and an open reading frame (ORF) of1005nucleotidesthat encoded for a polypeptide of334amino acid. Gene homology comparisonsshowed that S. maximus shared74%amino acid identity with Paralichthys olivaceusand Epinephelus coioides, and59%with Takifugu rubripes, indicating a highconservation of pIgR gene. Multiple sequence alignment demonstrated the pIgR ofteleosts was composed of two Ig-like domains (ILDs), corresponding to the ILD1andILD5of Mammal’s pIgR. The phylogenetic tree was constructed from the amino acidsequences of pIgR of18vertebrates and indicated that pIgR amino acids in S.maximus were clustered together with P. olivaceus.The expression of pIgR gene was analyzed by semi-quantitative RT-PCR indifferent tissues of flounder, which revealed that pIgR gene was expressed in almost all tissues of healthy S. maximuss, with higher levels in the mucosa-associatedlymphoid tissues, and the expression was highest in skin, gill, stomach and intestine,and lower level in liver, spleen, kidney and muscle, indicating that this gene wasclosely related to transfer of mucus Ig in flounder.. Meanwhile, the dynamics changesof pIgR expression in S. maximu were evaluated following bath immunization withinactivated Vibrio anguillarum by real-time PCR, and the results showed that therelative expression amount increased firstly and then decreased within72h in all thetested tissues and reached its maximum expression within48h and the peak appearedearlier in mucosa-associated lymphoid tissues.The ORF was successfully cloned and expressed in Escherichia coli BL21(DE3).The recombinant protein showed that the molecular mass was58.8kDa in SDS-PAGE.Furthermore, the protein was recombined and polyclonal antibody againstrecombinant pIgR was developed. Western blotting results showed that the polyclonalantibody can react with the recombinant protein antigen.The distribution of pIgR was studied using polyclonal antibody against pIgR inskin, gill, stomach and intestine via immunohistochemical technique. Results showedthat positive signals were found in the epithelial cells of the four tissues, whichindicated that pIgR existed in these tissues, corresponding to its function of bindingand transporting pIgs, and implying the importance of pIgR in mucosal immunity inteleost fish.All the results suggested pIgR played a critical role in the mucosal immunity ofteleost, which probably involved in the pIgs transport. These provided insights intothe roles of fish pIgR in the mucosal immunity.