Polymeric Immunoglobulin Receptor (pIgR) In Grass Carp (Ctenopharyngodon Idellus): Genecloning,expression Analysis And Binding Activity With IgM

Mucosal immune system,as the first line of immune defense,can recognize and neutralize pathogens,which plays an important role in immune defense.Polymeric immunoglobulin receptor(pIgR)is an important immune molecular in vertebrate mucosal immune system.Grass carp,Ctenopharyngodon idellus,is an important commercial fish,which suffers morediseases in recent years.In this study,gene cloning and expressing analysis of pIgR,as well as combining with IgM were studied in grass carp,the aim is to clear molecular characteristics and expression difference in grass carp and to provide some basic knowledge to improvethe immune level.In this study,the pIgR cDNA complete sequence was achieved through RT-PCR,3'RACE and 5'RACE,with the full-length sequence of1667 bp,which contains a 1008 bp open reading frame(Open reading frame,ORF),encoding a 336 amino acid,5 'untranslated region is 77 bp,and 3' untranslated region is 579 bp.The deduced pIgR amino acid sequence consists of extracellular domain,a transmembrane domain,intracellular domain,with the predicted molecular weight of 37.22 kDa,the signal peptide of 22 amino acids and two ILDs inthe extracellular domainBased on the extracted total RNA,of eight kinds of tissue(skin,muscle,gill,liver,spleen,intestine,head kidney andheart),using real-time quantities PCR to research the pIgR mRNA expression in different tissues.The result showed that the mRNA expression level ofpIgR was the highest in intestine,followed by higher expression in spleen,and it also expressed in skin,gill,liver and head kidney and muscle.It was suggested that the intestineenrich the more intestinal mucosa and the spleen is the main peripheral immune organs infish,therefore,the expression levels of pIgR will be higher.We constructed the pIgR prokaryotic expression vector pET-32 a,to express pIgR encoding region,and the recombinant proteins pIgR was induced to express using Rosetta E.coli expression system.The recombinant pIgR protein was purified to be used to make polyclonal antibody by injecting mice.The pcDNA3.1-pIgR eukaryotic expression vectorwas built and transfected into grass carp ovary cell line GCO(Grass carp ovary).The expression of pIgR protein was detectedusing the polyclonal antibody(mouse anti fish)in this study as the first antibody,and goat anti-mouse antibodylabeledby FITC as secondary antibody.Using the grass carp natural IgM and the antibody(rabbit anti grass carp IgM),the activity of pIgR to bind IgM was detected in the pcDNA3.1-pIgR transfected in GCO.