Molecular Cloning Of Common Carp Toll-like Receptor5and Its Responses Against Aeromonas Hydrophila Infection

The immune system of fish is relatively primitive, and compared withmammals, the studies about their molecular mechanisms of immune system arefewer, they depend primarily on the innate immune responses, which are mostlymediated by different kinds of PRRs. For each PAMPs and DAMPs, TLRs alwayshave specific ligands whether in vertebrate or in mammal, they are importantmoleculars which could bridge innate and adaptive immune, with a broad spectrumof biological activity. TLR5is one kind of PRRs that plays a vital role in protectingfish against bacterial infection, it can recognize flagellin in mammals and fish,which is a component of gram negative bacterial flagella and responsible forflagellin-mediated NF-kB activation[1]. At the same time, TLR5palys a significancerole in adaptive immune system by causing the differentiation of T helper cells,helping natural B cells transform into immunoglobulin A, and improving theproduction of plasma cells[2]. There are two TLR5variants in fish: TLR5M andTLR5S. A significant feature between the variants is that TLR5S has notransmembrane domain.In this study, TLR5M of common carp as research object was cloned based on itsEST sequences with the rapid amplification of cDNA ends (RACE) method, thus, thegenomic sequence including intron was obtained. Then, according to the cDNAsequence of TLR5M, the primers were designed and they were used in Real TimePCR to analyse TLR5M expression level in peripheral blood leucocytes which wereseparated from carp and stimulated with mitogen. At last, Aeromonas Hydrophila wasused to infect health common carp, after12h, total RNA from kidney, muscle, brain,spleen, gills and liver was extracted to analyse TLR5M expression changes. Theresults showed that TLR5M cDNA was3182bp in length, this sequence wassubmitted to GenBank and assigned the accession number KC347572. Sequenceanalysis showed that the full-lenth cDNA sequence contained a2658bp open readingframe, which encoded a protein of885amino acids (aa). The5′and3′untranslatedregions (UTRs) were38and486bp, respectively. Wherein,3′UTR contained a typical poly (A) structure and three polyadenylation signal AATAAA. Comparing thecDNA sequence of TLR5M and its genomic sequence, the results showed that theentire coding region of the TLR5M gene contained a single exon, this may indicatedthat the process of gene could express without intron involved. Real Time PCR resultsshowed that TLR5M transcriptional level in peripheral blood leucocytes wasup-regulated after mitogen stimulation, especially with LPS, but as the time went onthe amplitude was gradully decline. With different stimulant, the transcriptional levelof TLR5M was changed, this maybe indicated that TLR5M play a vital role in earlyimmune system in fish. The induction of TLR5M was analyzed in various organsinfected with Aeromonas hydrophila. In immune tissue like kidney, spleen and gill,the gene expression level was significant increased. In the liver and musle, theexpression variation was very little. This suggested that after bacterial stimulation,TLR5M gene could improve its expression level in a number of organizations,particularly in immune tissues to participate in immune course.