Molecular Cloning And Characterization Of Toll-like Receptor 22 In Common Carp, Cyprinus Carpio L.

Toll receptors were first characterized in Drosophila melanogaster and have a role in both embryonic patterning and innate immunity. More recently, a related family of Toll-like receptors (TLRs) was discovered encoded in vertebrate genomes. TLRs have a central role in innate immunity and are also required for the development of an adaptive immune response, bridging innate and adaptive immunity. And the interleukin-1 receptor/toll-like receptor (IL-1R/TLR) superfamily signaling involves myeloid differentiation factor 88 (MyD88) that acts as an important adapter protein in TLRs singaling pathway.In this paper, we have isolated, sequenced and characterized the full-length cDNA of TLR22 from common carp by the method of molecular cloning and rapid-amplification of cDNA ends (RACE) as well as studied their expression pattern in tissues in relatively health common carp by RT-PCR; TLR22 gene and MyD88 gene (cloned by our lab) expression level were detected by real-time quantitative PCR in common carp which were stimulated by Vibrio anguillarum; TLR22 gene, which encodes partial TLR22 protein of common carp, was cloned by the method of recombinant technics. The recombinant expression vector pET-28a(+)/TLR22 was constructed and E.coli BL21(DE3) was transformed to express the TLR22 protein. The protein was purified and used as an antigen to immune animals to gain the antiserum. The results are showed as follows:(1) The cloned TLR22 cDNA exhibited 3325bp in length and contains a 56bp 5'-untranslated region (UTR), a 2838bp open reading frame (ORF) and a 431bp 3'-UTR. TLR22 with 945 amino acids has a calculated molecular mass of 108.24kDa and a theoretical pI of 8.733, has a putative signal secretion peptide 22aa in its N-terminal region. Structurally, TLR22 has a Toll/interleukin-receptor (TIR) domain, a transmembrane domain and leucine-rich repeat (LRR) domain which are the hallnarks of TLR family. TLR22 and MyD88 non-constitutively detectable in various tissues and organs selected, both are highly expressed in the hindgut, spleen, kidney, gill and smouth epithelium; moderately expressed in the foregut and skin; and not expressed in musle. Protein organization, multiple alignment and signal peptide prediction shows that TLR22 shares highly identity with known TLR22 members, which achieves to 88.6% with goldfish TLR22.(2) Expression of TLR22 and MyD88 were measured in previously non-exposed fish, 0.5, 1, 3, 5, and 7 days after experimental Vibrio anguillarum challenge to determine if they are associated with host response to V. anguillarum infection by real-time quantitative PCR. The mRNA copy numbers of carp TLR22 and MyD88 were increased in some organs including intestine, gill and skin. These results imply that TLR22 and MyD88 have an important role in the mucosal immunity in common carp.(3) The first strand of cDNA was served as the template to amplify part of the gene TLR22. The product of amplification was treated by double endonuclease digestion (EcoRâ… and Hindâ…¢) and linked to pET-28a(+) expression vector. The recombinant expression vector pET-28a(+)/TLR22 was then transformed into E.coli BL21(DE3). SDS-PAGE results showed that a protein with a molocular weight of 38.17kDa was expressed within the host strain. The expressed TLR22 protein was purified primarily and the concentration of TLR22 was adjusted for later use as an antigen. Antiserum was gained from animal immunization, then double agar diffusion experiments and Western blotting were conducted afterwards. And the titer of the antiserum was measured as 1:4 according to the results of double agar diffusion experiments.