Analysis On Goose TLR15 Primary Characteristics And Expression Induced By LPS

Toll like receptors(TLRs)are a class of important transmembrane signaling molecules in the innate immune system.They can recognize molecular structures of conserved pathogen-associated molecular patterns(PAMPs)expressed by microorganisms,which have effects in the early phase of infection and provide activating molecules for acquired immunity.TLR15 is a unique pattern recognition receptor in poultry.The study on TLR15 will contribute to the understanding of anti-infection mechanism and provide basic materials for immune preparations.In this study,the TLR15 gene of Northeast White Geese was cloned by reverse transcription PCR,and the structural characteristics,taxonomic status and evolutionary relationship were analyzed by bioinformatics software.First,recombinant protein of Northeast White Goose TLR15 was induced to express after the prokaryotic expression vector was constructed and rabbit anti-goose TLR15 polyclonal antibody was prepared.Secondly,the inflammatory reaction model was induced by lipopolysaccharide(LPS).The transcription level of TLR15 gene and related immune factors in different tissues of Northeast White Goose was analyzed by realtime PCR,and the expression level of TLR15 in different tissues was detected by immunohistochemical technique.The results were as follows:1.The full-length cDNA of Northeast White Goose TLR15 contained an open reading frame(2 598 bp).Prediction of protein domains revealed that the putative amino acid sequence consisted of a signal peptide sequence encompassing the first 24 of 859 amino acid residues,a transmembrane domain at positions 650-672 of the carboxy-terminus.There were 78 phosphorylation sites,of which 13 are located in the LRRs and 9 in the intracellular region.In all,16 glycosylation sites were found.six leucine-rich repeat(LRR)domains,There are 5 variations at positions 357,451,720,1120 and 1 401 in the extracellular fragment(1 845 bp,615 amino acid residues).An "horseshoe" structure is located in the extracellular domain with 6 LRRs.Multiple sequence alignment showed that nucleotide sequence homology between Northeast White Goose and Anseranser was highest,up to 99.85%,and from 40% to 70% with reptiles.The resulting phylogenetic tree was composed of three major branches,gTLR15 were in the same subgroup with sequences from chicken,birds.Reptilian TLR15 was in the another subgroup.2.The molecular weight of recombinant protein was 43 kD,and the titer of polyclonal antibody after immunization was above 1:20 000;The expression of gTLR15 and immune factors(IFN-α,IFN-γ,IL-1β and IL-6)in Northeast White Geese organ tissues were analyzed by quantitative RT-PCR by LPS.The level of experimental group were higher than that of the control group,and the levels of TLR15,IFN-α and IFN-γ mRNA were highest in Bursa of Fabricius(P< 0.01)and the expression levels in the heart and lung were relatively low.The transcriptional level of IL-6 was highest in thymus,but lowest in liver and spleen.It indicated that there are different expression levels of TLR15 and immune factors in inflammatory response.The levels of different factors were also different in the same tissues,IFN and TLR15 were highest in Bursa of Fabricius and IL and TLR15 were highest in bone marrow.The studies of immunohistochemistry showed that the expression of TLR15 was very significant different(P< 0.01)in the Bursa of Fabricius,spleen and liver,and were significant different(P< 0.05)in the lung and kidneys;The results indicated that by LPS,the transcription and translation levels of Northeast White Geese TLR15 gene are consistant.In this study,Northeast white geese TLR15 was cloned,expressed and polyclonal antibody was prepared.The immune function of TLR15 induced by LPS was analyzed.It provides a theoretical basis for the anti-infection mechanism of TLR15 in northeast white geese,and also lays a foundation for the development of immune reagents.