| Toll like receptor is a natural immune factor.In the early stage of infection,it can play an anti infection role by identifying pathogen related molecular patterns and initiating signal transmission,so as to eliminate the invasion of pathogenic microorganisms.Goose is one of the more waterfowl in China.Because of the influence of the growing environment,it is easy to have infectious diseases such as avian influenza.The existing vaccines can not provide all the protective effects,so it has become a research hotspot to enhance the disease resistance from the perspective of natural immunity.At present,TLRs are widely studied in mammals,but less in poultry,especially in geese.In this study,the extracellular region of TLR4 gene in Sanhua goose was cloned and sequenced.Under the induction of H5 subtype avian influenza vaccine,the expression of TLR4 and related immune factors in Sanhua goose was analyzed by immunohistochemistry and semi quantitative PCR.The results are as follows:(1)The extracellular region of TLR4 gene of Sanhua goose was cloned,which was998 bp in size and encoded 304 amino acid residues.The analysis of nucleotide and amino acid evolutionary tree showed that TLR4 of Sanhua goose had the highest homology with Langde goose and Magang goose,and it was more conservative in poultry,clustered into a large branch,and had low homology with mammals and fish,with species specificity.(2)The recombinant expression vector of TLR4 extracellular region of Sanhua goose was constructed,and the recombinant protein was about 38 kD in size.The Rabbit anti TLR4 extracellular region polyclonal antibody was obtained by immunizing rabbits with specific protein obtained by gel cutting purification method.The titer of serum was 1:10~4 by ELISA,and the specific TLR4 band was confirmed by Western Blot.(3)The extracellular domain of TLR4 protein in goose was analyzed by bioinformatics software.The extracellular domain was composed of six leucine repeats LRR with signal peptide,belonging to secretory protein,with 30 phosphorylation sites.The secondary structure was mainly composed ofα-helix and irregular convolution.(4)Under the induction of H5 subtype avian influenza vaccine,semi quantitative PCR was used to detect the transcription level of TLR4 and related immune factors mRNA in different tissues of Sanhua goose.The results showed that the expression of TLR4 in spleen,bone marrow,heart and thymus of experimental group was higher than that of control group,and the control group in the lung and bursa was higher than that in the experimental group;the expression of IL-1β,IL-6 and IFN-γof experimental group was higher than that of control group;the expression of IFN-αin bursa of Fabricius of experimental group was slightly lower than that of control group,other tissues of experimental group was higher expression than that of control group.The results showed that TLR4 and related immune factors were activated and expressed after H5subtype Vaccine Immunized geese,which participated in the process of anti infection.(5)The expression of TLR4 Protein in different tissues was detected by immunohistochemistry method under the induction of H5 subtype avian influenza vaccine.The results showed that the expression of TLR4 Protein in spleen was the highest.In heart,liver and kidney,the expression of TLR4 Protein in experimental group was higher than that in control group.In lung and bursa of Fabricius,the expression of TLR4 Protein in control group was higher than that in experimental group.To sum up,the extracellular domain of TLR4 gene of Sanhua goose was cloned and the recombinant protein and polyclonal antibody were obtained.The expression of TLR4 and related immune factors in different tissues of Sanhua goose were analyzed under the induction of H5 subtype avian influenza virus vaccine.This study laid a foundation for the study of immune function of TLR4 in geese,and also provided a theoretical basis for the analysis of TLR signal transduction pathway in poultry. |
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