Study On The Mechanism Of TLR2 And Relatedimmune Factors In Duck After Immunizationwith Avian Influenza Vaccine

Toll-like receptors(TLRs)are an important pattern recognition receptor,which can initiate innate immunity by recognizing pathogen,and are also associated with acquired immunity.It plays a role in resisting pathogenic microorganism.Different pathogens are recognized by TLRs.TLR2 recognizes a wide range of ligands.The studies on duck TLR2 have been a hotspot in birds.In this study,the extracellular domain of TLR2 was cloned and expressed in prokaryotica vector,polyclonal antibody was prepared.The expression and tissue localization of TLR2 were analyzed in the lv-tou duck after immunization with avian influenza vaccine.The results are as follows:Firstly,based on the published sequence of extracellular domain of TLR2 gene from peking duck,a pair of specific primers were designed,and total RNA was obtained from duck spleen tissue.The extracellular domain of TLR2 was amplified by RT-PCR,and aquired 1788 bp.The target gene was inserted to the pEASY-T1 cloning vector.After the positive recombinant cloning plasmid was sequenced,and there are 100%identity between the extracellular sequence of lv-dou duck TLR2 and the same sequence of the Peking duck TLR2.It indicated extracellular sequence of duck TLR2 was obtained successfully.positive recombinant cloning plasmid was obtained after the extracellular domain was ligated with pET-32 a prokaryotic expression vector.The protein was induced by IPTG and detected by SDS-PAGE,the specific recombinant protein was detected,at 110 KDa.The recombinant protein was mixed with Freund's complete adjuvant to prepare immunogen,and rabbits were immunized to obtain a rabbit polyclonal antibody anti-duck TLR2 extracellular domain.A 110 KDa hybridization band was obtained by Western blot,indicating the protein is a gene expression product of the extracellular domain of duck TLR2.The expression of TLR2 mRNA in the heart,liver,spleen,lung and thymus tissues of the experimental group was known as Avian influenza vaccine immunization and the control group was detected by semi-quantitative PCR.The results showed TLR2 in the experimental group was higher than that in the control group.TLR2 is highly expressed in the spleen,expressed moderately in the thymus,liver,andlung,and expressed lowly in the heart.The IFN-? mRNA in the experimental group was higher than that in the control group,and IFN-? in the spleen was also expressed highly,expressed moderately in the thymus and liver,and expressed lowly in the heart and lung.It indicated that the expression of TLR2 and IFN-? increased in five tissues after immunization with avian influenza vaccine,and TLR2 was located mainly in the spleen.The expression of TLR2 was detected by immunohistochemistry in spleen,heart and liver.The expression of TLR2 in ducks in the experimental group was higher than that in the control group.The spleen was the main part of TLR2 produced by Image pro software,and it was mainly distributed in the cytoplasm and cell membrane.These results indicated that the expression of TLR2 and IFN-? in Lv-tou duck increased after immunization with avian influenza vaccine.TLR2 played an important anti-infective role in the immune response to antigen invasion.The analysis of these factors put a foundation for further study of its function,and also provide a theoretical basis for prevention and treatment of duck-related diseases.