Cloning,Recombinant Expression And Related Immunology Of Syk In Japanese Pansy

Syk is a protein tyrosine kinase consisting of 629 amino acids and is involved in the signaling of various signaling pathways in the body.In the past,most of the researches on Syk were selected from higher mammals.So far,Syk has been rarely studied in lower vertebrates,because Qiqi is not only an important representative of the existing lower jawless vertebrates.And it is also an important group of invertebrates that have a bearing on the evolution of vertebrates.Therefore,this paper selects the scorpion-free vertebrate of Qijing as the research object,and explores the role of Laj-Syk molecule in the immune response of the seven ticks.The purpose is to reveal the molecular evolution of Lja-Syk molecules from low to high vertebrates,and to further lay the experimental foundation for understanding the molecular mechanism of immune responses in non-mandibular vertebrate.Based on the research of this experiment,the Syk truncated sequence was constructed to express the expression vector,and the soluble recombinant protein of Lja-Syk was obtained after purification.Then,the rabbit polyclonal antibody against Lja-Syk was prepared by subcutaneous injection of New Zealand rabbit(Oryctolagus cuniculus)with the recombinant protein Lja-Syk as antigen.The ELISA method was used to detect and analyze the antibody,and the titer of the antibody reached 1:521 000 after the third booster immunization.The purified antibody was tested by Western blotting to have strong specificity for Lja-Syk recombinant protein and natural protein.To further understand the function of Lja-Syk,we examined the expression profile of Lja-Syk in immune-related tissues with changes in immune response time at mRNA and protein levels by means of reverse transcription PCR and Western blotting.The results of reverse transcription-PCR and immunoblotting showed that Lja-Syk was up-regulated in mononuclear leukocytes and myeloid bodies after stimulation by mixed bacteria and LPS,indicating that Lja-Syk molecules are involved in the immunization of scorpion Respond to the reaction.Since the VLRB+ lymphocyte subsets develop in the myeloid bodies,we speculate that the Lja-Syk molecules are mainly involved in the immune response associated with the VLRB+ receptor.The siRNA interference technique was used to perform the seven-in vivo interference knockdown assay on Lja-Syk molecules.The results showed that the designed three siRNA mixtures could effectively knock down the transcription of Lja-Syk mRNA at the time of immune activation at 7 h.Protein translation,but the repression was reduced after 36 h,and returned to normal after 48 h.This result lays the foundation for further exploration of the function of the Lja-Syk molecule.