Cloning,Recombinant Expression And Related Immunology Of Lja-SHP2 In Japanese Pansy

SHP2 is a protein tyrosine phosphatase encoded by the ptpn11 gene that regulates signal transduction in a variety of signaling pathways in the body.In the past,mammals were mostly used as experimental subjects,and there were few studies in lower vertebrates.This paper used the non-jaw vertebrate hermites as experimental subjects to select and clone molecules that are homologous to the higher vertebrate SHP2,and to study SHP2 molecules.In the process of the immune response of the sea slugs,it is clear that the phylogenetic tree of the SHP2 molecular family provides some clues for exploring the early occurrence and evolution of the adaptive immune system in higher vertebrates.The homologous sequence of SHP2 gene was searched in Lampetra japonica transcriptome database,and the primers were designed according to Primer5 software.Successful Cloning of a Complete Open Reading Frame(ORF)of the Scorpion SHP2 Molecule(Lja-SHP2)Using Nested PCR.In the open reading frame(ORF),the Lja-SHP2 ORF sequence is 1761 bp and encodes a 587 amino acid protein.Multiple sequence alignment display,The L.japonicum Lja-SHP2 molecule has greater than 65% sequence identity from fish to mammalian SHP2 molecules in other higher vertebrates and less than 60% sequence identity with other higher vertebrate SHP1 molecules.By constructing phylogenetic relationships between the SHP1 and SHP2 molecules of each class of representative animals,it was found that Lja-SHP2 molecules and SHP2 molecules from higher vertebrates cluster in the same large cluster,while SHP1 molecules from higher vertebrates are clustered.Another large cluster.In addition,homologous molecules of the SHP1 molecule were not found in other types of sea bream genome databases.These results indicate that the SHP family has not yet differentiated into SHP1 and SHP2 molecules in jawless vertebrates.Inserting restriction sites at both ends of the Lja-SHP2 gene sequence was ligated to the pET-32a(+)vector and transformed into E.coli Rosstte competent cells for expression,resulting in purified Lja-SHP2 soluble recombinant protein(rLja-SHP2).The recombinant protein rLja-SHP2 was used as antigen to immunize New Zealand rabbits(Oryctolagus cuniculus)to prepare polyclonal antibodies.By ELISA analysis,after the fourth booster immunization,the titer of the antibody could reach 1:256000.The purified antibody was highly specific to rLja-SHP2 and the native protein by Weasten blot assay.Vibrio parahaemolyticus and Aeromonas hydrophila were made into inactivated mixed vaccine immune healthy hermits.After two times of booster immunization,the expression of Lja-SHP2 was detected by PCR;In the immune Lja-SHP2 mRNA in white blood cells,sputum and pith in all tissues;after stimulation with mixed bacteria,Lja-SHP2 expression was not significantly different in leukocytes,there was no significant downregulation in the pith tissue,and there was a significant up-regulation in expression in the pupal tissue(p <0.05).It is suggested that Lja-SHP2 is mainly involved in the immune response of iliac tissues after immunostimulation,and it is speculated that it is positively related to the activation of VLRA immune cells.Using siRNA interference technology to knock down Lja-SHP2 molecules in vivo,the results showed that the designed three siRNA mixtures can effectively knock down the transcription of Lja-SHP2 mRNA at the time of immune activation Lampetra japonicum12,24 h.After 36 hours,the repression was reduced,and it basically returned to normal after 48 hours.This result laid the foundation for further exploration of the function of Lja-SHP2 molecule.