Cloning,Expression,Antibody Production And Immunological Related Research On PAK1 Molecule Of Lampetra Japonica

Adaptive immunity and innate immunity,as two immune pathways in vertebrates,carry the role of the body’s defense and protection.T cell receptor(TCR),B cell receptor(BCR)and the main histocompatibility complex(MHC)play major roles in the adaptive immune system of jawed vertebrates.p21-activated kinase 1(PAK1)is a class of serine/threonine kinases,and previous studies have found that the noncatalytic region of tyrosine kinase adaptor protein 1(NCK1)can act as a linker protein for TCR and cytoskeleton.PAK1,on the other hand,has been shown to participate in the TCR signaling pathway by binding to the second SH3 domain on NCK through the first proline-rich SH3 binding motif at its amino terminus.Through previous research,we discovered a PAK1 molecule for the first time in the transcriptome of Lampetra japonica,and named it Lja-PAK1 after bioinformatics analysis.The ORF region of Lja-PAK1 totaled 1497 bp,encoding and translating 498 amino acids,and it was found that the functional domain of this molecule was also consistent with the PAK1molecular family of higher vertebrates,consisting of the P21 binding domain(PBD)and the serine/threonine kinase catalytic domain(S＿TKc).By comparing the amino acid sequence of Lja-PAK1 with the PAK1,PAK2 and PAK3 sequences of some vertebrates and invertebrates and constructing an evolutionary tree,it was found that the sequence identity among Lja-PAK1 and PAK1 molecules of higher vertebrates was significantly higher than those among Lja-PAK1 and PAK2 molecules or PAK3 molecules of higher vertebrates.It could be clearly seen from the genetic distance that its relationship was closer to other PAK1molecules in the evolutionary tree.And its affinity with PAK1 homolog of sea lamprey is closest,so we have determined that this PAK1 homologous gene exists in jawless vertebrate lamprey.In order to prepare antibodies to Lja-PAK1 protein,we analyzed the digestion site of the cDNA sequence of the Lja-PAK1 molecule,and successfully constructed a recombinant plasmid using pET-32a(+)expression vector.The recombinant plasmid was introduced into E.coli Rosetta competent cells and induced their prokaryotic expression,and the Lja-PAK1recombinant protein was purified using Ni~+column affinity chromatography.Polyclonal antibodies were prepared by multiple immunostimulated rats(Rattus norvegicus)using purified protein as antigens,and murine anti-Lja-PAK1 polyclonal antibodies with a titer of1:16 000 were obtained.In order to investigate the role of Lja-PAK1 in the immune pathway of jawless vertebrates lamprey,the relative expression of Lja-PAK1 gene in the immune-related tissues of Japanese lamprey eel after bacterial stimulation was detected at the transcription level and protein level using real-time quantitative PCR(qPCR)and Western blotting assay(WB)experimental methods,respectively.Through the results of qPCR,we found that the relative expression of Lja-PAK1 in lymphocytes,gills and myeloid bodies was significantly upregulated compared with the blank control group under the stimulation of mixed bacteria;Through analysis,the results of Western blotting were also consistent with qPCR,indicating that the Lja-PAK1 molecule of lamprey was involved in the antibacterial immune response of lamprey.In this study,qPCR and western blot experiments were used to determine the changes in the expression of Lja-PAK1 at the transcription level and protein level after immune stimulation by phytohemagglutinin(PHA)and B-cell mitopolysaccharide(LPS),respectively,to confirm which lymphocyte group immune response response in the adaptive immune system of Japanese lamprey.Through the analysis of experimental results,it was found that Lja-PAK1 may participate in the immune response in VLRB~+c-ells.Through immunofluorescence experiments,Lja-PAK1 and VLRB molecules were colocalized,and the mixed flora had a significant effect on this colocalization phenomenon.Finally,through co-immunoprecipitation experiments,it was found that Lja-PAK1 also interacted with several other proteins,but further research was needed for the identification of protein types.Our research has laid an important foundation for later exploring the mechanism of Lja-PAK1molecule involvement in the immune response response of VLRA~+,VLRB~+and VLRC~+lymphocyte subsets.