Cloning,Expression,Antibody Production And Immunological Related Research On PAK4 Molecule Of Lampetra Japonica

P21-activated kinases(PAKs)are evolutionarily highly conserved serine/threonine protein kinases that serve as effector proteins for the small guanosine triphosphates of Rho family(Rho GTPases)such as recombinant cell division cycle protein 42(Cdc42)and ras-related C3 botulinum toxin substrate 1(Rac1)are involved in many signaling pathway to mediate signal transduction processes.As one of the representatives of jawless vertebrates,lampreys exist an adaptive immune defense system mediated by variable lymphocyte receptor(VLR),which has received much attention from immunologists.To explore the existance and function of PAK4 molecules in lamprey,we conducted the following research works.In this research,we successfully amplified the open reading frame(ORF)sequence of the lamprey PAK4 gene(Lja-PAK4),which is 2544 bp in length and encodes a protein molecule containing 848 amino acids,by using total RNA from the supraneural myeloid bidies of the Lampetra japonica as a template.This molecule is a cytosolic protein without transmembrane domain and is unstable,containing a P21-binding domain(PBD domain)at the N-terminal and a serine/threonine kinase catalyticdomain domain(S＿TKc domain)at the C-terminal.The phylogenetic analysis of the type Ⅱ of PAK family molecules showed that PAK6 originated earlier than PAK4 and PAK5;and the cloned PAK molecule from l.japonica has a high sequence identity with PAK4 molecules of the type Ⅱ of PAK family in higher vertebrates,so it was named Lja-PAK4.The results above indicated PAK4 homolog exists in lamprey.By antigenic epitope prediction,the amino acid sequence from 151th to 370th was selected as the antigen fragment for truncated expression,and after a small amount of induction mapping conditions;finally the induction expression was conducted at 30℃and IPTG concentration of 1 mmol/L.The truncated Lja-PAK4 protein after induction expression was purified by Ni~+column affinity chromatography,and the Lja-PAK4 murine-derived polyclonal antibody was successfully prepared by using of the purified Lja-PAK4 protein as antigen to immunize the adult healthy rats(Rattus norvegicus).The expression of Lja-PAK4 in the immune tissues and cells of lamprey was detected at the molecular and protein levels by quantitative real-time PCR(qPCR)and Western Blot(WB),and the expression of Lja-PAK4 in various immune tissues and cells stimulated by mixed bacteria showed differential expression at the mRNA and protein levels.It is also up-regulated in peripheral blood lymphocytes and myeloid bodies.In order to further verify whether Lja-PAK4 participated in the immune response of lamprey,the expression of Lja-PAK4 at the molecular and protein levels in immune-related tissues was detected after stimulation of lipopolysaccharide(LPS)and phytohemagglutinin(PHA).The results of qPCR and WB after LPS stimulation showed that Lja-PAK4 showed up-regulated expression in peripheral blood lymphocytes and myeloid bodies,indicating that Lja-PAK4 was involved in the immune response process mediated by VLRB in lamprey.The localization and distribution of Lja-PAK4 and VLRA,VLRB and VLRC in immune-related tissues after stimulation with mixed bacteria were further examined at the cellular level using immunofluorescence assays.It can be seen that the distribution of Lja-PAK4 and VLRB all can be detected in the peripheral blood lymphocytes and supraneural myeloid bodies.In addition,the fluorescence intensity of Lja-PAK4 and VLRB is significantly increased after the stimulation of mixed bacteria,and there is a co-localization phenomenon in the peripheral blood lymphocytes and myeloid bodies.However,the fluorescence signals of VLRA and VLRC can be detected in the gills,but the fluorescence signal of Lja-PAK4 can not be detected.The above results can further illustrate that Lja-PAK4 is involved in the LPS-mediated immune response of VLRB~+lymphocyte subpopulations in peripheral blood lymphocytes and myeloid bodies in lamprey.Several proteins interacting with Lja-PAK4were also identified by immunoprecipitation experiments,and their characterization still need to be further determined.Our study lays the foundation for further in-depth investigation of the Lja-PAK4 involved in immune response of lamprey VLRB~+lymphocyte subpopulation.