Cloning,Recombinant Expression And Related Immunology Of BCAP In Lampetra Japonica

B cell adaptor protein(BCAP)is also called PIK3AP1(phosphoinositide-3-kinase adaptor protein 1),BCAP contains four YxxM motifs.When the BCR signaling is activated,Tyr phosphorylation on the YxxM motif can recruit the P85?subunit of Phosphoinositide3-kinase(PI3K)and activate PI3K.The activated PI3K can mediate phosphorylation of its downstream kinase Akt and then Akt translocates into the nucleus resulting in the regulation of the transcription and expression of genes and ultimately in the regulation of cell proliferation,survival,metabolism,cytoskeletal reorganization and membrane trafficking.Lamprey is a model organism with high research value because it is the representative of the most primitive jawless vertebrate that is evolutionarily connection of vertebrates and invertebrates.At the same time,it not only has an innate immune system,but also has an adaptive immune system.Whether there is a BCAP homolog or not and what function it played in jawless vertebrates have not been reported yet.In the present study,a BCAP like gene was first discovered in Lampetra japonica,and the cDNA of lamprey BCAP gene was cloned by nested PCR amplification technology high-fidelity enzyme.The open reading frame of BCAP molecule of L.japonica(Lja-BCAP)is 2181 bp in length and encodes a protein containing 726 amino acid residues.By alignment of the protein sequences of several species of vertebrate s'BCAP molecules,the sequence identity of Lja-BCAP is about 34%with the BCAP molecules of the jawed vertebrates.In order to further understand their evolutionary relationship,the method of constructing phylogenetic tree was used.The results showed that the Lja-BCAP of the jawless vertebrate L.japonica is close to those of fishes.To better understand the role of Lja-BCAP played in immune regulation,we have prepared a rabbit-derived Lja-BCAP polyclonal antibody.Firstly,the recombinant plasmid of pET-32a(+)-Lja-BCAP was constructed,and the recombinant plasmid was transformed into expressing bacterium E.coli Rosetta for inducing protein expression.The purified Lja-BCAP protein was used as antigen to stimulate the generation of antibodies in New Zealand rabbits.The antiserum was isolated and purified to obtain a rabbit anti-Lja-BCAP polyclonal antibody with better specificity.In vivo interference knockdown experiments were performed on Lja-BCAP molecules using siRNA interference technology.The results showed that in the lymphocytes and supraneural myeloid bodies,the transcription levels of Lja-BCAP mRNA were effectively knocked down at 12-36 h after antigen stimulation by a siRNA pool containing three groups of siRNA mixtures.This result lays a foundation for further exploration of the function of Lja-BCAP molecules.Reverse transcription PCR and Western blotting experiments were used to detect the mRNA transcription and protein translation levels of Lja-BCAP.The results showed that the mRNA of Lja-BCAP was expressed in the lymphocytes,gills and supraneural myeloid bodies before immunization,and the expression level in lymphocytes is significantly higher than in the gills and supraneural myeloid bodies Lja-BCAP mRNA and protein levels showed significant up-regulation in lymphocytes after 36 h of PHA immune stimulation.respectively.These results demonstrate that Lja-BCAP may be involved in the immune response of lamprey VLRB~+lymphocytes(B-like cells).Our results lay a foundation for further study of the role of BCAP molecules in the immune response of the lamprey VLRB~+lymphocytes.