Molecular Cloning,Expression Pattern,Antibody Production And Immunological Studies Of The Lja-NICIR In Lamprey,Lampetra Japonica

In higher vertebrates,there are humoral and cellular immune pathways,which are respectively mediated by B cells and T cells.The immunoreceptor tyrosine-based activation motif(ITAM)is present on the B cell receptor(BCR)complex CD79a/b,and the tyrosine residue on the ITAMS motif is transiently phosphorylated,which can generate a temporary binding site to activate internal signal molecule to achieve transmembrane signal transduction of BCR.Although there is no adaptive immune system dominated by TCR,BCR and MHC has been found in jawless vertebrate,they form another type of adaptive immune system through the novel variable lymphocyte receptor(VLR)which can product a diverse array of antigen recognition receptor molecules required by the immune system.However,the molecular mechanism of how VLR receptors achieve transmembrane signal transduction has never been reported.In the current study,a novel ITAM-containing immunoglobulin superfamily receptor(NICIR)was first discovered in Lamprey japonica,and molecularly cloned to obtain its complete open reading frame(ORF)named Lja-NICIR.The ORF of Lja-NICIR is 864 bp and encodes a protein of 267 amino acids.The Lja-NICIR restriction enzyme cleavage sites were analyzed,and the expression vector pET-32a(+)was used to construct a recombinant plasmid,which was transformed into E.coli Rosstte competent cells for prokaryotic expression of Lja-NICIR protein.The recombinant protein of Lja-NICIR(rLja-NICIR)was purified as soluble protein.New Zealand rabbit(Oryctolagus cuniculus)were boosted several times by using rLja-NICIR as an antigen to prepare high titer polyclonal antibodies.The purified antibody has strong specificity for the rLja-NICIR and the natural protein in the tissue by immunoblotting assays.The siRNA interference method was used to knockdown the mRNA transcription of Lja-NICIR gene in vivo.The results of reverse transcription PCR detection showed that the transcription levels of Lja-NICIR mRNA in mononeuclear leukocytes was knocked down in 48 h,and the lowest knockdown effect was greater than 50%.The transcriptional level of Lja-NICIR gene in the myeloid body was not significantly knocked down.It is possible that the reaction of scorpion venom inhibited knockdown.The mRNA transcription level of Lja-NICIR gene in gill tissue was knocked down from 12 h to 24 h.The expression level of Lja-NICIR protein in mononeuclear leukocytes and myeloid bodies was also decreased at 24 h after siRNA interference.These results indicated that siRNA interference method is usable in Lja-NICIR gene knockdown in vivo.After LPS and PHA immunization,the transcription levels of Lja-NICIR molecules in mononeuclear leukocytes,myeloid bodies,and gill tissues were upregulated.The Lja-NICIR protein expression levels were increased in mononeuclear leukocytes and myeloid bodies 24 h post-immunization by LPS and PHA.No expression of Lja-NICIR protein was detected in the gill tissues.These results indicate that Lja-NICIR should be involved in VLRB mediated immunoreaction.Our results lay a basic foundation for further exploration of the function of the Lja-NICIR molecule.