Cloning,Expression,antibody Preparation And Immunological Study Of Vav3 Homolog From Lampetra Japonica

The most important functions of Vav family proteins are as the guanosine nucleotide exchange factor of Rho/Rac protein.Vav guanine nucleotide exchange factor 3(Vav3),a member of Rho family GTPase,is widely expressed in various tissues,and regulates a variety of cellular signal transduction pathway in vertebrates,including of the T and B cell receptor signaling mediated by Rho family.Thus far,the functions of Vav3 are more deeply studied in higher vertebrates,but little is known in jawless vertebrates.Lamprey is an important representative model animal of lower jawless vertebrates.Thus,the current study aimed to reveal the existence and the function of Vav3 through the cloning of Vav3 open reading frame of Lampetra japonica,prokaryotic expression of Vav3 protein,and preparation of antibody of lamprey Vav3.Our results will lay basic foundation for in-depth understanding of immune response of lamprey lymphocyte-like cells.The open reading frame of Vav3 gene of L.japonica(lja-Vav3)is 2568 bp long in length,encoding a protein of 855 amino acids,with 8 typical conserved domains of Vav family protein.Through sequence analysis of 26 protein sequences of Vav3s and Vav2s,Lja-Vav3possesses higher sequence similarity with jawed vertebrates'Vav3s(about 53%)than with the jawed and jawless vertebrates'Vav2s(about 51%).To further understand the evolutionary relationship of Vav2 and Vav3 molecules,the methods of phylogenetic tree reconstruction and conserved motif analysis were used.The results showed that the most significant differences of 8 conserved domains in Vav3 and Vav2 protein sequences were reflected in their SH3 domains.Our results revealed that Vav3 and Vav2 may derive from a common ancestor through a gene duplication event,and devolved into two different molecules under the selection pressure of specificity of their substrates.In order to betterly understand the Vav3 function in immune regulation,the polyclonal antibody of Lja-Vav3 was prepared.Firstly,the Vav3-pET32a(+)recombinant plasmid was constructed,and the recombinant plasmid was transformed into the expression host strain of E.Coli BL21 and the recombinant protein was expressed by induction with IPTG and purified,the purified Lja-Vav3 recombinant protein was used as antigen to stimulate New Zealand rabbits for antibody production.The blood was taken from carotid artery,the antiserum was isolated and the high specificity rabbit polyclonal antibody was purified by affinity histochemistry.The relative expression levels of lja-vav3 were detected at gene transcriptional level and protein level by real-time quantitative PCR and Western blotting methods.The results showed that the lja-vav3 in white blood cells and myeloid bodies upregulated significantly in mRNA and protein level after immune stimulation.In addition,the relative expression level of Lja-Vav3 was upregulated after stemultion by LPS,while it was not changed obviously after stimulation by PHA.Our results indicated that lja-vav3 may be involved in the process of immune response of lamprey VLRB~+lymphocytes(B-like cells)and has laid a basic foundation for further study of lamprey cell signaling pathway.