Cloning, Expression And Monoclonal Antibodies Preparation Of Vav3Gene From Lampetra Japonica

Vav3, a Rho GTPase guanine nucleotide exchange factor, is highly conserved in evolution. Vav3participates in many signal pathways in T and B cells through regulating the Rho members and plays important role in various biological functions, including cell proliferation, differentiation and immune response. Lampreys as the oldest extant jawless vertebrates, has the unique adaptive immune system. Therefore, lampreys are the important model organisms for investigations of early vertebrate evolution and adaptive immune origin. At present, researches focus mainly on Vav3in mammalian, little is known about the existence of Vav3in lampreys. In this study, molecular biology and bioinformatics methods were used to analyze the expressed sequence tags (ESTs) of the leukocytes cDNA library of Japanese lampreys. Cloning, prokaryotic expression and polyclonal antibody preparation were contribute to reveal the origin and evolution of Vav3.Here, an EST homologous to Vav3was found from the leukocytes cDNA library of Japanese lampreys. Full-length cDNA was obtained by RACE, and the ORF was2,511bp, which encodes a protein of837amino acids with a predicted molecular mass of92.07kDa. By the further analyse of primary sequence, sequence alignment and senior structure, three typical tyrosine phosphorylation site were found in Vav3of Japanese lamprey. Phylogenetic tree was constructed by neighbor) oining method to expound the genetic relationship among Japanese lamprey and other vertebrate. The result showed that the evolutionary position of Japanese lamprey Vav3was primitive and it is consistent with biological objective laws of natural evolution.Real-time PCR was used to study the expression level of Vav3in heart, supraneural body, kidney, intestine, liver, gill and leucocyte of Japanese lamprey. Vav3was widely expressed in a variety of organs, and the expression levels in supraneural body, gill and leucocyte were raised significantly after injection with LPS. All these results showed Vav3in Japanese lampreys took part in lymphocytes immune response and pathogen defense. Further more, a255bp fragment of Japanese lamprey Vav3conserved domain was amplified based on the full-length gene. The amplified products were cloned into a pET-32a expression vector. The soluble recombinant protein was expressed in Escherichia.coli BL21(DE3). Then the recombinant protein was purified by histidine affinity chromatography column. Polyclonal antibodies against Vav3recombinant protein were preparation in a New Zealand white rabbit. The expression level of Vav3in leucocyte was detected by flow cytometry. The results further demonstrated that Vav3played an important role in immune response of Japanese lamprey.In conclusion, Vav3may exist in jawless vertebrate, and the speculation of Vav3function provided an important clue for the mechanism of adaptive immune response of jawless vertebrate.