Cloning, Expression, Antibodies Preparation And Immunological Studies Of IRF From Lampetra Japonica

IRF was first named for its binding and regulation of interferons when infected withvirus. As multiple transcription factor, IRF is involved in regulating transcription of interferon,signal transduction of cytokines, differentiation of hemopoietic stem cells, tumor suppressionand played important roles in immunoregulation during virus infection. This makes IRFbecomes the significant immunoregulator and potential drug target of oncotherapy.The evolutionary status of lamprey is between invertebrates and vertebrates. As therepresentative of the oldest jawless vertebrates, lamprey has the unique evolutionary statusand immune systems. As the special defence mechanism of IRF in vertebrates was identified,what kind of immune responses of Lampetra japonica IRF, whether it has the ability ofinhibiting tumorigenesis? Presently，the study of Lampetra japonica IRF is still unknown. Theprimary research of Lampetra japonica IRF will be a significant research in their origin andevolution.In this study, the length of550bp nucleotide s equences of Lampetra japonica IRF wasamplified. Then the ORF of Lampetra japonica IRF was cloned and the prokaryoticexpression vector was constructed. After prokaryotic expression and recombinant proteinpurification, we got pure IRF protein. New Zealand rabbit was immunized with purifiedprotein to produce anti-IRF polyclonal antibody. The ELISA and Western blot assays weremade to detect the valence and specificity. The result showed that the valence of anti-IRFantidy was1:510000and the antidy has specific recognition ability. Further immunologystudy of IRF was maded. The Western blot result showed that IRF was mainly expressed inheart, gill, kidney and marrow of Lampetra japonica. In addition, Immunohistochemistryresult identified the same result that IRF was distributed in those four tissues. After thelampreys were stimulated with HMGB1, Immunohistochemistry showed that IRF wasexpressed in heart, gill, kidney and marrow. It also expressed in intestine. This indicated thatHMGB1might effected the expression of IRF, IRF might interact with HMGB1in theJak/Stat inflammation signal transduction pathway. Immunoprecipitation was conducted toprecipitate the potential protein that might interacted with IRF. The above data provided animportant clue for the origin and evolution of IRF in adaptive immunity and the speculationof IRF function.