Prokaryotic Expression, Antibodies Preparation And Immunological Studies Of Btk From Lampetra Japonica

Bruton’s tyrosine kinase (Btk) is a cytoplasmic non-receptor tyrosine kinase which is essential for B cell development and function in advanced vertebrates. Btk participates both in adaptive immunity and innate immunity. The innate immune response in vertebrates is the first line for resisting the invading microorganisms. Phagocytes, such as neutrophils, macrophages and dendritic cells, play important roles in the innate immune response. In the adaptive immunity, the most critical function and most studied is the function in B cell signaling.What species did Btk initially play its adaptive immune function in? Homolog of BTK has also been identified in the cartilaginous fish Raja eglanteria, and it is believed to be involved in B-cell development. Recent research suggested the origin of the adaptive immune system dated back to the jawless vertebrate lamprey. The study of jawless vertebrates provides a theoretical basis for the origin of adaptive immunity. Early works in our laboratory had certified that BTK is present in Lampetra japonica. While how Btk function in L. japonica’? This will be a breach to study the adaptive immune system in jawless lamprey.In this paper, a conservative kinase domain of L. japonica Btk was linked with the vector pET-32a. The fusion protein was expressed in the E.coli BL21and purified from it. Rabbit anti-Btk polyclonal antibodies were purified after the rabbit was immuned by L. japonica Btk recombinant protein and some immunological studies were carried out after that. Real-time PCR showed broad distribution of BTK in various immune-associated tissues, and after L. japonica was stimulated with LPS, the level of BTK mRNA was notably up-regulated in leukocytes.Flow cytometric analyses and western blot demonstrated that Btk was expressed in the leukocytes, and the level of Btk in leukocytes was notably up-regulated after L. japonica was stimuated with LPS. FACS results showed that VLRB and Btk present in the same leukocytes population. These results indicated that Btk may play an important role in immune response and may participate in the signaling started by variable lymphocyte receptors (VLR) in L. japonica. These may provide some reference for further studies about the origin and evolution of adaptive immunity.