Gene Cloning And Bioinformatics Analysis Of Non-receptor Protein Tyrosine Kinase BTK From Lamprey

Content: Bruton's agammaglobulinaemia tyrosine kinase belongs to a subfamily of non-receptor protein tyrosine kinases.It is characterized by five structural modules: Pleckstrin homology domain,Tec homology domain, Src homology3 domain,Src homology2 domain and Tyrosine kinase domain.BTK is thought to be a type of key signal molecules in various signal pathways, it plays a crucial role in immune system. Recently mechanism of how BTK plays an crucial role in B cell signaling pathway,TLR signaling pathway and mast cells degranulation is a hotspot.BTK plays important role in regulating cell proliferation, differentiation and apoptosis. Mutation of BTK could cause X-linked agammaglobulinemia, which clinically characterized by recurrent bacterial infections and markedly reduced numbers of B cells .In this work,we collected the leucocytes of lamprey, extracted the total RNA, measure concentration of RNA by ultraviolet spectrophotometer and agarose gel electrophoresis.We have successfully cloned BTK gene using RACE method and BTK gene was constructed into pET32a(+) vector. Then we study on primary structure alignment, advanced structure prediction,reconstructing a phylogenetic tree and conserved motifs distribution of this sequence.These mainly results were as follow:1 The intercomparison results of primary structure shows that its amino acid sequences are very conservative with other species,such as chimpanzee,human,horse,cattle,mouse,dog, ornithorhynchus,chicken,zebra fish,hagfish and drosophila and so on.2 We predicted the advanced structure of BTK amino acid from lamprey using the MEME system.It included the prediction result of PH domain advanced structure, the prediction result of SH3 domain advanced structure and the prediction result of kinase domain advanced structure.The prediction results show that advanced structure of Lamprey BTK is similar to Temple BTK.3 The MEME system shows that amino acid sequences of Lamprey BTK contains 10 conserved motifs totally.4 The phylogenetic tree contains two clusters:one including mammals, birds, fishes and lamprey,the other including hagfish and insects. These above results demonstrate that non-receptor protein tyrosine kinase BTK may exist in lampreys, the precise immune mechanism of lamprey remained to be elucidated.