Researching Of Cloning, Expression And Monoclonal Antibodies Preparation Of VLRB Gene From Lampetra Japonica

As a representative of jawless vertebrates Lamprey is the ancestor of vertebrates,it is an important species which has an important status of contact invertebrates and vertebrates, so it is in bound to played a very important role in the study of the evolution of vertebrates. As an important immune molecules, VLRB gene's found provides important clues to the evolution of the adaptive immune system, This shows that early in jawless organisms, there is a molecule which is similar to human immune molecules Ig, As the main immune receptors of adaptive immunity, it can identify a variety of different proteins and initiate immune response. To further understand the features and functions of the immune system in lamprey, this study will has VLRB gene cloning, protein expression, and preparation of monoclonal antibodies, and getting VLRB monoclonal antibody, Prepare for further understand of the lamprey adaptive immune system.The main contents are as follows:The experiment was based on published Genbank sequence, design two pairs of primers (conserved regions primers and full-length primer), using reverse transcription - polymerase chain reaction (RT-PCR), full-length gene sequence (1036bp) and the conserved sequence (252bp) were amplified from immuned lamprey, then connect to the pMD-18T vector and transformed into E. coli strain DH5α, positive clones were finally identified by double digestion. The sequencing results show that there is indeed exist VLRB genes in Japanese lamprey. Comparison and analysis of VLR sequence between various types of lampreys and hagfish, showed that they have not only a certain similarity, but also a conserved region of stability.Connected the cloning VLRB gene into the expression vector pET-32a, through DNA sequencing to detect positive clones, then expression in BL12 through IPTG induction. After protein purified, SDS-PAGE analysis showed that VLRB gene expression in E. coli achieve integration, and obtain a certain biological activity protein.The protein mixed with Freund's complete adjuvant and Freund's incomplete adjuvant to immune Balb/c mice, and fused spleen cells from immune mice with SP2/0 myeloma cells using PEG4000. Coated with the corresponding antigen, then screened positive hybridoma through indirect Elisa. By limited dilution subcloning 3 times, until the positive hole of monoclonal cells was 100%. Picked clones were frozen in liquid nitrogen after extended culture. Preparation of a large number of monoclonal antibody through ascites, and then purified by affinity purified. Finally, through Western-blotting to detecte specific of VLRB antibodies.In summary, this study received a large number of lamprey VLRB monoclonal antibodies, antibody preparation provides a crucial test materials for further study of lamprey adaptive immune system.