Prokaryotic Expression,Antibodies Preparation And Immunohistochemical Studies Of CD82 Molecule In Lamprey,Lampetra Japonica

CD82 is a member of the superfamily of type ? four transmembrane(TM)glycoprotein and widely expressed in many tissues of human body.It participates in a series of physiological processes,such as cellar migration,cell adhesion and signaling.Originally,CD82 is identified as an accessory molecule in T cell activation,which participates in the formation of immune synapse both in T cells and antigen-presenting cells(APCs)in jawed vetebrates.The lamprey CD82 cDNA sequence have been achieved in our lab.In the present study,we firstly analysed and compared the CD82 and CD37 molecules with the help of bioinformation measures,by which the lamprey CD82 we obtained was identifided really belonging to vetebrates CD82 molecule.The pCold TF is a cold shock vector with a trigger factor(TF)and was modified by short deletion processes based on Overlap PCR technique with primers,which is named GpCold TF.Then,we built prokaryotic expression vectors GpCold TF-CD82 and successfully expressed in E.coli BL21.The soluble recombinant protein was purified by histidine affinity chromatography column.And the recombinant CD82 protein removed of TF was obtained by digesting the recombinant protein with protein enzyme Factor Xa.Here,the specific antigenic epitope of CD82 was synthetized and polyclonal antibodies against CD82 was preparation by stimulating a New Zealand rabbit with the antigenic epitope.Through Western blotting,the expression level of CD82 in lymphocyte-like cells,gills,supraneural myeloid bodies,heart,liver and kidneys of lampreys before and after mixed-antigens stimulation and at different time after second stimulation were also deteced.The ?-actin was as control.The results showed that the natural CD82 protein was stablely expressed in lymphocyte-like cells,gills,supraneural myeloid bodies and kidneys and its expression were increased significantly in lymphocyte-like cells,gills and supraneural myeloid after stimulation.And the expression of CD82 in lymphocyte-like cells,gills and supraneural myeloid were highest at 12 h,6h and 12 h after second stimulation respectively.These results indicate that CD82 can participate in the immune response of lamprey lymphocyte-like cells and play a role in defense mechanism.Further more,immunofluorescent and confocal microscopy were used to determine the distribution and location of CD82 in lamprey lymphocyte-like cells.The results reveal that CD82 distributes mainly on the cytomembrane and slightly in the cytoplasm.After stimulation,the expressionof CD82 is up-regulated and the distribution pattern tends to be clustered beneath the cell surface of cells.The results further illustrates that CD82 may play an important role in immune response of lamprey.In conlusion,CD82 can paticipate in the immune response of the existing and most ancient jawless vertebrate,which provides us the clue to better explore the origin and evolution of adaptive immune system in ancient vetebrate from the protein level and lays the foundation for further studing the specific functions of CD82 in the immune response of jawless vertebrate.