Protein Expression,Antibodies Preparation And Immunological Research Of CD81 In Lamprey,Lampetra Japonica

CD81 is a member of the tetraspanin family and widly express on the surface,it is an important bridge to communicate the cell signals inside and outside.CD81,on the molecular comples,exists in the surface of B cell,and relates with CD4 and CD8 on the surface in the T cell,it defines the maturation stages of immunological synapses and plays an important role in innate immunity and adaptive immunity.CD81 is the receptor of HCV,because of CD81 has the binding sites of HCV-E2.Therefore,CD81 become the target site in studying HCV and provide scientific basis for prevention and treatment.Now,studies of CD81 mainly limited in higher vertebrates and had few researches in the ancient vertebrates.Lamprey is a jawed vertebrate and regard as an important animal in research the lower vertebrates for a long time.It has been extensive concerned in its variable lymphocyte receptors(VLRA VLRB VLRC),VLRs may be the origin of adaptive immunity.So,to study CD81 in immune response in lamprey has great significance.In this paper,because of the need of proteases cut and purification,we transformed the vector pCold TF.Using the multipoint mutation to replace His-tag on the vector and added the His-tag to 3 'end of the ORF of Lja-CD81.Lja-CD81 connected to the transformed vector pCold TF and induced soluble protein successfully.We got the Lja-CD81 without Trigger Factor with Factor Xa protease.By antigen design software,selecting suitable polypeptide and preparation of polyclonal antibody.Through performing western blot,we detected the relative expression of Lja-CD81 in a single nucleus leucocytes,supraneural myeloid bodies,gills and so on.At the same time,we used flow cytometry to observe the expression and colocalization between Lja-CD81 and VLRs in immune stimulation before and after.At last,we tried to seek the interaction between a protein and Lja-CD81 in lymphocyte cells,supraneural myeloid bodies and gills in lamprey by immunoprecipitation.The results show that the relative expression of Lja-CD81 is increase obvious in lampreys were immunized by two injections at 7-day intervals,and then sacrificed to take tissue samples at the third day after the second immunization.When we choosed the different time after the second immunization in immune tissues,we find the highest expression in lymphocyte cells and gills at the 12 hours after the second immunization,and the highest expression in supraneural myeloid bodies at the 18 hours after the second immunization.Lja-CD81 may be involved in the biological process of immune system.The result ofimmunofluorescence show that Lja-CD81 is distribution on the cell membrane,the expression of Lja-CD81 is uneven and forms polarized clusters after immunization.That maybe forms a structure similar to the immune synapse in lamprey.Meanwhile,the ratio of the colocalization between Lja-CD81 and VLRs is increase obvious,this means Lja-CD81 participate in the immune response in lamprey.