| Classical swine fever(CSF),one of OIE-listed diseases,is a highly contagious and economically important disease of pigs.Classical swine fever virus(CSFV)is the causative agent of classical swine fever(CSF),the capsid protein C and the glycoproteins Erns,E1,and E2 are structural components of the virion E2 is the most immunogenic of the CSFV glycoproteins,inducing neutralizing antibodies that provide protection against lethal CSFV challenge.At present,The antigenic properties of E2 have been characterized by using a number of monoclonal antibodies(MAbs).It is very important for differential diagnosis,antigenic variation and antigen epitopes of CSFV.As the deepening of antibody research,efficient and stable gene engineering antibody technology has gradually become the main method of generateing antibodies.In a previous work,we developed a murine MAbHQ06 against the E2 protein of CSFV.HQ06 was proved to be CSFV-specific and recognize a linear epitope on the E2 protein of CSFV C-strain or Shimen strain and was identified to be Ig G1 subtype and kappa light chain.However,production of MAbs in hybridoma cells is not stable and even lead to loss of activity.In this study,the variable region gene from HQ06 and constant region gene of swine antibody are fused and then cloned into the eukaryotic expression vectors to establish a cell line which can express a chimeric recombinant porcinized MAb against E2(r HQ06Sw)stably in suspension HEK293 cell.The purified r HQ06 Sw antibody protein is determined that successfully generated,which exhibited high recognition of rabbit anti-swine Ig G.We next evaluated the reactivity between r HQ06 Sw and E2 protein of CSFV by ELISA,Western blotting,IPMA.In addition,we used SPR to determine the high binding affinities of the CSFV E2 protein for r HQ06 Sw.The specific reactivity of r HQ06 Sw with CSFV was subsequently determined by cross-reactivity assay.Most importantly,we investigated the activity of rHQ06 Sw in neutralizing CSFV.In conclusion,this study aimed at generating r HQ06 Sw for efficient and stable production which can be used to develop diagnostic assays,investigate the structure and function of the E2 protein and generate novel preparations of diagnosis and treatment. |
