Production And Characterization Of The Monoclonal Antibodies Against Envelope Protein Of Japanese Encephalitis Virus

Japanese encephalitis virus(JEV),being a member of the family Flaviviridae,is usually infected to the human body and animal through mosquitoes and then may cause acute viral encephalitic and neurologic disease.Therefore,it is commonly regarded as a serious public health problem,with JEV being the infectious agent in many domestic and wild animals,especially with swine(viral associated abortions). Swine is considered the main vertebrate host and represents an important amplifier and reservoir for JEV. Therefore prevention of JE in swine can benefit for swine production and health of human being.Hence,it is essential to prevent swine JE,which is useful for swine production as well as health of human being.The envelope glycoprotein,a major structural antigen,is associated with viral attachment,fusion, hemagglutination,cellular tropism,viral virulence,and induction of protective immune response.In this study,Fifteen specificity monoclonal antibodies(McAb)against Japanese Encephalitis Virus(JEV)E protein of SA14-14-2 strain were prepared by fusing BALB/c mice myeloma cell line SP2/0 with B lymphocytes from spleen of BALB/c mice immunized with prokaryotic expressed E protein.There were named as 1A9,1G12,1H2,2A6,2B3,2G3,3D1,4E6,4H1,5B3,5B7,5D4,5D7,5Gll and 5G12,respectively,5B7 of the monoclonal antibody subtype of IgG2b,5D4 and 5G12 of the monoclonal antibody subtype of IgG2a,the remaining 12 for IgG1,all the light chains of the antibodies wereκtype. Western blot confirmed that the monoclonal antibody from the hybridoma production can be specifically response with JEV E protein.McAb reported in this work may provide valuable tools for accurate and rapid diagnosis of JEV.The clinical application of mouse mAbs is limited because of the serious HAMA reaction(human anti-mouse antibody)in the McAb cure process.In order to dismiss the immunogenicity of mouse mAbs,it is necessary to make its molecule as similar as human's.Therfore it can escape from the identification of human immunity system.Then the HAMA reaction could be reduced or avoided.Using the genetic engineering technique to produce Single-Chain Variable Fragment can dispense with the complicated selecting of hybridoma technique,it expanded the scope of antibody application,and achieved great progress of antibody engineering.VH gene were amplified by RT-PCR.Then the PCR product was ligated to pMD18- T vector and the positive clones were analyzed.The amplified VH genes of 5D4 was 328 bp, then sequence was analyzed by GenBank Data.The results showed VH gene contained four FRs,three CDRs and two characteristic cysteine residues,which lay a good foundation for constructing a diversity of engineering antibodies.