Production And Identification Of Monoclonal Antibodies Specific For Rabbit Haemorrhagic Disease Virus And Immunobiological Characteristics Of The VP60 Expressed Products In E.Coli

Rabbit haemorrhagic disease (RHD) is a fatal acute infectious disease caused by the rabbit haemorrhagic disease virus(RHDV). In this study, BALB/C mice were immunized by injection two kinds of immunogens, which were RHDV virion purified from the livers of experimentally infected rabbits and recombinant capsid proteins VP60 expressed in E.coli. After three immunization , McAbs were prepared by fusing SP2/0 myeloma cell with spleen cells . Specific McAbs were detected in hybridoma culture supernatant and mouse ascites by indirect ELISA. Positive hybridomas were cloned by technique of limiting dilutions .5 McAbs were raised against recombinant VP60 proteins and frozen due to their lower OD490 by ELISA. Another 5 McAbs, RH-1 RH-2,16D6-1,16D6-2,16F2 against intact virion were produced and characterized . The liters of the five McAbs in ascites were ranging between 1:819200 and 1:25600. The 1g subclass of these McAbs was IgG(n). Western blot test showed that the 4 McAbs RH-2 16D6-1, 16D6-2 16F2 specifically reacted with the virion not previously denatured by boiling .except for RH-1; but they all recognized the truncated peptides denatured by boiling. The HI titres(71og2,8Iog2) of RH-1 and RH-2 were higher than the other three McAbs, 16D6-1 16D6-2, 16F2 (2!og2, 31og2) . Dot-ELISA and TAS-ELISA proved all McAbs were specific for intact RHDV antigen, not for other antigens such as liver tissue, NDV and A1V.The major capsid protein VP60 gene of RHDV was cloned in two plasmids: pET5a and pGEX-6P-l; the recombinant plasmids pET5a-RHDV-VP60 and pGEX-6P-RHDV-VP60 were Separately transformed into E.Coli-JM109(DE3) and E.Coli-BL21. After induced by 0.4mM IPTG, the recombinant bacteria pET5a-RHDV-VP60/JMI09(DE3) expressed apeptide with molecule weight of 62kd on 12%SDS-PAGE; After induced by l.0mM IPTG, the recombinant bacteria pGEX-6P-RHDV-VP60/BL21 also expressed a peptide with molecule weight of 87kd, being consistent with that designed. The expressed products were undissolvable fusion protein, and most of them were inclusion bodies. In immunoblotting, the recombinant protein pET5a-RHDV-VP60 could be recognized by three McAbs (RH-1 16D6-1 16F2), and the recombinant protein pGEX-6P-RHDV-VP60 could be recognized by McAb 16F2. Mouse polyclonal antibodies specific for two recombinant VP60 proteins were detected positive (OD490 0.50, 0.33) by indirect ELISA . Antiserum from RHDV infected rabbit also could react with the E.Coli-expressed VP60 fusion protein. Our test proved the recombinant proteins had good antigenicity, immunogenicity and specificity. The expression and detection of VP60 gene laid a foundation of further study on RHDV VP60 protein and gene engineering vaccine.