Cloning And Expression Of F Gene Of Peste Des Petits Ruminants Virus Nigeria75/1Strain And Preparation Of Monoclonal Antibodies

Peste des petits ruminants(PPR)is a exotic animal epidemics infectious disease of small ruminantssuch as sheep and goats，caused by peste des petits ruminants virus(PPRV).PPR was listed as one of theanimal epidemic that must be reported by OIE. PPR outbreak for the first time in Tibet in China，in July2007.Therefore，it becomes more necessary for us to do some researches on the viral etiology，diagnos isand epidemiology.1. Expression and identification of peste des petits ruminants virus F gene in the prokaryoticexpression system.In this study，the useful of the E.coli expression system and Bac-to-Bac baculovirus expressionsystem to express F protein of peste des petits ruminants virus respectively. Total RNA was extractedfrom the Vero cells which infected by Nigeria75/1vaccine strain peste des petits ruminants virus. In theE.coli expression system， the PPRV F gene fragment without N-terminal signal peptide andtransmembrane domain were analysised by DNAStar and were amplified by reversetranscription-polymerase chain reaction(RT-PCR) technology and were inserted into the prokaryoticexpression vector pET-32a(+) and pGEX-6P-1，finally，the constructed recombinant plasmid weretransformed into E.coli BL21(DE3) for expression under induction of IPTG. The expressed protein werepurified by urea and identified by SDS-PAGE and Western blot，it showed that the fusion protein couldbe recognized by the goat positive sera.The recombinant pET-32a-PPRV-F protein as the coatingantigen to detect antibody in serum by Indirect ELISA.2. Expression and identification of peste des petits ruminants virus F gene in the Baculovirusexpression system.In Bac-to-Bac system，firstly，the recombinant baculovirus transferring vector pFastBacTMHTA-Fwas constructed and was used to transform E.coli DH10Bac competent cells.Through Tn7transpositionsystem，F gene was inserted into Bacmid.The recombinant baculovirus was obtained transfect insectcell Sf21and obtained the recombinant baculovirus containing PPRV F gene. The recombinant proteinwas expressed in insect cells could be recognized by the goat positive sera.3. Preparation and identification of monoclonal antibodies against peste des petits ruminantsvirus F protein.To generate stable hybridoma cell lines secreting monoclonal antibodies(McAbs) against PPRV Fprotein，the purified pET-32a-PPRV-F2protein and PPRV were used as antigens for immunization ofBalb/c. The spleen cell of the Balb/c mice immunized were collected to fused with the murine myelomacells SP2/0.Prokaryo-expressed pGEX-6P-1-PPRV-F2protein and PPRV were used as the detectionantigen in indirect ELISA.The classes and subclassed of the McAbs were identified by isotypingkit.Western blot and indirect immunofluorescence test analysis showed that the McAbs against F proteincould combine with recombinant protein and the whole virion-antigen of PPRV Nigeria75/1strain. Therefore，it provides a basis for the establishment of rapid detection methods based on the monoclonalantibody to peste des petits ruminants.