Cloning And Prokaryotic Expression Of F Gene Of PPRV GS Strain And Preparation Of Monoclonal Antibody

Peste des petits ruminants(PPR)is a disease of major economic importance and imposes a significant constraint upon sheep(Ovis aries)and goat(Capra hircus),because of its high fatality rate,often caused huge economic losses to livestock in sheep.For the serious situation of peste des petits ruminants,the rapid diagnosis method was need to establish which can effectively distinguish between natural infection and vaccination.Because F gene is highly conserved in the Morbillivirus,F protein is distributed on the surface of the envelope of the virus and promotes the fusion between the viral envelope and the cell membrane,which is related to the invasion and adhesion of the virus.Therefore,carrying out the related research of the pathogen is of great significance to the monitoring and prevention of PPR epidemic situation.However,the establishment of diagnostic methods for F protein is limited to vaccine strains,but less in new strains.Based on this,the research was carried out in our study as following:(1)In order to study the F gene sequence characteristics and gene evolution of PPRV GS strain,The primers were designed according to the F gene sequence of PPRV Nigeria 75/1 strain in GenBank(GenBank No.HQ197753),and the F gene of PPRV GS strain was amplified by RT-PCR.Based on sequencing and sequence analysis,the F gene characteristics was completed.The results showed that the complete CDs of F gene(GenBank No.KX822738)was 1 641 bp,encoding 546 amino acids.It is a transmembrane protein with a signal peptide.The highest level of nucleotide homology was 99%.And the nucleotide homology with the vaccine strain Nigeria75/1 was only 93%.The phylogenetic tree based on F gene showed that PPRV GS strain was spectrum with 26 strains in Asia and 2 strains in northwestern Morocco.(2)In order to study the immunogenicity of GS strain fusion protein of PPRV,the primers were designed and the Fa fragment of F gene without the signal peptide and transmembrane domain was amplified,Then the recombinant plasmid pET-PPRV-Fa was constructed through cloning Fa fragment into pET-32a(+)and the recombinant plasmid was transformed into Escherichia coli Transetta(DE3).After induced by isopropyl?-D-1-thiogalactopyranoside(IPTG),the recombinant protein was expressed,and the New Zealand rabbit(Oryctolagus cuniculus)was immunized with purified protein,then the polyclonal antibody against PPRV fusion protein was prepared.Subsequently,the analysis of its immunogenicity was accomplished using indirect enzyme-linked immunosorbent assay(ELISA)and Western-blot.The results show that the signal peptide and The Fa fragment of F gene was successfully expressed in E.coli,whose molecular weights approximately was 59 kDa and mainly existed in the form of inclusion body.The indirect ELISA and Western blot results showed that the prokaryotic expression products of Fa gene of PPRV GS strain had good immunogenicity.(3)The BALB/c mice was immuned with purified protein to prepare immune spleen cells which would fusioned with SP2/0.The positive hybridoma cells were tested through indirect ELISA and then subclone screening should be in process,and then prepare the ascites,titers and reaction specificity were detected at the same time.The resulted showed than the titers of monoclonal antibody is 1:51 200 and reaction specificity is good.The classes and subclassed of the McAbs were identified by isotyping kit,and the antibody subtypes are IgG2 b,light chain for?.Western-blot showed that the McAbs against F protein could combine with recombinant protein.In conclusion,the research results provide theoretical basis for the study on the function of fusion protein from PPRV GS strain,and for developing a rapid diagnosis method.