Cloning And Expression Of F Gene Of Peste Des Petits Ruminants Virus Nigeria 75/1 Strain And Applied Research

Peste des petits ruminants (PPR) is an acute and highly contagious infectious disease of small ruminants such as sheep and goats, caused by peste des petits ruminants virus (PPRV). PPR was listed as one of the A disease by OIE, and was also classified as type A diseases by Chinese government. According to announcement of the Ministry of Agriculture, in July of 2007, the first outbreak of peste des petits ruminants in Tibet was confirmed. Therefore, it is very necessary to do some researches on the viral etiology, epidemiology and diagnosis of this disease.Total RNA was extracted from the vero cells which infected by peste des petits ruminants virus vaccine strain Nigeria 75/1. The F gene was amplified by RT-PCR and then cloned into pMD18-T vector, the recombination vector was named pMD18-F. After analysis of F protein structure, the F gene fragment deleted N-terminal signal peptide and transmembrane domain was amplified by PCR using plasmid pMD18-F as template and was subcloned into pET-30a(+) vector. The identified recombinant plasmid pET30-F-1was transformed into E. coil BL21 (DE3) subsequently, which was then induced by IPTG. The expressed recombinant protein was examined by SDS-PAGE and Western-blotting. The antibody against the expressed recombinant protein was prepared by immunization of rabbits for an indirect immunofluorescence assay, and titers of serum antibody were detected by ELISA. The result showed that the cloned fragment was inserted into the expression vector correctly. The F gene was expressed efficiently in the form of inclusion bodies. ELISA showed that the titer of specific antibody against the expressed protein in sera from the immunized rabbits was significantly higher than that of the unimmunized rabbits. Western-blot analysis showed that the expressed recombinant protein had reactogenicity. Indirect immuno- fluorescence assay showed that the sera from the rabbit immunized with the recombinant protein could recognize the whole virion-antigen of PPRV Nigeria 75/1 strain. The results revealed that the recombinant protein had good reactogenicity. The recombinant proteins purified were used to immunize goats of three months, and the specific antibody was assayed by indirect ELISA every week. In conclusion, this recombinant protein could induce humoral immune responses in goats.Five stable hybridoma cell lines secreting monoclonal antibodies (McAbs) against F protein, were developed by fusion between the murine myeloma cells SP2/0 and spleen cells of BALB/c mice immunized with the recombinant protein, after several times cloning and selection with indirect-ELISA. The classes and subclasses of the McAbs were identified by using mouse monoclonal antibody isotyping kit as IgG2a, IgG2b and IgA. Western blot analysis showed that the monoclonal antibody against F of hybridoma cell lines secreted could combine with recombinant protein specifically. The abilities of hybridoma cell lines secreting McAb remained the same after identification of its biological characters. So it laid a foundation for the further research of established a diagnosis method of PPR.