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Generaiton of Recombinant Peste Des Petits Ruminants Viruses with Foreign Epitopes Fused to the Carboxy Terminus of N Protein

Author: YinZuoZuo
Tutor: BuZhiGao
School: Chinese Academy of Agricultural Sciences
Course: Preventive Veterinary Medicine
Keywords: PPRV Nucleocapsid protein Foreign epitope Marker vaccine
CLC: S852.65
Type: Master's thesis
Year: 2013
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Abstract


Peste des petits ruminants (PPR) caused by PPR virus (PPRV) is a highly contagious andinfectious disease in some small ruminants with high morbidity and mortality rates. Since it was firstreported at1942, PPR spread dramatically and lead to a wide range of economic loses in endemicarea. Vaccination is the main way to control PPR since there is no effective medical therapy to PPR.Although the PPR live-attenuated vaccines are efficacious to control PPR, it could not differentiatenatural infected animals from vaccined animals (DIVA) and would interfere with the serologicalsurveillance of it. Therefore it is necessary to develop the marker vaccine (DIVA vaccine) since itcould cover this shortage.PPRV with the single-stranded negative-sense RNA genome is a menber of Morbillivirus genusthat belongs to Paramyxoviridae family. Nucleocapsid (N) protein is the most abundant and the mostimmunogenic structural protein of PPRV. Despite its failure of inducing protective immunity againstPPRV, N protein has been wide used in diagnostic tests and it is a good candidate target protein tointroduce foreign epitope to construct marker vaccine.In order to develop marker vaccine, the first thing is to know whether the foreign fusion epitopeswould affect the function of PPRV N protein, PPRV rescue was done respectively with the helperplasmid expressing N protein fused with foreign epitope (HA epitope, FLAG epitope,5B19epitopeof the S2glycoprotein of murine hepatitis virus) at the carboxyl terminus instead of natural Nprotein, and the successful rescue of recombinant PPRV showed that foreign epitopes fusion had littleinfluence on the function of N protein. Subsequently, the N gene in the full-length infectious clone ofPPRV was replaced by fusion gene NHA, NFLAG, N5B19respectively and the recombinant virusesexpressing N protein fused with foreign epitopes were rescued. The expression of N protein fusedwith foreign epitopes by recombinant viruses was detected by western blot and indirectimmunofluorescence assay. The growth curves showed that the highest growth titer of recombinantviruses was lower than that of parent virus, however the titer is high enough for it’s future applicationas marker vaccine. In this study, the immunogenicity of rPPRV/GFP/5B19was evaluated in mice, theresults showed that3of5mice were positive for5B19ELISA detection in3weeks after immunizedwith rPPRV/GFP/5B19, and the5B19ELISA antibody response could be detected in all mice after asecond dose. Similar to rPPRV/GFP, rPPRV/GFP/5B19could induce high level of PPRVneutralizing antibodies in mice serum.In conclusion, this study has established a good foundation on developing marker vaccine ofPPRV.

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CLC: > Agricultural Sciences > Livestock, animal medicine,hunting,silkworm,bee > Animal Medicine ( Veterinary Medicine) > Basic Veterinary Science > Animal Microbiology ( Veterinary Microbiology, ) > Livestock Virology
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