Preparation Of Monoclonal Antibodies To Nucleocapsid Proteins Of PPRV Wuwei Strain

Pestedes Petits Ruminants Virus(PPRV)belongs to the genus Morbillivirus in the family Paramyxoviridae,it primarily affecting sheep,goats and wild small ruminants.Pestedes Petits Ruminants(PPR)is an acute,severe and highly contagious disease,which is characterized with nasal and ocular discharges,gastroen-teritis,necrotic stomatitis,pyrexia,and erosion of the pulmonary tract mucosa.The disease spreads rapidly and has a high mortality rate,PPR is responsible for severe economic losses in the countries where it is endemic.In China,the first PPR epidemic occurred in July 2007 in the Ali area of Tibet,but in the following years,it was limited to the Tibet area.On November 30,2013,a new epidemic occurred in the Xinjiang Yili Kazakh Autonomous Prefecture,and it spread quickly.So far,25 provinces,autonomous regions,and municipalities arised PPR,which have caused tremendous economic losses to China's sheep-rearing industry.At present,the prevention and control of PPR is concentrated on the strengthening of legal supervision,epidemic monitoring,and early warning capabilities.In the event of an outbreak,measures such as blockades and exclusions will be adopted.Therefore,establish a fast,efficient,specific and sensitive PPRV detection method,will provide technical support for rapid diagnosis and effective prevention and control of PPR.Based on this,this study mainly completed the following work:(1)Amplification and sequence analysis of N gene of PPRV Wuwei strainThe primers were designed according to the N gene sequence(HQ197753)of PPRV Nigeria 75/1 strain in GenBank.The full-length N gene of PPRV Wuwei strain was obtained by RT-PCR amplification.On the basis of sequencing,bioinformatics analysis of 53 strains of PPRV N gene sequences in Gen Bank was completed.(2)Prokaryotic expression of N gene of PPRV Wuwei strain and preparation of polyclonal antibodiesThe N gene of PPRV Wuwei strain was cloned into pET-28a(+)to construct a prokaryotic expression plasmid pET-PPRV-N,which was then transformed into E.coli Rosetta(DE3).After induced by IPTG,the soluble expression of the recombinant protein was obtained.The relative molecular weight was about 57.8 kDa.Immunize BALB/c mice with the purified expressed protein.The titer and immunogenicity of the antiserum were detected by ELISA and Western blot.The results showed that the N protein of PPRV Wuwei strain had good immunogenicity.(3)Preparation of Monoclonal Antibody to N Protein of PPRV Wuwei StrainFusion of spleen cells of immunized BALB/c mice with myeloma cells(SP2/0).Screening for two hybridoma cell lines 2B7 and 3I4 that stably secrete monoclonal antibodies against PPRV N protein.The results of ELISA showed that the monoclonal antibodies produced by hybridoma cells 2B7 and 3I4 were specific;The results of subtype identification showed that the two cell secretory McAb heavy chains were IgG2 b type and light chain were ? type.Injecting cells into the abdominal cavity of BALB/c mice,collect and test hydroperitonium titers,and the test results show that the titer is 1:51 200 and 1:25 600,respectively.