Expression Of Truncated Nsp2 Of HP-PRRSV (HuN4) In E.coli And Preparation Of Monoclonal Antibodies Against Nsp2

Porcine Reproductive and Respiratory Syndrome(PRRS) characterized by severe reproductive disorders in sows and respiratory diseases in young pigs,was first recognized in the US in 1987. Since 2006,highly pathogenic porcine reproductive and respiratory syndrome(HP-PRRSV) outbreaks and the prevalence in our country had brought enormous economic losses.In the highly pathogenic porcine reproductive and respiratory syndrome virus(HP-PRRSV) genome,it was occurred deletion in Nsp2 gene which was the first 1447～1449 and 1597-1698 nucleotide.The nucleotide homologies of Nsp2 gene was 69.9%～86.2%compared with PRRSV isolated from our country before 2005.In this study,the Nsp2 gene of HP-PRRSV(HUN4 strain) was cloned, identificated and expressed in Escherichia Coli.On the base of it,the monoclonal antibodies against Nsp2 were successfully prepared,which reported here may provide valuable tools for luther investigation of the structure and function of HP-PRRSV Nsp2 protein.1 Clone and prokaryotic expression of the PRRSV Nsp2 protein geneThe nonstructural protein 2 gene of HP-PRRSV(HUN4 strain) was amplified by RT-PCR with a pair of specific primers,and then the product was cloned into pET-30a(+) vector and sequenced. The plasmid pET-H-Nsp2 was transformed into Escherichia coli BL21(DE3) for expression under induction of IPTG.The result of SDS-PAGE analysis revealed that the molecular weight of the expressed product was 63.5 ku.The expressed protein was purified in one step using Ni-NTA affinity chromatography.Western blot analyses showed the recombinant protein had good immunoreactivity with the reference positive serum.2 Preparation and characterization of the monoclonal antibodies against Nsp2 of HP-PRRSV (HUN4 strain)Three monoclonal antibodies(McAbs) against PRRSV nonstructural protein(Nsp2) of HuN4 strain were produced.These McAbs were generated by fusing BALB/c mice myeloma cell line SP2/0 with spleen lymphocytes from BALB/c mice immunized with prokaryotic expressed Nsp2 and were designated as 1A5,4B3,3C2,respectively.The McAb 1A5 belonged to the subtype of IgG2b,while 4B3,3C2 were identified as IgGl subtype,and the light chains of all antibodies wereκchain.The antibody titers of ascite were 1:10240.1:10240 and 1:20480 respectively.Western blot showed that the antibodies were specific to Nsp2 protein.IFA showed that 3 McAbs recognized Nsp2 protein in PRRSV(HuN4 strain) infected Marc-145 cells.McAbs reported here may provide valuable tools for futher investigation of the structure and function of PRRSV Nsp2 protein.