Expression Of Truncted NSP2 Protein Of Porcine Reproduction And Respiratory Syndrome Virus In E.Coli And The Development Of Monoclonal Antibodies Against NSP2 Protein

Porcine reproductive and respiratory syndrome virus(PRRSV) is a member of the Arteriviridae,can cause reproductive failure in sows and respiration disturbance in piglets.The genome is a positive sense,15 kb single strand of RNA consisting of 8 open reading frames(ORFs).The ORF1 is predicted to be anto-proteolytically cleaved at 12 sites,producing 13 non-structural proteins ultimately(NSP_1-13).NSP2 contains a larger number of linear B-cell epitopes,indicating that NSP2 protein of PRRSV is an immunogenic protein capable of eliciting specific antibody production during viral infections.In the study,NSP2 protein was partly expressed in E.coli and two NSP2-specific monoclonal antibodys were developed.The process was as following:1.Expression of NSP2 gene of PRRSV in prokaryocyte system:In order to express the NSP2 gene of PRRSV,a pair of primers that have EcoRI and NotI sites were designed according to the sequence of PRRSV S1 strain.The NSP2 gene was amplified from recombinant plasmid pET-NSP2 by PCR using the primers,and cloned into a prokaryotic expression plasmid pGEX-6p-1.The ligated product was transformed into E.coli DH5αand then picked the positive clone,a recombinant plasmid named pGEX-6p-1-tNSP2 was constructed and identified with restriction enzyme digestion and sequenced.The plasmid pGEX-6p-1-tNSP2 was transformed into E.coli BL₂₁ and induced by 0.6mM isopropyl-β-D-thiogalactoside(IPTG) at 37℃for 4h.SDS-PAGE of the bacteria revealed that a fusion protein GST-tNSP2 with molecular weight of 50 kDa was expressed in E.coli.The purified GST-tNSP2 protein showed a Strong reaction with the PRRSV-positive sera in Western blot assay.2.The development of monoclonal antibodies against NSP2 protein:Six to eight -week-old Balb/c mice were hypodermically immunized three times at 2-week interval with 50ug of the GST-tNSP2 protein emulsified in Freund'scomplete(first injection) or incomplete adjuvant(second and third injections).The mice received a final injection of 50 ug of the protein in PBS before fusion.The splenocytes of the immunized mice were fused with murine myeloma cells SP2/0,and the rate of fusion was 80%,the positive rate of the first screen was 60%.After subcloning by 3 times, two hybridoma clones which produced McAbs steadily were screened by ELISA, named 3H3 and 2B5.Both of the McAbs belong to IgG1 isotype,and their light chains belongκtype.They all reacted strongly with the PRRSV S1 infected Marc-145 cells in IFA,but not with the PRRSV SY0608 strain.The ELISA titers of 2B5 and 3H3 in the ascites were 1:102400 and 1:204800 respectively,meanwhile the ELISA titers in cell cultural supernatant were 1:512 and 1:640 respectively,and the titers of 5,10,20 generations were also the same.In summary,in this experiment we get two hybridoma clones which produced McAbs steadily,and the expressed GST-tNSP2 protein and McAbs could be used for identification of PRRSV isolates and functional analysis of NSP2.