Porcine IgG Chimeric PRRSV Nsp9 Nanobody Inhibits Virus Replication

Porcine reproductive and respiratory syndrome virus is the causative agent of PRRS and can induce respiratory diseases and reproductive failures in sow.At present,vaccination is the most prevalent way of controlling PRRSV infection,but the current commercially vaccines fail to provide sustainable disease control and prevention.And there are no commercial anti-PRRSV drugs.The nonstructural protein(Nsp)9 of PRRSV coded the viral RNA-dependent RNA polymerase(Rd Rp)is essential for viral replication,and it possess a relatively conserved sequence,making it a logical antiviral target for PRRSV.In our previous study,a Nsp9-specific nanobody,Nb6,were isolated and identified as an antiviral nanobody.Furtherly,cell-penetrating peptide fused Nb6(TAT-Nb6)were produced and form prokaryotic expression system.Nb6-p Fc could be uptake by Marc-145 and PAM and suppresses PRRSV replicating regardless of genotypes.Nevertheless,TAT exhibits several disadvantages including short half-life in blood and nonspecific delivery to normal tissue.PRRSV shows a strong tropism for cells of monocyte-macrophage lineage.The virus firstly infection and replication in PAM after hosts were infected.Fcγ receptors(FcγRs)expressed on the surface of leukocytes,which bind to the Fc portion of Ig G,played a prominent role in receptor-mediated phagocytosis.Based on the above backgrounds,in this study,we produced a pig Fcγ and Nb6 fused chimeric nanobody(Nb6-p Fc),which can enter PRRSV susceptible cells via FcγR-mediated endocytosis and inhibit virus infection efficiently.We also constructed suspended 293 cell line express Nb6-p Fc stably,and provided a basis for the development of anti-PRRS drugs.The main research contents and results are as follows: 1.Expression and activity verification of Nb6-p FcThe gene of Nb6-p Fc was firstly amplified by overlap PCR,then inserted into p PICZαA vector.The recombinant plasmid p PICZαA-Nb6-p Fc was electro-transformed into X-33 strain of Pichia pastoris.After induction by methyl alcohol,the recombinant protein Nb6-p Fc were successfully expressed in culture medium and then purified by protein G affinity chromatography.After running of reduced and non-reduced SDS-PAGE,Nb6-p Fc were showed as dimer.The results if indirect ELISA,IFA and immunoprecipitation experiments showed that Nb6-p Fc can bind with prokaryotic expressed Nsp9 and PRRSV encoded Nsp9.2.Nb6-p Fc enter PAM by FcγR-mediated endocytosisDifferent concentrations of Nb6-p Fc was diluted by cell culture medium and incubated with PAM for different times for cellular uptake.IFA,Western blot,and flow cytometry were used to detect the entrance of Nb6-p Fc.The results showed that Nb6-p Fc firstly absorbed to the cell membrane then were endocytosed into the PAM.The endocytosis was dose-dependent from 5 μM to 20 μM;the chimeric antibodies could be persistent detected out for 24 h.We subsequently tested the cytotoxicity of Nb6-p Fc with CCK8 to PAM,the results showed that Nb6-p Fc had no affect for cell viability at concentrations below or equal to 50 μM.3.Exploration of the anti-PRRSV effect of Nb6-p Fc on PAMDifferent genotypes of PRRSV were selected for exploring antiviral effect of Nb6-p Fc.The results showed that Nb6-p Fc could inhibit the replication of PRRSV 1 strain GZ-11,PRRSV 2 strain GD-HD,SD-16,JX-A1,CH-1A and infectious clone r SD16/TRS2/Clover on PAM.The inhibition effect of Nb6-p Fc was gradually enhanced with the increasing of antibody concentration.Meanwhile,it was proved that Nb6-p Fc up-regulated several inflammatory factors expression on PAM and helped viral inhibition.Multiply administration of Nb6-p Fc can improve the inhibition efficiency for r SD16/TRS2/Clover replication on PAM.4.Construction of suspension 293 cell line which express Nb6-p Fc stablyNb6-p Fc gene with signal peptide was firstly inserted into p Lvx-IRES-Zs-Green plasmid.After transformation of p Lvx-Nb6-p Fc-IRES-Zs-Green to HEK-293 T cells,the lentivirus with Nb6-p Fc were packed.Then,the packed lentivirus was added to suspended 293 cells.According to ELISA and Western blot identification,Nb6-p Fc were verified successfully expression in suspended 293 cells culture medium.Finally,the positive expressed cells were separated by flow cytometry.In summary,a PRRSV Nsp9 specific chimeric nanobody Nb6-p Fc were produced.The Fcγ ligated Nb6 can enter PRRSV susceptive cells and inhibit PRRSV replication of different genotypes.can suppress PRRSV replication targeting PRRSV Nb6-p Fc is potential for developing as an anti-PRRSV drug.