Preliminary Validation Of Swine Reproductive And Respiratory Syndrome Virus Nsp9 Nanobody For Antiviral Infection In Vivo

Porcine Reproductive and Respiratory Syndrome is a viral infection caused by porcine reproductive and respiratory syndrome virus,which is characterized by abortion of pregnant sows and respiratory diseases of pigs of different ages.Because PRRSV has the characteristic of escaping the host humoral immune response mechanism,the commercialized vaccines can not provide a complete protection for the herd,and no effective treatment has been known yet.This disease causes heavy economic losses to the global swine industry.In the previous study,the PRRSV Nsp9 specific nanbody Nb6 gene was fused with the cell transmembrane peptide TAT gene by the（G4S）3 Linker,cloned into the pET21b vector,and the pET21b-TAT-Nb6 expression vector was successfully constructed,and the TAT-Nb6 recombinant protein was obtained.It has been shown to have an interaction with Nsp9 recombinant protein,and can enter MARC-145 cells and PAM cells,and inhibits multiple strains of PRRSV（including genotype 1（GZ11-G1）and genotype 2（SD16,JX-A1）,GD-HD,VR-2332）replication in MARC-145 cell line and primary PAM cells.Therefore,TAT-Nb6 has the potential to be developed as a novel anti-PRRSV Nanobody drug.Based on the above background,this study determined the optimal expression conditions of TAT-Nb6,including induced expression temperature,IPTG addition and induction time,and prepared a large number of recombinant TAT-Nb6 proteins,and verified the antiviral function of TAT-Nb6 in pigs.This study provides a theoretical basis for the development of anti-PRRSV Nanobody drugs.The main research contents and results are as follows:1.Optimized expression conditions of TAT-Nb6 recombinant protein:To find out the maximal expression of TAT-Nb6 in E.coli BL21（DE3）,we cultured the bacteria containing expression vector pET-21b-TAT-Nb6 constructed by our lab in different conditions,such as temperature,the concentration of IPTG and duration of induction.After analysis of SDS-PAGE and western blot,the highest yields of TAT-Nb6 recombinant protein were observed with 0.2 mM IPTG at 37℃for 6 h.2.Preparaed TAT-Nb6 recombinant protein for animal experiment:TAT-Nb6recombinant protein was induced with 0.2 mM IPTG at 37°C for 6 h,purified by Ni-NTA affinity chromatography,dialyzed in urea gradient dialysis buffer,and concentrated with ultrafiltration tube.Then a total of 350 mg（6.25 mg/ml）of TAT-Nb6 recombinant protein was successfully prepared.The result of ELISA showed that TAT-Nb6 had strong binding ability to Nsp9 protein,and the result of Immunohistochemistry showed that TAT-Nb6 could successfully enter all the detected tissues.3.Anti-PRRSV function of TAT-Nb6 recombinant protein in pigs:In this study,after artificial infection of PRRSV JXA1 strain(10^4.5TCID50)of piglets aged 28-35 days without specific pathogens,the TAT-Nb6 recombinant protein was injected to detect the changes of body temperature,antibody and viremia of piglets on different days.The results showed that the body temperature and PRRSV antibody levels in the treatment group were significantly lower than those in the challenged control group,while there was no significant difference in the load of virus in the serum.The antiviral activity of TAT-Nb6 in vivo needs further investigation.