Recombinant Monkey MYH9 Protein C-Terminal Domain Blocks Porcine Reproductive And Respiratory Syndrome Virus Infection

Porcine reproductive and respiratory syndrome(Prorcine reproductive and respiratory syndrome,PRRS)is a widely spread pig reproductive and respiratory system disease.Porcine Reproductive and Respiratory Syndrome Virus(PRRSV)is an enveloped,single-stranded positive-strand RNA virus.Spherical or elliptical particles with a diameter of about 50-60 nm have a relatively smooth appearance.PRRSV has erupted in the world for more than 30 years,and every year brings huge economic losses to the pig industry.Therefore,the research on the pathogenic mechanism of PRRSV and efficient vaccines is urgent.Pigs are the only natural host of PRRSV known so far.In addition,the virus infection process has a strong cell tropism.Fully differentiated porcine alveolar macrophages(PAM)are the main target cells for PRRSV infection.Among all PRRSV putative cell receptors,MYH9 and CD163 proteins are the receptor proteins necessary for PRRSV infection.MYH9 provides a bridge for the process of PRRSV particles adsorbing on the host cell surface,unpacking the virus,and releasing the genome into the cytoplasm.Therefore,it has important research value as an inhibitor of PRRSV.In this paper,the prokaryotic E.coli expression system is selected,and the p Clod-SUMO plasmid is used as the expression vector.The soluble expression of the protein is promoted through the SUMO tag and low temperature induced culture conditions.Through the design of primers,the PCR amplification of the target gene,double enzyme digestion,ligation and transformation success The recombinant plasmid p Cold-SUMO-Monkey PRA was constructed.After that,the recombinant plasmid was introduced into BL21(DE3)competent cells for expression,purified by means of nickel column affinity chromatography,and the SUMO tag was removed by r TEV enzyme.After dialysis and concentration,the monkey PRA high-purity protein was expressed.Afterwards,the binding of Monkey PRA protein to the anti-unique antibody Mab2-5G2 was verified by ELISA and Western blot.To further investigate whether Monkey PRA has the function of blocking PRRSV infection of PAM and MARC-145 susceptible cells.First detect the virulence of Monkey PRA protein on cells.Western blot,relative fluorescence quantitative PCR,TCID50 and other methods were used to verify the viral protein expression level and transcription level,as well as the cell supernatant virus titer to verify that Monkey PRA protein blocked PRRSV SD16 infection on PAM and MARC-145 susceptible cells..Finally,the above method was used to verify the effect of Monkey PRA protein on PAM and MARC-145 susceptible cells to block the infection of different PRRSV strains.The results of the study found that the highly purified protein Monkey PRA was successfully obtained through the prokaryotic expression system.The results of ELISA and Western blot showed that the Monkey PRA protein has good biological activity and can be combined with the PRRSV GP5 protein anti-unique antibody Mab2-5G2 Indirectly prove that Monkey PRA protein can be combined with PRRSV.Blocking test results showed that Monkey PRA protein can effectively reduce PRRSV SD16 infection of PAM and MARC-145 cells.At the same time,Monkey PRA protein has the effect of blocking infection of different strains of PRRSV,indicating that it has a broad spectrum of blocking viral infection.It was finally verified that the monkey-derived PRA protein can have a similar inhibitory effect on PRRSV infection as that of swine-derived PRA,which has enlightening significance for further exploring the deeper function of monkey-derived MYH9 in PRRSV infection at the cellular level.