The Research Of Expression Of PNGase F Protein In Yeast,Purification And Its Antiviral Activity

The frequent outbreak of viral infectious diseases has become a serious threat to global public health and economic development.At present,the measures to deal with viral diseases are mainly vaccination for epidemic prevention,which has the problems of long cycle,low titer and high cost.In the face of the outbreak of new viral diseases,efficient targeted drugs have become the first choice,including antiviral polypeptide drugs.In antiviral peptide drugs,the narrow scope of application has become the main limiting factor for the application of some drugs,and it is urgent for researchers to develop broad-spectrum antiviral drugs.The membrane glycoproteins of enveloped viruses are widely distributed and play an important role in the process of virus infection.In our previous study,we found that PNGase F(Peptide Nglycosidase F,PNGase F)peptide can inhibit the activity of a variety of enveloped viruses.PNGase F is a deamidase with a molecular size of 34.8 k Da,which can catalyze the cleavage of N-glycans on glycoproteins.Therefore,we speculate that PNGase F can inhibit viral infection by deglycosylation of envelope glycoproteins.This study intends to analyze the molecular mechanism of PNGase F deglycosylation in the process of envelope virus infection,and lay a theoretical foundation for the comprehensive discussion of the broad-spectrum antivirus application of PNGase F.Firstly,the yeast expression vector of PNGase F was successfully constructed and transformed into Pichia pastoris host bacteria according to conventional operation.After the positive strains were successfully screened,the PNGase F protein was highly expressed,and the content of the exogenous protein in the yeast supernatant was 194.8 mg / L.The deglycosylation activity of PNGase F expressed in yeast was verified by using RNase B as substrate.Pseudorables virus(PRV)and Porcine epidemic diarrhea virus(PEDV)were treated with appropriate concentration of PNGase F.The experimental results confirmed that PNGase F could significantly inhibit the infection of the above two viruses in cells,and 15 μM PNGase F could completely inhibit the proliferation of the virus in cells.The results of cytotoxicity experiments showed that PK-15 and MDCK cells still had 100 % viability after treatment with27 μM PNGase F at 37 °C for 36 h,and Vero-CCL81 cell viability also exceeded 90 %.Further evaluation of the effect of PNGase F on different infection stages of virus infection showed that the inhibitory effect of PNGase F on the two viruses at specific stages was quite different.In the process of anti-PEDV infection,PNGase F had a significant inhibitory effect on the adsorption,invasion and replication stages of the virus,and had the best inhibitory effect on the replication stage.PNGase F at a concentration of 6μM could prevent more than 50 % of the virus from successfully infecting.At the same time,the inhibitory effect of PNGase F on PRV was detected mainly in the two stages of virus adsorption and invasion.In view of the preventive effect of PNGase F on two viral infections,it was found that PNGase F only showed a maximum inhibitory effect of about 20 % on PRV,while the preventive effect on PEDV was not obvious.In this study,the inhibitory effect of PNGase F on PRV and PEDV was detected in cells by establishing a model of large-scale expression of PNGase F protein in yeast,and its inhibitory mechanism was preliminarily discussed.By interpreting the large-scale preparation method of PNGase F and the inhibition mechanism of virus,it can provide a new reference for the development of broad-spectrum antiviral drugs based on PNGase F.