The Expression And The Antiviral Activity Of Recombinant Griffithsin Protein On Porcine Epidemic Diarrhea Virus

Background: The Porcine epidemic diarrhea(PED)disease caused by Porcine epidemic diarrhea virus(PEDV)has caused serious economic losses to pig farmers.Due to the high variability of PEDV and other reasons,the attenuated virus vaccine and inactivated vaccine of current market can not fully meet the requirements of effectiveness and safety.Therefore,there is an urgent need to develop new PEDV vaccines,or antiviral drugs for the prevention and treatment of PED.The Griffithsin(GRFT)protein is a broad-spectrum lectin resistant to coronavirus extracted from the red alga Griffithsia sp.The lectin inactivates the virus by directly binding to the sugar chain on the viral envelope protein,inhibiting its binding to the host cell receptor.Because the sugar chain on the envelope protein of coronavirus is generally more conservative,the antiviral effect of GRFT protein is not affected by the easy mutation of amino acids on the virus envelope membrane,and thus it is expected to overcome the problem of the mutant virus strain caused by the amino acid variation of the virus.Research purposes: This article attempts to detect the activity of GRFT protein against PEDV infection,and provides a new idea for the prevention and treatment of PED.Research content: According to the GRFT protein sequence,the gene sequence was deduced and the double-stranded DNA was synthesized.The recombinant expression vector pET28a-SUMO-GRFT was constructed to express and purify the recombinant GRFT.Then,the GRFT protein was incubated with PEDV to study the effect of GRFT protein on PEDV infection.The inhibitory effect of the cells was determined and their effective doses for inhibition of viral infection were determined;to further explore the role of GRFT in PEDV targets,PNGase F was used to treat PEDV;finally,the antiviral activity of the GRFT protein in the heating,acid bathing,the effects of trypsin digestion,pepsin digestion,and storage conditions were evaluated initially.Research Methods: Protein expression and purification,SDS-PAGE,immunoblot experiments,cell plaque assayResearch results: Prokaryotic expression of recombinant GRFT protein has a good effect on the inhibition of PEDV infection,and can be used as a candidate drug against PEDV infection.Vero cells infected with PEDV treated with expressed recombinant GRFT protein,the experimental results show that recombinant GRFT protein could significantly inhibit the infection of Vero cells by PEDV,and the anti-infective effect of the protein was dose-dependent.The relative plaque inhibition rate of the recombinant GRFT protein of 1 ?M was 95.8%,and the GRFT protein concentration required to achieve 50% relative plaque inhibition was 39.22 nM.The results of GRFT protein stability experiments show that the recombinant GRFT protein expressed herein has good thermal stability,acid stability,trypsin tolerance,and good pepsin tolerance.The storage experiment of GRFT protein showed that the recombinant GRFT protein can be stored at 4? for at least 6 months and at room temperature(25?)for at least 3 months.Significance of the study: This article demonstrates for the first time that the recombinant GRFT protein has a good anti-infective effect on PEDV and provides a new idea for the prevention and treatment of PED.