Establishment Of Drug Screening Method Targeting WSSV Envelope Protein And Screening Of Chinese Herbal Medicine

In recent years,White spot syndrome virus disease（WSD-RRB-caused by White spot syndrome vir WSSVWSSV）has seriously threatened the sustainable development of shrimp industry in our country.Immunoenhancers,vaccines,RNAi,Chinese herbal extracts and stress-resistant seed breeding are the hot research fields of WSD prevention and control,but there is no substantial breakthrough Because of the shortcomings of shrimp in vaccine prevention and control,drug prevention and control is still the most direct and effective means in production.Virus cell line culture and animal in vivo model have been traditional screening methods.Because of the lack of replicable cell lines for shrimp viruses,it is difficult to achieve high-throughput screening in vivo due to the long cycle,high cost and instability of experimental animals,makes the drug development process slow.Therefore,the construction of shrimp cell-independent in vitro drug screening system has important theoretical value for new antiviral drug innovation.WSSV envelope proteins VP26 and VP28 are involved in the key steps of virus replication,such as adhesion,infection,assembly and release,and can be used as targets for antiviral drug screening.In this study,we constructed an in vitro drug screening system independent of shrimp cells by eukaryotic expression model targeting the envelope proteins VP26 and VP28 of WSSV,the anti-WSSV activities were screened from 100 Chinese herbal medicines,and the anti-WSSV activities of the main active components were further verified.The results were as follows:1.Construction of drug screening system targeting VP26 and VP28 of WSSV in vitroThe gene fragments of envelope protein VP26 and VP28 were cloned from WSSV by PCR,and the gene fragments of Luciferase（Luc）were obtained from Plasmid p MT-Fluc.The recombinant plasmids pcDNA3.1-VP26-Luc and pcDNA3.1-VP28-Luc were obtained by inserting the LUC gene fragment into the eukaryotic expression vector pcDNA3.1-VP26-Luc and pcDNA3.1-VP28-Luc respectively.The fusion proteins VP26-Luc and VP28-Luc were obtained by eukaryotic expression after transfection of the two recombinant plasmids into 293T cells.The eukaryotic expression system was optimized with different transfection cells（s 2,Sf9,293T）and different transfection conditions（incubation time and transfection time）,the fusion proteins VP26-Luc and VP28-Luc were expressed in 293T with the highest expression efficiency.The screening system in vitro was verified by the positive drugs genipin（GN）,Genipin（GP）and genipin acid（GPA）,the results showed that the inhibition rates of GN,GP and GPA on VP26-LUC Fusion protein were 67.16%,59.12%and 46.81%,respectively.The inhibition rates of GN,GP and GPA on VP28-Luc fusion protein were 85.18%,53.79%and65.49%,respectively,the results of 12 experiments showed that the z’factor of 12 experiments was>0.4,which indicated that the system was stable,can be used for subsequent drug screening.2.Discovery of anti-WSSV drugs based on in vitro screening system of WSSV envelope protein VP26 and VP28The methanol extracts of 100 Chinese herbal medicines were screened by the drug screening system in vitro.The recombinant plasmids pcDNA3.1-VP26-Luc and pcDNA3.1-VP28-Luc were transfected into 293T cells,respectively,and then methanol extract of Chinese herbal medicine was added.The results showed that the methanol extracts of Prunella vulgaris L.,Senecio scandens Buch.-Ham.ex D.Don and Perilla frutescens（L.）Britt.could inhibit the expression of VP26,and the methanol extract of Prunella vulgaris L.had the highest inhibitory activity on the expression of VP26,the inhibitory rate was 92.29%,IC₅₀ was 7.88 mg/L,and the methanol extract of Prunella vulgaris L.had strong inhibitory activity on the expression of WSSV envelope protein VP28,the inhibitory rate was 96.67%,IC₅₀ was 7.34 mg/L.The main active component ursolic acid of Prunella vulgaris L.was studied by the same method.The results showed that ursolic acid also inhibited the expression of VP26 and VP28.The inhibitory rate of ursolic acid on the expression of VP26 was 96.61%,IC₅₀ was 1.47 mg/L,and the inhibitory rate of ursolic acid on the expression of VP28 was 98.80%,IC₅₀ was 1.01 mg/L.3.Validation of anti-WSSV activity of ursolic acid in vivoThe anti-WSSV activity of ursolic acid was tested in vivo in a Procambarus clarkii model.The results showed that the survival rate of the Procambarus clarkii Procambarus clarkii treated with ursolic acid was 45.45%after 10 days,while all the Procambarus clarkii in the control group died.The results of RT-qPCR showed that ursolic acid could significantly inhibit the proliferation of WSSV in Procambarus clarkii gill at a concentration of 40mg/kg for 24h with an inhibition rate of 92.56%.Ursolic acid showed good antiviral activity in the muscles,hepatopancreas,intestines and claws of the Procambarus clarkii.The inhibition rates of ursolic acid on WSSV were 90.17%,85.45%,94.20%and 91.99%,respectively.In order to further explore the antiviral mechanism of ursolic acid,the expression of immune related genes in Procambarus clarkii was detected by relative quantitative experiment.The results showed that ursolic acid could significantly inhibit the expression of inflammatory genes（cox1 and cox2）,the expression of COX1 was down-regulated by about 40%,the expression of COX2 was down-regulated by about 65%,and ursolic acid could significantly up-regulate the expression of antioxidant related genes（c Mnsod,m Mnsod,cat,GST）,the expression of GST was up-regulated about 11-fold.The results indicated that ursolic acid had good anti-WSSV activity,which was screened by the screening system in vitro targeting VP26and VP28 of WSSV.In conclusion,this study constructed an efficient and rapid in vitro drug evaluation model that does not depend on the in vivo model and targets the envelope protein of WSSV,and verified its reliability and stability.At the same time,ursolic acid with good anti-WSSV activity was successfully screened by this model.This study is helpful to improve the theoretical system of drug control of shrimp viral disease,and has important theoretical guiding value in the prevention and control of aquatic animal diseases.