Establishment And Application Of Antiviral Drug Screening Model Based On NSN Of WSSV

White spot Disease（WSD）caused by White spot syndrome virus（WSSV）is a viral disease with rapid onset,high fatality rate and strong infectiality,which brings serious economic losses to the global prawn culture every year.As a low invertebrate animal,shrimp do not have adaptive immune system.Therefore,antiviral drugs become an effective means of WSD prevention and control.Since some medical apis such as ribavirin have been banned,there has been no effective drug for prevention and control of WSD in clinic so far,and the development of anti-WSSV drugs has become an urgent demand of the industry.Viral cell line culture and in vivo animal models have been the traditional screening methods for antiviral drugs.Due to the lack of proliferative cell lines of shrimp-like viruses and the long cycle,high cost,unstable laboratory animals and other factors that rely on animal in vivo model screening,the difficulty and cycle of shrimp drug innovation research have been increased.Therefore,it is of great theoretical significance to construct a new high-throughput drug screening technology platform independent of shrimp cells and animal models,and research on new drug innovation against shrimp viroids such as WSSV.With the analysis of the genomic function of WSSV protein,targeting key functional enzymes in the process of WSSV replication has become a new choice for drug screening.Moreover,enzyme targeted drug screening has the advantages of short cycle,clear target,cost saving and large-scale screening.In this study,non-specific endonucrenases,key enzymes in WSSV replication,were used as targets to establish anti-WSSV drug screening models,and the interaction between the active monomer and the target protein was preliminarily studied.The results of this study are as follows:1.Prokaryotic expression of WSSV-NSNIn this study,the non-specific endonucrenase（WSSV-NSN）sequence of white spot syndrome virus was optimized,and the recombinant prokaryotic expression plasmid p ET32a-NSN was synthesized.After expression and purification by Escherichia coli BL21（DE3）,WSSV-NSN recombinant expression protein with the size of about 60 k Da was obtained.2.Establishment of WSSV-NSN drug screening modelThe activity of WSSV-NSN enzyme was determined.The specific activity of WSSV-NSN enzyme was 166.7 U/mg.Mg²⁺had a strong activation effect on WSSV-NSN enzyme activity,and the Mi constant Km value was 0.106 mol/L.The concentration of recombinant protein was 0.11 mg/m L.According to the WSSV-NSN enzymatic reaction principle,the enzymatic reaction system was established,and the WSSV-NSN enzymatic reaction conditions were optimized.The results showed that the optimal enzymatic reaction conditions were as follows:reaction temperature37℃,p H=7,substrate concentration10mmol/L,reaction time 20 min,Mg²⁺concentration 10 mmol/L.A drug screening model with WSSV-NSN as target was established.The statistical index Z’factor was used to evaluate the stability of the screening model,Z’=0.70,which met the requirements of the drug screening model.3.Application of WSSV-NSN sieve model36 antiviral drugs in the laboratory drug library were selected,and the established WSSV-NSN drug screening model was used for activity evaluation.The results showed that:Five compounds,luteolin,β-sitosterol,genipine side,cinnamic acid and1-hydroxyphenazine,had certain inhibitory effects on WSSV-NSN,with inhibitory rates of96.6%,84.1%,82.3%,80.4%and 58.5%,respectively.It was found by circular dichroism（CD）that both luteolin andβ-sitosterol could interact with WSSV-NSN protein and cause the change of the secondary structure of the protein.It is speculated that this may be the key pathway for the antiviral effect of drugs.In conclusion,this study successfully established an effective anti-WSSV drug screening model targeting WSSV-NSN,and found that luteolin,β-sitosterol,genipine side,cinnamic acid and 1-hydroxyphenazine can effectively inhibit the enzyme activity of WSSV-NSN and have the potential to resist WSSV,which provides a scientific basis for the innovation of shrimp viroid new drugs.