Antiviral Function Study Of Fusion Protein Of Duck Tembusu Virus Capsid And Staphylococcus Aureus Nuclease

Tembusu virus（TMUV）belongs to the Flavivirus genus in the family Flaviviridae.It is an enveloped,non-segmented,single positive-stranded RNA virus transmitted by mosquitoes.It main infects ducks.Ducklings and laying ducks are susceptible,growing ducks have a certain resistance,it can also infect chickens,geese,etc,mainly causing a decline in egg production of egg poultry,bringing great economic losses to the poultry industry.In this study,according to the gene sequence of duck tembusu virus（DTMUV）CQW1 strain that was isolated from clinical duck disease material and submitted to NCBI（accession No.KM233707.1）by our research group and staphylococcus aureus nuclease（SNase,accession No.CP006630.1）sequence,specific primers encoding DTMUV Capsid protein（Cap）and SNase gene were designed,the Cap and SNase genes were amplified and fused,the fusion recombinant plasmids were constructed,fusion protein stable cell lines were constructed,and anti-duck tembusu virus was studied in vitro and in vivo.The results are reported as follows:1.Biological characteristics analysis of DTMUV Cap and SNase fusion proteinTo explore the antiviral effect of SNase,the DTMUV Cap and SNase were fused to obtain target fragments Cap-SNase and Cap-Linker-SNase.Eukaryotic recombinant plasmids pc DNA3.1（+）-Cap-SNase and pc DNA3.1（+）-Cap-Linker-SNase were constructed,and the expression and localization of fusion proteins were detected in BHK21 and DEF cells.The enzymatic activity and cytotoxicity of fusion proteins were detected by DNA degradation and MTT assay,respectively.Anti-DTMUV assay was performed in BHK21 and DEF cells.The results showed that the fusion proteins Cap-SNase and Cap-Linker-SNase were mainly expressed in cytoplasm.The DNA degradation assay of heat-treated proteins showed that Cap-SNase and Cap-Linker-SNase showed more obvious DNA degradation with the increase of protein amount.At the same protein amount,Cap-Linker-SNase was better than Cap-SNase in degrading DNA.The morphology and death of BHK21 and DEF cells expressing fusion proteins Cap-SNase and Cap-Linker-SNase were not different from those of the negative control group.MTT assay showed that there was no difference in the activity of BHK21 and DEF cells expressing Cap-SNase and Cap-Liner-SNase compared with the negative control group.Antiviral results showed that at 48 h,60 h,72 h,84 h after virus infection,the Cap-SNase and Cap-Linker-SNase groups in BHK21 and DEF cells had different degrees of decrease in viral load than those of the negative control groups.These results indicate that the fusion proteins Cap-SNase and Cap-Linker-SNase have thermostability and nuclease activity but no cytotoxicity,and show anti-DTMUV effect.2.Construction and characterization of stable cell line of DTMUV Cap and SNase fusion protein and exploration of anti-DTMUV effect in vitroIn order to continuously explore the anti-DTMUV effect of the fusion protein,the monoclonal cell lines were constructed using lentivirus,and its stability at the gene and protein levels was detected.The enzyme activities of the fusion protein Cap-SNase and Cap-Linker-SNase expressed by the cell lines were detected by DNA degradation method,and the anti-DTMUV test was carried out on the monoclonal stable cell line.The results showed that the target gene and target protein could still be detected in the positive monoclonal cell lines even after 80 generations.The DNA degradation test showed that with the increasing amount of fusion protein（2.5,3.0,3.5μg）,the DNA degradation phenomenon was more obvious.There was no difference in cell morphology and state between the stable cell line and BHK21 cells.Antiviral test showed that the viral load of BHK21/Cap-Linker-SNase group was 10^0.5 times lower than that of the control group at 36h,48 h and 60 h,while the antiviral effect of Cap-SNase group was not obvious.The above results indicate that the successfully constructed monoclonal cell lines BHK21/Cap-SNase and BHK21/Cap-Linker-SNase can be stably passed and expressed,and the expressed fusion proteins Cap-SNase and Cap-Linker-SNase have heat resistance and nuclease activity.The fusion protein integrated into BHK21 is cell tolerant and has no cytotoxicity,but the anti-DTMUV effect of the fusion protein expressed by the cell line is not obvious.3.Anti-DTMUV of fusion protein with lentivirus as vector in vivoIn order to further explore the anti-DTMUV effect of fusion proteins Cap-SNase and Cap-Linker-SNase in ducks,we used lentivirus as vector,the concentrated lentivirus with fusion protein infected 7-day-old duck embryos,duck embryos were hatched into ducklings,DTMUV was used to infect 7-day-old ducklings,and then duck feces,blood and visceral tissues（heart,liver,spleen,lung and kidney and brain）were collected 13 days after DTMUV infection.The anti-DTMUV effect of the fusion protein in ducks was explored by pathological observation,RT-q PCR,body weight monitoring and other tests.The results showed that the DTMUV infection caused pathological symptoms,such as hemorrhage in heart,liver,spleen,lung kidney and brain,could be alleviated by the fusion proteins from lentivirus-Cap-SNase and lentivirus-Cap-Linker-SNase.The results of RT-q PCR showed that the viral load in blood,tissue（brain,spleen）and fecal virus emission in the fusion protein group were significantly lower than those of the 1640 and lentivirus-PHBLV groups.The results of body weight monitoring showed that DTMUV infection caused growth retardation in ducks,which was alleviated by the fusion protein.These results indicate that the fusion proteins Cap-SNase and Cap-Linker-SNase have anti-DTMUV effect in ducks.In conclusion,in this study,DTMUV Cap and SNase were fused to study the anti-DTMUV effect in vitro and in vivo.The transient expression of fusion protein showed anti-DTMUV effect,while the fusion protein expressed by the stable cell lines showed no obvious anti-DTMUV effect,but the fusion protein with lentivirus as the vector had obvious anti-DTMUV effect in duck.