Cloning And Identification Of Duck TRM32 And Analysis Of Its Mediated Anti-Duck Tembusu Virus

Our country is a big country of duck breeding.In recent years,duck virus disease has caused serious harm to duck breeding industry in our country.Duck tembusu virus(Duck Tembusu virus,DTMUV)caused by duck tembusu virus has the characteristics of acute disease and fast transmission.It is a serious epidemic disease that endangers laying duck and meat duck breeding.The DTMUV belongs to the family Flaviviridae and the genus Flaviviridae,and is a single strand RNA virus.TRIM32(triplite motif 32),an important member of the E3-ubiquitin ligase family,was discovered in 1995 and consists of two B-Box、Coil-coiled domains conserved at the N end and variable domains at the C end.TRIM32 studies in mammals and fish have found that they regulate central nervous system function and antiviral effects.There are few studies on TRIM32 in avian antiviral activities,especially in waterfowl,for further understanding of the role of TRIM32 in the anti-duck tembusu virus in cherry valley duck,the gene TRIM32 cherry valley duck was cloned and its inhibitory effect and mechanism on DTMUV were analyzed.The study consists of four parts:1、Amplification of the TRIM32 Gene of Cherry Valley DuckThe primers were designed and identified according to the prediction sequence of duck TRIM32 gene published on the NCBI,and the RNA of spleen of healthy cherry valley duck was extracted and retracted as a template for PCR amplification reaction.the target gene fragment was obtained by PCR reaction.At last,the genes of duck TRIM32 were obtained by electrophoresis and gel recovery.The CDs region size of duck TRIM32 is 1950 bp,encoding649 amino acids.phylogenetic tree analysis showed that TRIM32 had higher homology with Taeniopygia guttata in birds,and lower homology with reptiles.2、The distribution of TRIM32 in the tissues of cherry-grain duckThe RNA of heart,liver,spleen,lung,kidney,brain,cerebellum,brainstem,thymus,pancreas,bursa,duodenum,jejunum,ileum,cecum,skin,muscle,glandular stomach,muscle stomach,trachea,esophagus and other 21 tissues of healthy cherry valley duck were extracted and retracted as c DNA template.The ileum was used as reference tissue and fluorescence quantitative PCR technique was used to determine the distribution of TRIM32 in these 21 tissues of cherry valley duck.According to the results,the expression of TRIM32 in trachea was the highest,34.3 times higher than that of reference tissue,duodenum,lung,skin,pancreas,muscle,brain and thymus.The expression was higher in the bursa and lower in jejunum,spleen and heart.3、Detection of antiviral effects of TRIM32 and related factorsTo explore the antiviral effect of TRIM32,this study constructed a TRIM32 eukaryotic expression vector and synthesized interference against TRIM32 RNA,The results showed that the viral content was significantly lower than that of the blank control group after overexpression TRIM32,but the knock-down TRIM32 had the opposite effect.The fluorescence quantitative PCR DTMUV,infection after overexpression TRIM32 showed that the expression of IFN-α、IL-1β、RIG-I、Mx、OAS was up-regulated,and it was the most significant 12 h after infection.Among them IFN-α the largest increase,IFN-α expression was16.9 times higher at 12 h,Mx up 7.1 times,OAS 13.2 times,RIG-I 2.9 times,IL-1β2.4times.Knock low TRIM32 have opposite effect,among them IFN-α down-regulation is most significant.These results suggest that TRIM32 can enhance the up-regulation of IFN-α and other cytokines caused by DTMUV infection and may play an antiviral role in the early stage of viral infection.4、 Construction of Prokaryotic Expression Vector TRIM32 DuckThe preparation of a specific antibody for duck TRIM32 is necessary for further study of its function.In this study,the prokaryotic expression vector of duck TRIM32 was successfully constructed and verified by enzyme digestion,sequencing,SDS-PAGE,etc.The results showed that duck TRIM32 protein could be expressed in BL21 expression Escherichia coli in prokaryotic expression vector,which provided an effective antigen preparation method for the preparation of characteristic antibodies against duck TRIM32 in the future.The full-length CDs sequence of duck TRIM32 was obtained by cloning from cherry valley meat duck in this project.The homology evolution tree analysis was carried out,and it was found that duck TRIM32 had higher homology with Taeniopygia guttata in birds.These results suggest that TRIM32 can enhance the up-regulation of IFN-α and other cytokines caused by DTMUV infection and may play an antiviral role in the early stage of viral infection.TRIM32 was also widely distributed in other tissues.Over-expression and RNA interference experiments showed that TRIM32 could significantly reduce DTMUV replication in DEF cells.The upregulation of IFN-α、IL-1β、RIG-I、Mx、OAS expression in DTMUV infected DEF cells can be promoted early in the infection,while the interference experimenthad the opposite effect.The results will be helpful to TRIM32 further research on innate immune response and anti-infection mechanism in waterfowl,and also provide a theoretical basis for other biological functions of TRIM32 molecules,which is of great significance for the prevention and treatment of waterfowl infectious diseases.