Research On The Effect Of Stimulator Of Interferon Genes During The ORFV Infection

Stimulator of interferon genes(STING)is found as a key linker protein in natural immune signaling pathway in recent years,and it plays an important role in the signal transduction of cytoplasmic DNA and immune defense.STING may be involved in the innate immune response induced by a variety of pathogens' dsDN A or RNA stimulation,which can raise TBK1 and activate downstream IRF3 to induce the production of type I interferon and regulate the immune response against infection.Orf virus(ORFV)is an important zoonotic pathogen,it can infect sheep,goats,a variety of wild animals and humans.Sheep aphtha caused by O RFV has become one of the most important diseases which restricting the development of sheep industry and caused many difficulties in clinical prevention and treatment in C hina.In this study,we first obtained the sequence of goat antiviral gene STING and prepared the rabbit anti-goat polyclonal antibody.O n this basis we studied the molecular mechanism of STING during ORFV infection,which provides the basic data for exploring the role of STING in the prevention and control of goat disease.Details are as follows:Cloning and sequence analysis of STING gene in Hainan black goatThe STING gene of Hainan black goat was amplified by RT-PCR method and submitted to GenBank for Accession No.:KR060015.The analysis of sequences revealed that the STIN G gene encodes 378 amino acids.Homology analysis of STING protein of different species showed that the homology of STING gene in Hainan black goat and cattle was 97.4 % while the lowest homology with zebrafish was 47.1 %.The phylogenetic trees also revealed that STING was conservative within species while highly variant between species.Prokaryotic expression and preparation the polyclonal antibody of STING proteinThe pET-30a(+)-STING which got rid of the transmembrane domains were constructed successfully using the p ET-30a(+)plasmid,and the recombinant protein approximately 28 KDa was successfully expressed in E.coli.Rabbit anti-STING polyclonal antibody was prepared by immunizing the New Zealand white rabbits using the recombinant protein.The antibody can maintain a good specificity and antigen activity confirmed by western blot and ELISA methods.The specific expression and tissue distribution of STING proteinReal-time PC R was used to explore the expression of STING mRN A in different tissues of Hainan black goat.Results showed that the highest expression was found in mesenteric lymph nodes,whereas in the heart,spleen and brain,the expression was higher than that in lung,kidney and liver.And the locations of STING protein in different cells was detected by immunohistochemistry.Results showed that the STING protein was expressed in the cytoplasm of positive cells.In the cytoplasm of myocardial cells and liver cells,splenic germinal center and peripheral lymphocytes,bronchial epithelial cells and alveolar epithelial cells,part of mesenteric lymph node germinal center and peripheral lymphocytes were strong positive expression.While the renal tubular epithelial cells and cerebral cortex were weakly positive.Construction and expression of overexpression and interference vectors of STING proteinpEGFP-N1 and pSuper.Retro.Neo+GFP vectors were used to construct STING expression plasmid pEGFP-N1-STING and STING interference plasmid shRNA-STING.Results showed that the recombinant plasmids could be successfully expressed in the cells by transfection,observation of the green fluorescence and western-blot.The fusion protein of STING and GFP about 69 KDa can be detected,and the interference efficiency of STING protein is higher about 70 %.This indicates that the establishment of the special and negative expression of the STING protein of the cell lines.The antiviral research of STING when ORFV infected the OFTu cellsAfter ORFV infected the normal,overexpression and interference of STING protein cell lines,the levels of related signaling proteins,cytokines and the growth curve of virus were detected at different times.The results showed that it could activate a series of antiviral response which caused by a variety of receptors such as STIN G,DDX41,RNA Poly III,RIG-1,c GAS,TBK1 and IRF3 after the virus infected the normal cells.The antiviral ability of cells were significantly enhanced after overexpression of STING protein,while the cells resistance decreased after STING protein was interference.Analysis of the growth curve of virus showed that STING protein could effectively inhibit the appreciation of virus,and the interference of STIN G was beneficial to the appreciation of virus.These results suggest that STING protein plays an important role in antiviral process.