Cloning,Expression Pattern And Antiviral Activities Of OAS Gene In Chinese Giant Salamander,Andrias Davidianus

Chinese Giant Salamander(Andrias davidianus)is a national second-class protected animal which has been listed in the appendix of CITES Convention.In recent years,with the continuous rise of artificial breeding of giant salamanders,the harm of diseases to giant salamanders is becoming more and more serious,among which,Chinese Giant Salamander iridovirus(GSIV)is the most harmful,causing huge economic losses to the breeding industry.The innate immune system plays a vital role in resisting pathogen invasion and protecting the body from pathogen infection.The interferon-mediated 2’-5’-Oligoadenylate synthetase(OAS)is one of the most response proteins in antiviral infection.The enzyme of OAS is widely distributed in jawed vertebrates such as fish,reptiles,birds and mammals,and is related to immune regulation.At present,the antiviral function of OAS enzyme has not been reported in amphibian.Therefore,as an important part of the innate immune system,the characteristics and functions of OAS in Chinese giant salamander need to be investigated.In this study,the Open Reading Frame(ORF)c DNA of Chinese giant salamander OAS gene(AdOAS)was cloned and identified for the first time,and its expression pattern and antiviral function were detected in vivo and in vitro.The contents are as follows:1.cDNA cloning and sequence analysis of AdOAS geneUsing cDNA from spleen and kidney of Chinese giant salamander as template,the ORF sequence of AdOAS was 1185 bp in length and encoded 394 amino acids,including a nucleotide transferase domain(40-143 aa)and one conserved OAS1 C superfamily domain(165-341 aa).The molecular weight of AdOAS encoded protein was calculated to be 45 k Da and the theoretical isoelectric point was 7.968.The sequence alignment results selected by Gen Bank showed that AdOAS had 76% ～ 99%homology with common vertebrates.Phylogenetic tree showed that AdOAS clustered into a clade with Aminostoma mexicana OAS1 and had high homology.2.Expression patterns of AdOAS in vivo and in vitroQuantitative real-time PCR(q RT-PCR)showed that AdOAS m RNA was expressed in all tissues examined.Compared with spleen with the lowest expression level,the m RNA relative expression level of AdOAS in intestine was the highest,which was about 9 times that in spleen.The expression level was higher in heart,and the relative expression level was about 8 times that in spleen.After GSIV infection,the level of AdOAS m RNA in kidney increased significantly at 6 h and 12 h,reached the peaked at 48 h,which was significantly different compared to the control group.Finally,the m RNA level increased slightly at 72 h,and basically remained stable compared with the initial 0 h.In liver,the m RNA level of AdOAS increased significantly at 24 h and48 h after infection,and peaked at 72 h after infection,which was significantly different from the control group.In spleen,the m RNA level of AdOAS was slightly up-regulated at 6 h after infection.It reached the peak at 72 h and showed significant difference compared to the control group,while there was no significant change at 12 h,24 h and48 h.In vitro,after GSIV infection AdOAS m RNA level was significantly up-regulated at 24 h,and reached the highest level at 48 h.3.Construction and functional study of AdOAS gene eukaryotic plasmidEukaryotic plasmid pm Cherry N1-AdOAS was constructed and transfected into GSM cells.Western blot confirmed that the molecular weight of pm Cherry N1-AdOAS protein was 73 k Da,while the molecular weight of transfected pm Cherry N1 protein was 28 k Da.At 48 h post transfection,the GSM cells were cleaned and fixed in Confocal Dish,and the cell nucleus were stained by DAPI.The subcellular localization was observed under Laser Scanning Confocal Microscope showed that AdOAS protein was mainly expressed in cytoplasm,and few were expressed in nucleus.After pm Cherry N1-AdOAS was transfected into GSM cells for 48 h,and GSIV MCP m RNA levels were significantly decreased at 12 h,24 h,and 48 h post GSIV infection,compared with the normal cells and cells transfected with pm Cherry N1.Western blot analysis of MCP protein expression showed that GSIV MCP protein synthesis was significantly decreased in AdOAS overexpression group compared with the normal cells and cells transfected with pm Cherry N1 at 24 h,48 h and 72 h post GSIV infection.The effect of AdOAS on the copy number of MCP gene during virus infection was analyzed using Digital Droplets PCR(dd PCR).The results showed that the MCP gene copy number of GSIV was significantly decreased at 48 h and 72 h compared with normal group and pm Cherry N1 transfected group.All the above results showed that AdOAS overexpression could significantly inhibit the replication of GSIV and had significant antiviral function.