Characterization,Expression Pattern And Antiviral Activities Of Mx Gene In Chinese Giant Salamander,Andrias Davidianus

The Chinese giant salamander(Andrias davidianus),a second-class protected animal in China and the largest amphibian in the world,belongs to the species that migrate from aquatic animals to land animals,it has important scientific significance in the study of species origin,evolution,biodiversity and gene variation.Moreover,due to the rapid development of Chinese giant salamander artificial culture in recent years,the viral disease caused by Chinese giant salamander iridovirus(GSIV)has caused huge economic losses to the industry,so it has great significance to study the antiviral defense mechanism of Chinese giant salamander for the prevention and control of its viral diseases.Mx protein is an interferon-induced effector.It was named Mx(myxovirus resistance)gene due to the resistance to influenza virus of Mx gene encoded protein.Mx protein is widely found in vertebrates and invertebrates,which is widely expressed in various tissues,and has a broad spectrum of antiviral activity.At present,Mx gene has been studied in mammals,birds and fish,However,no more studies were conducted on Mx in amphibians.In this paper,the Fluorescence quantitative PCR was used to investigate the expression patterns of Mx gene in different tissues and cells after GSIV infection.The recombinant plasmid was constructed and transfected into GSM cells,and the cell morphology observation,dd PCR detection of MCP gene copies and Western blotting dection of MCP protein synthesis were analyzed to verify the antiviral function of Mx,so as to lay a foundation for the immune defense system and antiviral infection mechanism of Chinese giant salamander.The major results are as follows: 1.Cloning and structural analysis of Chinese giant salamander Mx(Ad Mx)geneRT-PCR was used to clone the full-length c DNA of Chinese giant salamander Mx(Ad Mx)gene.Bioinformatics was used to analyze the sequence features of Ad Mx.The full-length of the Ad Mx gene is 2848 bp,with a 5' UTR of 132 bp of and a 3' UTR of 604 bp.It has an open reading frame(ORF)of 2112 bp and encodes for 703 amino acids.Amino acid sequence analysis showed that the Ad Mx sequence had a signal peptide with a length of 33 amino acids,and the predicted molecular weight of the protein was 79.09 KD and a theoretical isoelectric point of 5.33.Similar to proteins encoded by Mx genes in other species,Ad Mx possesses a putative hydrophobic signal peptide at the N-terminus,a GTPase domain containing a triplet GTP-binding region and GTPase effector domain GED at the C-terminus.A dynamin family signature with the sequence of LPRGSGIVTR was also observed in Chinese giant salamander.The homology of nucleotide sequences was 55.0%-77.0% by comparing with the Mx gene sequences of vertebrates registered in Gen Bank.Phylogenetic tree results showed that Ad Mx and Xenopus tropicalis Mx1 formed a cluster.2.Tissue expression distribution and induced expression of Ad Mx geneq RT-PCR analysis indicated that the expression of Ad Mx was the highest expression levels in the brain,the intermediate levels in the thymus,heart,spleen and kidney and the lowest levels in the intestine,liver and muscle in healthy giant salamender.Following GSIV infection,Ad Mx expression level in spleen and kidney significantly increased and peaked at 48 h.In muscle,Ad Mx expression level significantly increased at 48 h and 72 h.In vitro,after GSIV infection in GSM cells,the expression of Ad Mx in GSIV-infected cells was significantly up-regulated and reached the highest expression level at 72 h,compared with the control GSM cells.3.Construction and functional study of recombinant expression plasmid of Ad Mx geneThe recombinant expression plasmid of p EGFP-N1-Ad Mx was constructed and transfected into Chinese giant salamander muscle cell(GSM).Western blotting and immunofluorescence experiments showed that the recombinant plasmid p EGFP-N1-Ad Mx was successfully expressed in GSM cells and located in the cytoplasm.The experimental results of GSIV-infected cells showed that Ad Mx could significantly inhibit the Cytopathic effect(CPE)of GSIV-infected GSM cells.In Ad Mx gene over-expressed GSM cells,after GSIV infection for 24 h and 48 h,the copy number of GSIV MCP gene was decreased by 6 times and 2.5 times,respectively,compared with the control group.Western blotting results showed that the synthesis of viral MCP protein in Ad Mx gene over-expressed cells was significantly lower than that in the control group.The above results showed that overexpression of Ad Mx gene can significantly inhibit the replication of GSIV and has significant antiviral function.