| Mx protein, induced by interferon, is a kind of GTP activated protein。 And it has been thought to have a broad spectrum antiviral function. Massive researches on Mus musculus and Homo sapiens Mx had been done. With those researches, antiviral function and antiviral mechanism of Mus musculus and Homo sapiens Mx is relatively clear. Some researches on fish Mx proteins have also been done. Some fish Mx were proved to have certain inhibition of viral replication capabilities, but that is not thorough. Antiviral function and antiviral mechanism of Fish Mx still need further researches. Mx is an important humoral factor in the nonspecific immune system. Therefore, its antiviral function research has become one of current hot topics in immunology studies. Disease-resistant breeding with Mx shows great potential, and has also become one of the hot spots of genetic breeding researches.In this study, full-length cDNA of Mx from Squaliobarbus curriculus had been cloned. Using β-actin as internal standard control, the regular of ScMx gene mRNA expression levels in health Squaliobarbus curriculus and Squaliobarbus curriculus after GCRV infection was investigated by fluorogenic quantitative PCR. Recombinant plasmids ScMxORF-cap and ScMxORF-Tgf2-L-R were constructed for further studies like transgenosis. The main results were as follows:1. Full-length cDNA of Mx from the spleen of Squaliobarbus curriculus was amplified using degenerate PCR and SMART RACE. The sequencing results showed that the full-length of Squaliobarbus curriculus Mx (ScMx) gene is2325bp composed of40bp5’-UTR,401bp3’-UTR and1884bp ORF, encoding627amino acid. The predicted Mx protein with typical dynamin family structure characteristics is about70.9kD molecular weight and a theoretical isoelectric point (PI) of8.25. The results forecasted by the DNA star subprogram Protean revealed that ScMx is a kind of hydrophilic protein and contains a lot of antigenicity site. ScMx is of the nearest homology with Carassius aruatus Mx3searched by NCBI Blast. The phylogenetic tree based on Mx amino acid sequences of some species showed the consistent result with their evolutionary relationship. The ScMx merges with Mx proteins of cyprinid fishes, then with Mx proteins of other fish species and finally with Mx proteins of Anas platyrhynchos, Gallus gallus, Mus musculus and Homo sapiens. 2. Fluorescence quantitative specificity primers were designed according to the sequences of ScMx gene. To investigate the expression profile of ScMx mRNA, Mx expression in different tissues collected at Oh (without infection),6h,12h,24h,48h and72h after GCRV infection was examined by RT-qPCR. The data revealed that ScMx was broadly expressed in all tested tissues (liver, spleen, intestinal, gill, head kidney and muscle). In healthy group, the relative expression in intestine and spleen were significantly higher than that in others (P＜0.05). After GCRV infection, the ScMx expression change in all tissues showed the similar tendence:The relative expression of Mx increased at first, then decreased. The expression level in intestinal and gill had no obvious differences at all tested time point (P>0.05), while in liver (6h,12h,24h), spleen (6h,12h,24h), head kidney (6h,12h,48h) and muscle (6h) were significant higher than the control group (Oh)(P<0.05). This result suggested that ScMx plays an important role in the host defense against GCRV infection.3. Primers with different enzyme loci were designed to obtain ScMx ORE ScMxORF-1was connected under the cap B-actin promoter site of plasmid pUC118-cap, While ScMxORF-2was connected under the efla-p promoter site of plasmid pTgf2-efla-eGFP. After reliability verification tests, the recombinant plasmids were converted to DH5a, and stored in-80℃. |
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