Cloning,expression and role of IFNE gene associated with mastitis in dairy cows
[Objective]The present study aimed to investigate the expression pattern of interferon epsilon(IFNE)gene and its potential mo-lecular functions in inflammation of bovine mammary epithelial cells(bMECs).[Method]The coding sequence(CDS)of IFNE gene was obtained by PCR and sequencing.The characteristics of IFNE gene and its protein were analyzed by bioinformatics methods.The expression of IFNE,Interferon-β(IFNB),Interferon-K(IFNK),Cystathione aspartase 3(CASP3)and Cystathione aspartase 9(CASP9)genes in li-popolysaccharide(LPS)-induced inflammatory response in bMECs were examined using RNAi and qPCR.[Result]IFNE gene had a CDS length of 582 bp,encoding a hydrophilic and unstable non-secretory protein consisting of 193 amino acids.Compared with control group(0 hours),the expression of IL-6 and IL-1β were significantly upregulated(P＜0.05,the same as below)and IL-8 was extremely significantly upregulated(P＜0.01,the same as below)in bMECs induced by LPS at 3,6,12 and 24 hours,indicating that the inflammatory cell model was successfully constructed.Based on the successful construction of the inflammatory cell model,qPCR was used to detect the expression of IFNE in bMECs.The results showed that the expression of IFNE was extremely significantly upregulated at 3,6,12 and 24 hours of LPS in-duction in bMECs compared with control group(0 hours),and its expression was the highest at 6 hours of induction,suggesting that the gene had a specific function in the inflammatory response of bMECs.In addition,RNAi experiment showed that interference with IFNE re-sulted in significant upregulation of IL-8,IFNB,IFNK and IL-10RB and extremely significant upregulation of IL-6,IL-1β,CASP3,CASP9 and BAD genes in inflammatory bMECs.[Conclusion]The expression of IFNE is extremely significantly upregulated in the inflammatory re-sponse of bMECs induced by LPS,and interference with IFNE can alleviate the inflammatory response of bMECs by regulating the expression of IL-6,IL-8,IL-1β,IFNB,IFNK,CASP3 and CASP9,which lays a theoretical foundation for the molecular treatment and resistance breeding of mastitis in dairy cows.
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