奶牛乳房炎相关IRF5基因CDS区克隆及表达分析

CDS cloning and expression analysis of IRF5 gene related cow mastitis

【目的】研究奶牛干扰素调节因子5(Interferon regulatory factor 5,IRF5)基因在奶牛乳腺组织及乳腺上皮细胞炎症中的表达模式。【方法】利用RT-PCR和测序技术克隆得到IRF5基因的编码区(Coding sequence,CDS)序列,并采用生物信息学方法分析IRF5基因及其特性;利用qPCR技术检测IRF5基因及相关干扰素调节因子3(Interferon regulatory factor 3,IRF3)和MyD88基因的表达量。【结果】IRF5基因的CDS长度为1500 bp,编码499个氨基酸,存在47个潜在磷酸化位点,氨基酸组成中脯氨酸(Pro)和亮氨酸(Leu)所占比例较高。IRF5蛋白分子式为C_(2524)H_(3901)N_(671)O_(728)S_(20),主要存在于细胞核和线粒体,理论等电点为5.38,是碱性亲水且不稳定的非分泌蛋白。蛋白互作分析结果显示,IRF5蛋白可能与骨髓分化初级反应蛋白(MyD88)、白细胞介素-1受体相关激酶1(IRAK1)、C-C基序趋化因子4(CCL4)、组蛋白乙酰转移酶p300(EP300)和核受体共激活因子3异构体X1(NCOA3)等蛋白相互作用,与机体或细胞的炎症反应有关。qPCR结果表明,与健康奶牛相比,IRF5基因在乳房炎奶牛的乳腺组织中的表达量极显著上调(P<0.01)。在乳腺上皮细胞中,与对照组(0 h)相比,IRF5及其相关的Myd88和IRF3基因在LPS诱导乳腺上皮细胞炎症反应的3、6和12 h的表达量均极显著上调(P<0.01)。【结论】IRF5基因在奶牛乳房炎过程中表达量上调,可能通过上述基因参与促进奶牛乳房炎症反应。

【Objective】The purpose of the present study was to investigate the expression pattern of the interferon regulatory factor 5(IRF5) gene in mammary tissues with mastitis and cow mammary epithelial cells during the inflammation.【Method】In the study, the CDS of the IRF5 gene was cloned using RT-PCR and sequencing, and the biological properties of the IRF5 gene and its encoded protein were analyzed using bioinformatics. The expression of IRF5 gene and its related interferon regulatory factor 3(IRF3) and MyD88 genes were detected by qPCR.【Result】IRF5 gene had a CDS length of 1500 bp and it encoded 499 amino acids with 47 potential phosphorylation sites. Proline(Pro) and Leu(Leu) accounted for a high proportion of amino acid composition. The molecular formula of IRF5 protein was C_(2524)H_(3901)N_(671)O_(728)S_(20), which mainly existed in the nucleus and mitochondria. The theoretical isoelectric point was 5.38, and it was an alkaline hydrophilic and unstable non-secreted protein and encoded 499 amino acids.The results of protein interaction analysis showed that IRF5 protein may be interacted with myeloid differentiation factor 88(MyD88), interleukin-1 receptor associated kinase 1(IRAK1), C-C motif chemokine 4(CCL4), histone acetyltransferase p300(EP300) and nuclear receptor co-activator 3 isoform X1(NCOA3), and were associated with the inflammatory response of the organism or cells. qPCR results showed that the expression of the IRF5 gene was significantly upregulated in the cow mammary tissues of cows with E. coli mastitis compared to healthy cows(P<0.01). In mammary epithelial cells, the expression levels of IRF5 and its related Myd88 and IRF3 genes were significantly up-regulated at 3, 6 and 12 hours after LPS induced inflammatory response compared with the control group(P<0.01). 【Conclusion】These results suggest that the expression level of IRF5 gene is up-regulated during dairy cow mastitis, which may be involved in promoting dairy cow udder inflammation through the above genes.