重组克柔念珠菌14-3-3蛋白诱导奶牛乳腺上皮细胞炎症反应的分子机制

doi:10.11843/j.issn.0366-6964.2023.04.031

摘要/Abstract

摘要： 旨在研究重组克柔念珠菌（Candida krusei）14-3-3蛋白（rCK14-3-3）对奶牛乳腺上皮细胞（MAC-T）炎性反应的分子机制，为系统研究克柔念珠菌损伤奶牛乳腺上皮细胞的机制提供理论依据。采用原核表达的方法构建克柔念珠菌BMH1基因重组质粒并转化入大肠杆菌BL21（DE3）中，经诱导表达纯化后进行Western blot鉴定；CCK-8法筛选rCK14-3-3蛋白对MAC-T的最适作用浓度和时间；Western blot检测rCK14-3-3蛋白对MAC-T的Toll样受体、MAPK信号通路、NF-κB信号通路主要蛋白相对表达量的影响；ELISA检测rCK14-3-3蛋白对MAC-T中相关炎性因子IL-1β、IFN-γ、IL-18、TNF-α、IL-6、IL-12相对表达量的影响。结果显示：成功构建了pET-21a-BMH1重组表达载体，经诱导表达纯化后获得有生物学活性的rCK14-3-3蛋白，纯度达90%以上，浓度为0.2 mg·mL^-1。与空白对照组相比，采用50 μg·mL^-1的rCK14-3-3作用MAC-T，TLR2、TLR4、MyD88在1、3 h时表达量极显著升高（P<0.01），6 h时，TLR4表达量显著降低（P<0.05），TLR2、MyD88表达量无显著变化；p-JNK在1 h时的表达量无显著变化，3、6 h时极显著降低（P<0.01）；p-ERK在1、3、6 h时的表达量显著或极显著升高（P<0.05，P<0.01）；p-p38在1 h时的表达水平极显著降低（P<0.01），3、6 h时极显著升高（P<0.01）；p-IκB-α、p-p65在1、3、6 h时的表达量均极显著升高（P<0.01）。IL-1β、IFN-γ、IL-18、TNF-α在作用1、3、6 h后均呈现出显著或极显著的上调趋势（P<0.05，P<0.01）；IL-6在作用1、3 h后极显著上升（P<0.01），6 h后显著降低（P<0.05）；IL-12在作用1 h后极显著上升（P<0.01），3、6 h后极显著降低（P<0.01）。本研究成功表达具有生物学活性的rCK14-3-3蛋白，该蛋白可通过TLR2/4-MAPK/NF-κB信号通路诱导MAC-T发生炎性反应，rCK14-3-3蛋白可作为引起MAC-T炎症的重要因子之一。

关键词: 克柔念珠菌, 14-3-3蛋白, 原核表达, 奶牛乳腺上皮细胞, 炎性

Abstract: This study was conducted to research the molecular mechanism of inflammatory response of recombinant Candida krusei 14-3-3 protein (rCK14-3-3) treat on the mammary epithelial cells (MAC-T), so as to provide a theoretical basis for the systematic study of the mechanism of MAC-T damage induced by Candida krusei. The recombinant plasmid of Candida krusei BMH1 gene was constructed by prokaryotic expression and transformed into E. coli BL21 (DE3), rCK14-3-3 protein was identified by Western blot after induction and purification; the optimal concentration and time of rCK14-3-3 on MAC-T were screened by CCK-8 method; the expression levels of the Toll receptors, MAPK and NF-κB signaling pathway major proteins of MAC-T was determined by Western blot;the expression level of the inflammatory factors IL-1β, IFN-γ, IL-18, TNF-α, IL-6 and IL-12 were determined by ELISA in MAC-T. The recombinant expression vector pET-21a-BMH1 was successfully constructed, the rCK14-3-3 protein has biological activity after expression and purification. The purity of rCK14-3-3 protein reached more than 90% and the concentration was 0.2 mg·mL^-1. Compared with the blank control group, MAC-T was treated by rCK14-3-3 of 50 μg·mL^-1, the protein expression level of TLR2, TLR4, and MyD88 was very significantly increased at 1 and 3 h (P<0.01), at the time of 6 h, the TLR4 expression level was significantly reduced (P<0.05), no significant changes in TLR2 and MyD88 expression occurred; No significant change of p-JNK expression at 1 h, it was very significantly decreased at 3 and 6 h (P<0.01); The expression level of p-ERK was increased significantly at 1, 3 and 6 h (P<0.05 or P<0.01); The expression level of p-p38 was very significantly reduced at 1 h (P<0.01), and it increased very significantly at 3 and 6 h (P<0.01); The expression levels of p-IκB-α and p-p65 were all significantly increased at 1, 3, and 6 h (P<0.01); MAC-T was treated with 50 μg·mL^-1 of rCK14-3-3 showed a significant or extremely significant upregulation in IL-1β, IFN-γ, IL-18, and TNF-α after 1, 3 and 6 h (P <0.05, P<0.01), IL-6 increased significantly after 1 and 3 h (P <0.01), and decreased significantly after 6 h (P<0.05), IL-12 increased significantly after 1 h (P<0.01), and decreased significantly after 3 and 6 h (P<0.01). In the study, the rCK14-3-3 protein of biological activity was successfully expressed, which can induce an inflammatory response in MAC-T through the TLR2/4-MAPK/NF-κB signaling pathway, and rCK14-3-3 protein can be one of the important factors causing MAC-T inflammation.

Key words: Candida krusei, 14-3-3 protein, prokaryotic expression, bovine mammary epithelial cells, inflammatory

中图分类号:

S852.661

蔡明玉, 张海龙, 海珍珍, 乔亚蕊, 杜军, 周学章. 重组克柔念珠菌14-3-3蛋白诱导奶牛乳腺上皮细胞炎症反应的分子机制[J]. 畜牧兽医学报, 2023, 54(4): 1679-1689.

CAI Mingyu, ZHANG Hailong, HAI Zhenzhen, QIAO Yarui, DU Jun, ZHOU Xuezhang. The Inflamed Molecular Mechanism Induced by Recombined 14-3-3 Protein of Candida krusei on Bovine Mammary Epithelial Cells[J]. Acta Veterinaria et Zootechnica Sinica, 2023, 54(4): 1679-1689.

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