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

The Inflamed Molecular Mechanism Induced by Recombined 14-3-3 Protein of Candida krusei on Bovine Mammary Epithelial Cells

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BMH 1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50μg·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。

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 BMH 1 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 50μg·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-αand p-p65 were all significantly increased at 1,3,and 6 h(P<0.01);MAC-T was treated with 50μg·mL^(-1) of rCK14-3-3 showed a significant or extremely significant upregulation in IL-1β,IFN-γ,IL-18,and TNF-αafter 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.