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