摘要The role of S-layer proteins(SLP),which form the outermost layer of cell walls in lactic acid bacteria(LAB),plays a crucial role in regulating immune-stimulating activity,thereby closely influencing LAB's ability to boost host immunity.In this study,the heterologous expression,purification,and characterization of Lactobacillus acidophilus CICC6074-derived slpX protein and the molecular mechanism of its anti-inflammatory effect on LPS-induced RAW264.7 cells were investigated.Initially,the PCR results were shown to successfully clone the slpX DNA sequence by homologous recombination to obtain the pet-32a-slpX recombinant plasmid.SDS-PAGE results revealed that slpX protein with a molecular weight of 54 kDa was successfully obtained under 0.7 mmol/L IPTG-induced conditions,which was further demonstrated by Western blot(WB)and LC-MS/MS.ELISA,WB and molecular docking results indicated that slpX protein can inhibit LPS-induced cellular inflammatory responses by linking to TLR4 and MD2 via hydrogen bonding and increasing the levels of anti-inflammatory factor IL-10 and decreasing the levels of inflammatory factors(TNF-α,IL-6,NO)and ROS via MAPK and NF-κB signaling pathways.The study is essential for the preparation of pure slpX protein and revealing its anti-inflammatory molecular mechanism.