Abstract: To explore the regulatory effect of Brucella secreted protein L（BspL）on the inflammatory response of macrophages，based on the BspL gene sequence（BAB1_1533）of Brucella abortus 2308 strain，an expression vector of pET‐32a‐BspL was constructed，and the BspL recombinant protein（rBspL）was expressed and purified. After identification and concentration determination by SDS‐PAGE，rBspL was used to stimulate RAW264.7 macrophages.The mRNA expression levels of inflammatory factors（IL‐1β and IL‐18）and inflammasomes（NLRP3 and Caspase‐1）were detected by qRT‐PCR，and the protein expression levels of inflammasomes（NLRP3 and Caspase‐1）were analyzed by Western blot.The bioinformatic analysis of BspL was performed using online softwares.The results showed that the pET‐32a‐BspL expression vector was successfully constructed，pET‐32a‐BspL expression product was purified to show a specific band at 36 ku.After stimulating RAW264.7 cells with rBspL，compared with the control group（DMEM was added），the experimental group（RAW264.7 cells were stimulated with 50 μg/mL rBspL）significantly increased the relative mRNA expression levels of NLRP3，Caspase‐1，IL‐1β and IL‐18 and the protein expression levels of inflammasomes NLRP3 and Caspase‐1 were significantly increased.Bioinformatics analysis showed that BspL protein was hydrophilic，with two transmembrane regions，two secretion pathway signal peptides（Sec signal peptides），26 phosphorylation sites and five antigenic epitopes.The secondary structure of BspL protein was dominated by random coil and α‐helix，and there were a few extended chains.In addition，the results of tertiary structure prediction showed that most of the BspL protein structures were random coil and α‐helix，which was consistent with the results of secondary structure prediction.In conclusion，Brucella rBspL expressed by Escherichia coli could induce host cells to release inflammatory factors，which provided references for further study of the function and molecular mechanism of BspL.

Key words: Brucella, BspL protein, Inflammatory factors, Bioinformatics analysis

摘要： 为探究布鲁氏菌分泌蛋白L（Brucella secretion protein L，BspL）对巨噬细胞炎症反应的调控作用，基于牛种布鲁氏菌（Brucella abortus）2308菌株的BspL基因序列（BAB1_1533），构建pET-32a-BspL表达载体，诱导表达并纯化重组BspL 蛋白（rBspL）。经SDS-PAGE 鉴定及浓度测定后，使用rBspL 刺激RAW264.7巨噬细胞，通过qRT-PCR检测炎症因子（IL-1β和IL-18）及炎症小体（NLRP3和Caspase-1）的mRNA表达水平，并通过Western blot分析炎症小体（NLRP3和Caspase-1）的蛋白质表达水平；利用在线软件对BspL蛋白进行生物信息学分析。结果表明，成功构建pET-32a-BspL表达载体，pET-32a-BspL表达产物经纯化后在36 ku处出现特异性条带；用rBspL刺激RAW264.7细胞后，与对照组（添加DMEM培养基）相比，试验组（添加50 μg/mL rBspL）NLRP3、Caspase-1、IL-1β和IL-18的mRNA相对表达量极显著提高，且炎症小体NLRP3、Caspase-1的蛋白质表达水平显著提高。生物信息学分析显示，BspL蛋白具有亲水性，存在2个跨膜区，有2个分泌途径信号肽（Sec信号肽），有26个磷酸化位点和5个抗原决定簇；BspL蛋白二级结构以无规卷曲和α-螺旋为主，还存在少量延伸链；三级结构预测结果显示，BspL蛋白中多为无规卷曲，与二级结构预测分析结果一致。综上，经大肠杆菌表达的布鲁氏菌rBspL可诱导宿主细胞释放炎症因子，为进一步研究BspL的功能及分子机制提供了参考。

关键词: 布鲁氏菌, BspL蛋白, 炎症因子, 生物信息学分析