Abstract: Listeria monocytogenes is a common food-borne pathogen that causes listeriosis and poses a tremendous threat to food safety. prsA1 is conservative and specific. Signal peptides, transmembrane regions and spatial antigen epitopes of PrsA1 were predicted in this study by SignalP 4.1 Server program, TMHMM Server V.2.0 program and SEPPA 2.0 program, respectively. The results showed that the N terminus of PrsA1 contained a signal peptide and a transmembrane region, and that the protein had good epitopes. Therefore, it could be used as a detection target. On this basis, non-transmembrane region sequence of prsA1, Δ84prsA1, was acquired through PCR. The recombinant plasmid pET30a-Δ84prsA1 was constructed and transferred into Escherichia coli to induce expression of Δ28PrsA1. Δ28PrsA1 was purified by Ni-IDA-Sefinose Column. The titer of polyclonal antibody prepared by purified Δ28PrsA1 was determined by indirect ELISA, and the titer was as high as 1∶128 000. Western blotting analysis showed that the polyclonal antibody could recognize PrsA1 protein extracted from Listeria monocytogenes. In this study, bioinformatics methods was used to screen the detection target and analyze the epitopes, and polyclonal antibody was prepared successfully, which provided a practical basis for the screening of detection target and immunological detection of Listeria monocytogenes.

Key words: Listeria monocytogenes, prsA1, bioinformatics, prokaryotic expression, polyclonal antibody

摘要： 单增李斯特菌（Listeria monocytogenes）是一种常见的食源性致病菌，能够引发李斯特菌病，对食品安全构成巨大威胁。prsA1具有保守性和特异性，利用SignalP 4.1 Server程序、TMHMM Server V.2.0程序和SEPPA 2.0程序预测了PrsA1的信号肽段、跨膜区域及空间抗原表位，预测结果显示PrsA1的N端含有信号肽段及跨膜区且该蛋白具有良好抗原表位结构，因而可作为检测靶标。在此基础上，采用PCR法获得prsA1的非跨膜区序列即Δ84prsA1，构建重组质粒pET30a-Δ84prsA1并转入到大肠杆菌中诱导表达Δ28PrsA1，Ni-IDA柱亲和纯化重组蛋白Δ28PrsA1，以纯化的Δ28PrsA1为抗原制备多克隆抗体。间接ELISA检测多克隆抗体的效价，高达1∶128 000。Western blotting分析结果显示该多克隆抗体能够识别从单增李斯特菌中提取的PrsA1蛋白。利用生物信息学筛选检测靶标并分析抗原表位结构，最后成功制备了多克隆抗体，为单增李斯特菌检测靶标的筛选和免疫学检测提供了实践基础。

关键词: 单增李斯特菌, prsA1, 生物信息学, 原核表达, 多克隆抗体