一种基于NanoLuc荧光素酶报告布鲁菌基因启动子活性质粒的构建及应用

背景]布鲁菌病是由布鲁菌感染引起的一种人兽共患传染病,对畜牧业发展和人类健康有着巨大的威胁。利用新型报告基因NanoLuc荧光素酶构建一种可以检测布鲁菌基因启动子活性的质粒,对于研究布鲁菌毒力基因的调控表达具有重要意义。目的]制备NanoLuc荧光素酶多克隆抗体,构建一种基于NanoLuc荧光素酶报告布鲁菌基因启动子活性的质粒,并通过测定bcsp31基因启动子和virB启动子活性验证该方法的可行性。方法]构建NanoLuc荧光素酶原核表达载体pET-Nluc,纯化蛋白免疫新西兰大白兔制备多克隆抗体;以广宿主质粒pBBR1MCS为骨架,构建质粒pNluc、pBcsp31-Nluc和pVirB-Nluc,通过电转化构建S2308(Nluc)、S2308(Bcsp31-Nluc)和S2308(VirB-Nluc)重组菌株,在体外培养条件下测定bcsp31基因启动子和virB启动子活性;比较分析virB启动子在胞内感染条件下和体外培养条件下的活性。结果]通过原核表达获得NanoLuc荧光素酶重组蛋白,并制备得到效价高于1:100 000的多克隆抗体;成功构建pNluc、pBcsp31-Nluc和pVirB-Nluc质粒以及S2308(Nluc)、S2308(Bcsp31-Nluc)和S2308(VirB-Nluc)重组菌株;体外培养条件下测定bcsp31基因启动子和virB启动子活性,结果显示pNluc质粒可以精确报告其活性;测定virB启动子在胞内诱导条件下和体外培养条件下的活性,结果显示virB启动子活性在胞内感染条件下明显增强。结论]构建了基于NanoLuc荧光素酶报告布鲁菌基因启动子活性的质粒,并验证其可以精确反映布鲁菌基因启动子活性,为研究布鲁菌毒力基因以及揭示其致病机制奠定了基础。

Construction and application of a novel plasmid for promoter activity determination of Brucella genes base on nanoLuc luciferase

Background] Brucellosis is a zoonotic infectious disease caused by Brucella spp. and threats the development of animal husbandry and human health. A novel plasmid was constructed for promoter activity determination in Brucella based on NanoLuc luciferase gene (nluc), which is important for research on regulatory mechanism of Brucella virulence genes. Objective] Preparation of rabbit polyclonal antibody of Nluc, construction of a Nluc reporter plasmid for promoter activation determination in Brucella, and verification of the Nluc reporter plasmid for Brucella bcsp31 gene and virB promoter. Methods] The nluc gene was ligated into prokaryotic expression vector pET-28a and constructed as recombinant vector pET-Nluc. The New Zealand rabbit was immunized to prepare polyclonal antibody of Nluc protein. The plasmids pNluc, pBcsp31-Luc and pVirB-Luc were constructed based on a broad-host-range vector pBBR1MCS. Brucella recombinant strains S2308(Nluc), S2308(Bcsp31-Nluc) and S2308(VirB-Nluc) were constructed by electrotransformation of plasmids. The promoter activity of bcsp31 and virB were detected in TSB. The activity of virB promoter in Brucella was compared in TSB and within RAW264.7 cells. Results] The Nluc protein was expressed and purified. The ELISA titer of polyclonal antibody was approximately to 1:100 000. The plasmid pNluc, pBcsp31-Luc, pVirB-Luc and the S2308(Nluc), S2308(Bcsp31-Nluc), S2308(VirB-Nluc) strains were constructed successfully. The results of bcsp31 and virB promoter activity in TSB showed that promoter activity can be detected accurately in pNluc plasmid. The result of virB promoter activity in intracellular Brucella showed that the activity of virB promoter is enhanced significantly. Conclusion] In this study, a plasmid for promoter activity determination of Brucella genes was constructed successfully. The results of this study showed that the promoter activity of Brucella genes can be detected accurately in pNluc plasmid. This study provides a novel strategy for determining promoter activity of Brucella virulence genes, which may benefit investigation of Brucella pathogenesis.