萤火虫荧光素酶标记的马尔尼菲篮状菌的构建与应用

Construction and application of firefly luciferase-tagged talaromyces marneffei

目的:构建稳定表达萤火虫荧光素酶(Luc)的马尔尼菲篮状菌(TM),并进行生长状况和毒力的评估,为TM研究提供新手段和新工具。方法:构建Luc基因表达质粒pCAMBIA1303-TrpC-Hygro-gpdA-Luc,通过琼脂糖凝胶电泳和测序手段验证质粒是否携带Luc基因。采用农杆菌介导转化法将Luc插入TM基因组,在含有潮霉素培养基上筛选转化成功的TM转化子(TM-Luc)。于PDA培养基(27°C)中连续培养TM标准株ATCC18224和TM-Luc 14 d,记录二者的生长情况,挑取单个菌落进行乳酸酚棉蓝染色,荧光显微镜下观察两种菌株的形态。利用RT-qPCR和流式细胞术检测TM标准株和TM-Luc对THP-1来源巨噬细胞的杀伤情况。结果:经潮霉素筛选与电泳验证,得到稳定表达Luc的TM-Luc转化子。TM-Luc与荧光素底物结合后可发出荧光。在PDA培养基(27°C)连续培养14 d后,TM标准株菌落直径为(6.54±0.22)cm,TM-Luc菌落直径为(6.45±0.23)cm。两者表面均呈扁平绒毛状,并产生红色色素,生长曲线(P=0.273)与生长速率曲线(P=0.49)均无统计学差异(均P>0.05)。荧光显微镜镜下TM标准株和TM-Luc形态相似。流式细胞术显示TM标准株对THP-1来源的巨噬细胞杀伤作用较强于TM-Luc,在48 h时差异具有统计学意义(P=0.0156),在72 h时差异无统计学意义(P=0.1033)。炎症因子TNFA(24 h P=0.1529,48 h P=0.1115)与IL1B(24 h P=0.1338,48 h P=0.6921)在24 h、48 h表达差异均无统计学意义(均P>0.05)。结论:TM-Luc与TM标准株形态学与功能学特征相似,可作为工具菌用于细胞感染和动物感染模型上,以便于更加直观地评估TM与宿主间的相互作用。

Objective:To construct talaromyces marneffei(TM)that stably expresses firefly luciferase(Luc)and evaluate its growth status and virulence,providing new tools for TM research.Methods:The Luc gene expression plasmid pCAMBIA1303-TrpC-Hygro-gpdA-Luc was constructed,and agarose gel electrophoresis and sequencing were conducted to test whether the plasmid carried Luc gene.Luc was inserted into the TM genome by Agrobacterium-mediated transformation,and the successful TM transformants(TM-Luc)were screened on medium containing hygromycin B.The growth of TM standard strain ATCC18224 and TM-Luc was recorded in PDA medium(27°C)for 14 d.Individual colonies were selected for lactophenol blue staining and the morphology of both strains was observed under fluorescence microscope.The killing of THP-1-derived macrophages by the TM standard strain and TM-Luc was detected using realtime reverse transcription-quantitative PCR(RT-q PCR)and flow cytometry.Results:TM-Luc transformants stably expressing Luc were obtained by hygro-mycin B screening and verified by electrophoresis.TM-Luc fluoresced upon binding to the fluorescent substrate.After 14 d of continuous incubation in PDA medium(27°C),the colony diameter was(6.54±0.22)cm for TM standard strain and(6.45±0.23)cm for TM-Luc.Both had a flattened fluffy surface and produced red pigment,and the differences in growth curves(P=0.273)and growth rate curves(P=0.49)were not statistically significant(both P>0.05).The morphology of TM standard strain and TM-Luc was similar under fluorescence microscopy.Flow cytometry showed that TM standard strain was more potent than TM-Luc in killing THP-1-derived macrophages,with a statistically significant difference at 48 h(P=0.0156)and no statistically significant difference at72 h(P=0.1033).The differences in expression of inflammatory factors TNFA(24 h P=0.1529,48 h P=0.1115)and IL1B(24 h P=0.1338,48 h P=0.6921)at 24 and 48 h were not statistically significant(all P>0.05).Conclusion:TM-Luc has similar morphological and functional characteristics to the TM standard strain and can be used as a tool in cellular and animal infection models to facilitate a more straightforward assessment of TM-host interactions.