摘要
目的构建稳定遗传的马尔尼菲篮状菌(TM)绿色荧光蛋白(GFP)标记菌株,评估其生长状况及毒力水平。方法构建GFP基因表达载体p CAMBIA1303-TrpC-Hygro-gpdA-GFP,通过琼脂糖凝胶电泳及测序验证GFP基因是否插入载体,采用农杆菌介导转化法构建GFP稳定表达的TM转化子。使用荧光显微镜、流式细胞术检测转化子荧光表达水平和qPCR检测细胞损伤水平,与标准株ATCC18224比较。结果TM转化子的潮霉素筛选浓度选择为80μg/mL,筛选后的转化子通过荧光显微镜和流式细胞术进行绿色荧光检测,确定获得稳定遗传的GFP标记菌株。在25℃马铃薯葡萄糖固体培养基上培养15 d后,转化子和标准株的菌落直径分别为(7.18±0.12)和(7.04±0.16)cm,均呈扁平绒毛状,表面多为灰褐色菌丝,同时两者的生长速率曲线差异无统计学意义(P>0.05)。进一步使用转化子和标准株感染THP-1巨噬细胞后,巨噬细胞的形态、细胞死亡以及炎症因子(肿瘤坏死因子-α、白细胞介素-1β、白细胞介素-10)的表达差异无统计学意义(P>0.05)。结论TM转化子与标准株在形态和功能上无差异,可以作为示踪菌应用于细胞感染模型上,更加直观地评估TM与细胞之间的相互作用、TM在细胞内定位以及细胞免疫通路的表达水平等,是研究TM感染的新工具。
Objective To construct a stable genetically inherited Talaromyces marneffei(TM)green fluorescent protein(GFP)-labeled strain,and evaluate its growth status and virulence.Methods The GFP gene expression vector pCA was constructed,and whether the GFP gene was inserted into the vector was verified by agarose gel electrophoresis and sequencing.Agrobacterium-mediated transformation method was used to construct TM transformants with stable expression of GFP.Compared with the standard strain ATCC18224,the fluorescent transformants were detected by fluorescence microscope and flow cytometry and the cell damage of transformants were detected by qPCR.Results The transformants were selected by 80μg/mL of hygromycin.The selected transformants were subjected to green fluorescence detection by fluorescence microscopy and flow cytometry to confirm that a stable genetic GFP-labeled strain was obtained.After culturing on potato dextrose agar medium at 25℃for 15 days,the colony diameters of the transformant and the standard strain were(7.18±0.12)and(7.04±0.16)cm,respectively.Both of them were flat villi.The surface was mostly gray-brown hyphae,and both grew at the same time.The difference in growth curve was not significant(P>0.05).After further using transformants and standard strains to infect THP-1 macrophages,there was no significant difference in the morphology,cell death and expression of inflammatory factors(tumor necrosis factor-α,interleukin-1β,interleukin-10)of the macrophages.Conclusions There was no difference in morphology and function between TM transformants and standard strains.It could be used as tracer bacteria in cell infection models to more intuitively evaluate the interaction between TM and cells,the localization of TM in cells and the expression level of immune pathways.TM transformants could be used as a new tool for researching TM infection.
作者
何锦豪
韦吴迪
王刚
张洪
罗强
李珍
赖菁贞
蒋俊俊
叶力
梁浩
HE Jin⁃hao;WEI Wu⁃di;WANG Gang;ZHANG Hong;LUO Qiang;LI Zhen;LAI Jing⁃zhen;JIANG Jun⁃jun;YE Li;LIANG Hao(Guangxi⁃ASEAN Collaborative Innovation Center for Major Disease Prevention and Treatment,Life Sciences Institute,Guangxi Medical University,Nanning,Guangxi 530021;Guangxi Key Laboratory of AIDS Prevention and Treatment,School of Public Health,Guangxi Medical University,Nanning,Guangxi 530021;China(Guangxi)⁃ASEAN Emerging Infectious Disease Joint Laboratory,Nanning,Guangxi 530021,China)
出处
《热带医学杂志》
CAS
2022年第4期513-517,603,共6页
Journal of Tropical Medicine
基金
国家自然科学基金(81971934)
中国博士后科学基金(2020M683212)
广西医科大学青年基金(GXMUYSF202106)
关键词
马尔尼菲篮状菌
绿色荧光蛋白
示踪菌
标准株
Talaromyces marneffei
Green fluorescent protein
Tracer Fungus
Standard strain
