摘要：

利用增强型绿色荧光蛋白基因(EGFP)表征鼠伤寒沙门氏菌株14028(Sal-14028),以研究其在猪肺泡巨噬细胞(3D4/21)中的示踪作用,首先将增强型绿色荧光蛋白基因通过电击法转入Sal-14028,经卡那霉素抗性培养基筛选和测序鉴定,获得了具有绿色荧光信号的阳性菌株并检测其荧光稳定性,命名为Sal-pPpagC-EGFP。接着在同等条件下对比荧光菌株和野生菌株(WT)的生长特性,通过小鼠感染试验检测EGFP基因的插入对荧光菌株毒力有无明显影响,以分析荧光菌株的生物学特性;应用荧光显微镜和间接免疫荧光法检测荧光菌株在猪肺泡巨噬细胞3D4/21吞噬溶酶体中的定位情况。结果显示,在不同时间点,EGFP标记的鼠伤寒沙门氏菌在荧光显微镜下发出强烈的绿色荧光,且所带荧光不受抗性选择的影响,可稳定遗传。相同培养条件下,荧光菌株Sal-pPpagC-EGFP生长曲线的变化趋势与野生株基本相同。另外,经过改良寇氏法计算出的LD₅₀与野生株相比无显著差异(P>0.05),说明EGFP基因的插入对荧光菌株毒力无明显影响。荧光菌株在感染猪肺泡巨噬细胞后,吞噬溶酶体内可观察到标记菌株,并随着时间增加菌株数量呈现增长趋势。表明荧光菌株除了具备发光特性外,其他生物学性状没有明显改变。

关键词: 鼠伤寒沙门氏菌, EGFP, 电转化, 生物学特性, 胞内示踪

Abstract:

The enhanced green fluorescent protein gene(EGFP)was used to characterize Salmonella typhimurium strain 14028(Sal-14028)to study its tracer role in porcine alveolar macrophages(3D4/21),and firstly,the EGFP was transferred into Sal-14028 by electroshock method and obtained green fluorescent positive and stable strain,named Sal-pPpagC-EGFP.Then,the growth characteristics of the fluorescent strain and the wild strain(WT)were compared under the same conditions,and the virulence of the fluorescent strain was examined by mouse infection test to determine whether the insertion of the EGFP gene had any significant effect on the virulence of the fluorescent strain,so as to examine the biological characteristics of the fluorescent strain,and analysis the localization of fluorescent strains in the phagocytic lysosomes of porcine alveolar macrophages(3D4/21)using fluorescence microscopy and indirect immunofluorescence.The results showed that EGFP-labeled Salmonella typhimurium emitted strong green fluorescence under fluorescence microscopy at different time points,and the fluorescence carried was not affected by resistance selection and could be stably inherited.The trend of the growth curve of the fluorescent strain Sal-pPpagC-EGFP was basically the same as that of the wild strain under the same culture conditions.In addition,the LD₅₀ calculated by the modified Kou method was not significantly different from that of the wild strain(P>0.05),indicating that the insertion of EGFP gene had no significant effect on the virulence of the fluorescent strain.After the fluorescent strains infected porcine alveolar macrophages,labeled strains were observed in the phagolysosomes,and the number of strains showed an increasing trend with the increase of time.It showed that the fluorescent strains had no significant changes in biological traits other than having luminescent properties.These results suggested that the construction of Sal-pPpagC-EGFP provided an effective tool to further reveal its infestation pathogenic process.

Key words: Salmonella typhimurium, EGFP, Electrical transformation, Biological properties, Intracellular tracing