基于GFP观察鼠伤寒沙门氏菌HilD介导ssrAB表达的初探

目的 以绿色荧光蛋白(GFP)标记的鼠伤寒沙门氏菌(Salmonella typhimurium,S.Typhi)为研究对象,秀丽隐杆线虫为模式生物,初步探究体内环境下HilD对ssrAB表达的调控作用。方法 通过基因工程技术分别构建携带gfp基因的亲本株S.Typhi(ssrAB-gfp+pcDNA3.1)、hilD基因缺失株S.Typhi(ΔhilD+ssrAB-gfp+pcDNA3.1)和hilD基因回补株S.Typhi(ΔhilD+ssrAB-gfp+pcDNA3.1-hilD) 标记菌。利用荧光显微技术,观察3株标记菌在饲喂感染秀丽隐杆线虫体内外的GFP表达状况及其定殖分布,并通过ImegJ软件进行荧光强度的比较。 结果 成功构建的3株标记菌在秀丽隐杆线虫体内外均呈现绿色荧光,荧光强度为S.Typhi(ssrAB-gfp+pcDNA3.1)高于S.Typhi(ΔhilD+ssrAB-gfp+pcDNA3.1-hilD)和S.Typhi(ΔhilD+ssr AB-gfp+pcDNA3.1),S.Typhi(ΔhilD+ssrAB-gfp+pcDNA3.1-hilD)高于S.Typhi(ΔhilD+ssrAB-gfp+pcDNA3.1);3株标记菌在线虫体内主要定殖分布于口咽部和肠道中,荧光强度为口咽部较肠道高。结论 GFP在鼠伤寒沙门氏菌中得到稳定表达,感染秀丽隐杆线虫后同样获得GFP的稳定表达,且在线虫的口咽部和肠道具有良好的定殖分布,首次初步证明体内环境下HilD介导ssrAB表达,但并非为单一的调控作用。

HilD mediated expression of ssrAB based on GFP in Salmonella typhimurium

Salmonella typhimurium (S.typhi) labeled with GFP was used as the research object and Caenorhabditis elegans as a model organism, initially the role of HilD in the regulation of the expression of ssrAB was explored in vivo. Genetic engineering technology was used to construct S.typhi(ssrAB-gfp+pcDNA3.1), hilD gene deletion strain S.typhi(ΔhilD+ssrAB-gfp+pcDNA3.1) and hilD gene reversion strain S.typhi(ΔhilD+ssrAB-gfp+pcDNA3.1-hilD) carrying GFP gene respectively. Fluorescence microscopy was used to observe the expression and distribution of GFP in 3 labelled strains in Caenorhabditis elegans in vitro and in vivo, and the fluorescence intensity was compared by ImegJ. Three labelled strains were successfully constructed and showed green fluorescence in Caenorhabditis elegans in vitro and in vivo. The fluorescence intensity of S.typhi(ssrAB-gfp+pcDNA3.1) was higher than that of (ΔhilD+ssrAB-gfp+pcDNA3.1-hilD)(P<0.05) and S.typhi(ΔhilD+ssrAB-gfp+pcDNA3.1)(P<0.01), the fluorescence intensity of S.typhi(ΔhilD+ssrAB-gfp+pcDNA3.1-hilD) was higher than that of S.typhi(ΔhilD+ssrAB-gfp+pcDNA3.1)(P<0.05). Colonization of 3 labelled strains in Caenorhabditis elegans in vitro was mainly distributed in oropharyngeal and intestinal tract, the fluorescence intensity of oropharynx was high that of intestinal (P<0.05). The expression of GFP was stable in Salmonella typhimurium, expressing the same in Caenorhabditis elegans. Three labelled strains colonized in oropharyngeal and intestinal very well in Caenorhabditis elegans. The study proved that HilD mediated the expression of ssrAB at the first time in vitro, not only a regulation role.