减毒沙门氏菌作为原核表达宿主菌和真核表达载体的研究

本研究以增强型绿色荧光蛋白(eGFP)为报告基因,探讨了减毒沙门氏菌作为原核表达宿主菌和真核表达载体的可行性.通过多功能分光光度仪测定细菌的OD600和eGFP在大肠杆菌和减毒沙门氏菌中的表达量,结果表明:eGFP在沙门氏菌中的表达量依赖于IPTG的浓度,最佳浓度为0.5 mmol·L-1,增加浓度至0.75 mmol·L-1不能增加eGFP的表达量;而该现象在大肠杆菌中不明显.当IPTG浓度等于或大于0.5 mmol·L-1时,沙门氏菌中eGFP的表达量显著高于大肠杆菌.若以相对荧光度(1个OD600单位的荧光值,即eGFP相对表达量)表示,两种宿主菌的最佳诱导时间均为3 h.Hela细胞侵袭力试验表明该减毒沙门氏菌仍具有侵袭力,同时以脂质体转染为对照,将携带重组质粒pcDNA3-eGFP的减毒沙门氏菌转染Hela细胞,48 h后可观察到绿色荧光,表明了该菌可作为载体将重组质粒携带到细胞内,并使外源基因得到表达.此外,利用基于微孔板的多功能分光光度仪可直接检测荧光强度和OD值,能快速、准确地进行多样本检测,可用于GFP在不同宿主菌或相同宿主菌在不同试验条件下,甚至不同表达载体在相同宿主菌中表达等方面的研究.

Attenuated Salmonella typhimurium as a carrier for prokaryotic and eukaryotic expression vectors

An attenuated Salmonella enterica serovar typhimurium strain as the host for recombinant prokaryotic and eukaryotic vectors containing the enhanced green fluorescent protein (eGFP) gene was examined by fluorimetric and fluorescent microscopic methods. Expression of eGFP was dependent on the concentration of IPTG used for induction, being optimal at 0.5 mmol. L-1 . Further increase to 0.75mmol. L-1 did not increase the fluorescence output. This dose-dependent induction was not apparent when E. coli was used as the host strain. The expression was higher in S. typhimurium than in E. coli typhimurium strain was invasive, though less than its parent strain, into the HeLa cells and able to deliver the recombinant eukaryotic plasmid pcDNA3-eGFP for expression of eGFP as shown by fluorescing cells 48 h after transfection. The results of this experiment also demonstrate the utility of direct measurement of fluorescence and optical density in a multifunctional microplate-based spectrophotometric reader, allowing high throughput multiple quantitative comparisons of eGFP expression by different host strains or the same strain under different conditions or even different expression vectors.