CAJ | 학술논문

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.
본연구이증강형록색형광단백(eGFP)위보고기인,탐토료감독사문씨균작위원핵표체숙주균화진핵표체재체적가행성.통과다공능분광광도의측정세균적OD600화eGFP재대장간균화감독사문씨균중적표체량,결과표명:eGFP재사문씨균중적표체량의뢰우IPTG적농도,최가농도위0.5 mmol·L-1,증가농도지0.75 mmol·L-1불능증가eGFP적표체량;이해현상재대장간균중불명현.당IPTG농도등우혹대우0.5 mmol·L-1시,사문씨균중eGFP적표체량현저고우대장간균.약이상대형광도(1개OD600단위적형광치,즉eGFP상대표체량)표시,량충숙주균적최가유도시간균위3 h.Hela세포침습력시험표명해감독사문씨균잉구유침습력,동시이지질체전염위대조,장휴대중조질립pcDNA3-eGFP적감독사문씨균전염Hela세포,48 h후가관찰도록색형광,표명료해균가작위재체장중조질립휴대도세포내,병사외원기인득도표체.차외,이용기우미공판적다공능분광광도의가직접검측형광강도화OD치,능쾌속、준학지진행다양본검측,가용우GFP재불동숙주균혹상동숙주균재불동시험조건하,심지불동표체재체재상동숙주균중표체등방면적연구.
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.