CAJ | 학술논문

为探讨肠炎沙门菌非编码小RNA (non-coding small RNA) IsrE在转录后水平是否参与对fimA的调控作用，在大肠杆菌中构建并表达FimA重组蛋白，并对其培养条件进行优化，制备高纯度FimA重组蛋白。利用原核表达载体pET-28a（+）构建出能表达fimA的重组质粒，并分别对IPTG浓度、诱导温度和诱导时间等条件进行优化，以提高目的蛋白的产量和增大其可溶性，最后经Ni-NTA亲和层析分离纯化FimA重组蛋白。通过酶切、测序和Western blot鉴定分析，成功构建并表达肠炎沙门菌I型菌毛主要亚单位FimA，蛋白分子质量约为19.3 kDa，且主要以包涵体的形式表达，经Ni-NTA亲和层析纯化后的重组蛋白在SDS-PAGE中显示单一条带。本研究构建、表达并获得纯度较高的重组蛋白FimA，为进一步研究和验证肠炎沙门菌非编码sRNA IsrE是否在转录后水平参与对fimA的调控奠定了基础。
위탐토장염사문균비편마소RNA (non-coding small RNA) IsrE재전록후수평시부삼여대fimA적조공작용，재대장간균중구건병표체FimA중조단백，병대기배양조건진행우화，제비고순도FimA중조단백。이용원핵표체재체pET-28a（+）구건출능표체fimA적중조질립，병분별대IPTG농도、유도온도화유도시간등조건진행우화，이제고목적단백적산량화증대기가용성，최후경Ni-NTA친화층석분리순화FimA중조단백。통과매절、측서화Western blot감정분석，성공구건병표체장염사문균I형균모주요아단위FimA，단백분자질량약위19.3 kDa，차주요이포함체적형식표체，경Ni-NTA친화층석순화후적중조단백재SDS-PAGE중현시단일조대。본연구구건、표체병획득순도교고적중조단백FimA，위진일보연구화험증장염사문균비편마sRNA IsrE시부재전록후수평삼여대fimA적조공전정료기출。
To investigate if the non-coding small RNA IsrE of Salmonella enteritidis was involved in regulating FimA at the post-transcriptional level, fimA gene was cloned in pET-28a(+) vector that was then transformed into E.coli BL21(DE3). The expression conditions of FimA were optimized and expressed FimA was purified using Ni2+-chelating chromatography. The recombinant FimA was visualized as a single clear band of a relative molecular weight at 19.3 kDa in SDS-PAGE. The availability of highly purified recombinant FimA provided the foundation and platform for further study on whether or not FimA acted as a candidate target gene for small non-coding RNA IsrE of Salmonellaenteritidis at the post-transcriptional level.