CAJ | 학술논문

对鼠伤寒沙门氏菌的鞭毛蛋白FliC的基因进行克隆、鉴定和原核表达，为鞭毛蛋白佐剂作用的研究奠定基础。以鼠伤寒沙门氏菌8014菌株基因组DNA为模板，PCR扩增Flic基因，产物与T载体连接，经测序鉴定正确后与表达载体pQE30（＋）连接构建重组表达质粒Flic—pQE30，将此重组质粒转化人表达宿主E．coliXLI—Blue菌株内抽提质粒，酶切鉴定正确后对转化菌株以1PTG进行诱导后，表达产物经镍离子亲和层析柱纯化后，进行SDS—PAGE和Westernblot分析。测序结果显示，FliC为完整的编码区基因1485bp，编码495个氨基酸残基，蛋白相对分子质量约为56kDa。经SDS—PAGE分析表明，以37℃、1mM的IPTG诱导5h，表达效果最好，诱导产物是与理论值相符的56kDa的融合蛋白。经western—blotting检测，表达的重组蛋白FliC可与小鼠抗鼠伤寒沙门氏菌全菌体多抗血清反应得到清晰的目的条带，表明表达的重组蛋白具有良好的反应原性。成功进行了鼠伤寒沙门氏菌鞭毛蛋白nic基因的克隆表达，为进一步研究其免疫佐剂作用奠定了基础。
대서상한사문씨균적편모단백FliC적기인진행극륭、감정화원핵표체，위편모단백좌제작용적연구전정기출。이서상한사문씨균8014균주기인조DNA위모판，PCR확증Flic기인，산물여T재체련접，경측서감정정학후여표체재체pQE30（＋）련접구건중조표체질립Flic—pQE30，장차중조질립전화인표체숙주E．coliXLI—Blue균주내추제질립，매절감정정학후대전화균주이1PTG진행유도후，표체산물경얼리자친화층석주순화후，진행SDS—PAGE화Westernblot분석。측서결과현시，FliC위완정적편마구기인1485bp，편마495개안기산잔기，단백상대분자질량약위56kDa。경SDS—PAGE분석표명，이37℃、1mM적IPTG유도5h，표체효과최호，유도산물시여이론치상부적56kDa적융합단백。경western—blotting검측，표체적중조단백FliC가여소서항서상한사문씨균전균체다항혈청반응득도청석적목적조대，표명표체적중조단백구유량호적반응원성。성공진행료서상한사문씨균편모단백nic기인적극륭표체，위진일보연구기면역좌제작용전정료기출。
The purpose of this study was to clone, express and identify FliC protein of Salmonella typhimurium in order to provide the foundation for the study of adjuvant effects of FliC. The gene encoding protein FliC was amplified from the genomic DNA of S.typhimurium 8014 by using PCR technique. The amplified product was cloned into pMD18-T. Plasmids containing the right insert were sequenced to confirm its identity,and then cloned into expression vector pQE30 (+). The constructed recombinant plasmid Flic-pQE30 was transformed to E.cali XL1-Blue and induced with IPTG, and the expressed product was identified by SDS-PAGE and Western blot. Sequence analysis showed that the full coding length of Flic was 1 485 bp,which could encode 495 amino acid residues with a molecular mass of 56 kD.The SDS-PAGE electrophoresis analysis showed that the best expression was induced in 5 hours by 37 ~C and 1 mM IFl'G,under which a relative molecular weight of 56 kD recombinant protein FliC was produced. The recombinant FliC showed specific reaction with sera of rats immunized with S.typhimurium by Western blotting,which showed that the recombinant protein has well reactogenicity. The prokaryotic expression vector for fliC gene was constructed successfully,and fusion protein was expressed in E.coli XLl-Blue,and lay the foundation for the study of adjuvant effects of FliC.