CAJ | 학술논문

目的原核表达及纯化绿脓杆菌鞭毛蛋白FlgE并进行初步活性鉴定.方法分析绿脓杆菌鞭毛蛋白FlgE的基因序列,设计带适当酶切位点的引物,用PCR方法扩增出 FlgE的编码DNA序列,与大肠杆菌表达载体pET24a同时经Nde Ⅰ/HindⅢ双酶切、纯化及连接后构建pET24a-FlgE原核表达质粒,挑选重组阳性质粒经DNA测序确认序列正确,转化至大肠杆菌BL21中进行诱导表达条件优化,使重组质粒表达目的蛋白,采用组氨酸标签亲和层析法对目的蛋白进行纯化,通过SDS-PAGE对纯化后蛋白相对分子质量进行鉴定,用试剂盒进行去内毒素化处理.在体外培养人角膜上皮细胞系细胞,加入纯化的FlgE重组蛋白,培养4h后应用实时定量PCR检测各种相关的炎性因子以反映FlgE蛋白活性.结果可用于大肠杆菌表达系统的重组pET24a-FlgE构建成功,其编码的蛋白在BL21中获得高效表达,当诱导剂异丙基-β-D-硫代吡喃半乳糖苷浓度为1 mmol/L,在16℃、225r/min振荡培养20 h,诱导目的蛋白表达量最高,经纯化、去内毒素处理和SDS-PAGE鉴定,获得纯化的重组FlgE蛋白.角膜上皮细胞用20 μg/ml FlgE处理后,细胞内炎性相关分子IL-6和IL-8的表达量明显上升,而灭活的FlgE蛋白则丧失该刺激活性.结论获得纯化的重组绿脓杆菌鞭毛蛋白FlgE,且可刺激角膜上皮细胞上调炎性因子IL-6、IL-8的表达.
목적 원핵표체급순화록농간균편모단백FlgE병진행초보활성감정.방법 분석록농간균편모단백FlgE적기인서렬,설계대괄당매절위점적인물,용PCR방법확증출 FlgE적편마DNA서렬,여대장간균표체재체pET24a동시경Nde Ⅰ/HindⅢ쌍매절、순화급련접후구건pET24a-FlgE원핵표체질립,도선중조양성질립경DNA측서학인서렬정학,전화지대장간균BL21중진행유도표체조건우화,사중조질립표체목적단백,채용조안산표첨친화층석법대목적단백진행순화,통과SDS-PAGE대순화후단백상대분자질량진행감정,용시제합진행거내독소화처리.재체외배양인각막상피세포계세포,가입순화적FlgE중조단백,배양4h후응용실시정량PCR검측각충상관적염성인자이반영FlgE단백활성.결과 가용우대장간균표체계통적중조pET24a-FlgE구건성공,기편마적단백재BL21중획득고효표체,당유도제이병기-β-D-류대필남반유당감농도위1 mmol/L,재16℃、225r/min진탕배양20 h,유도목적단백표체량최고,경순화、거내독소처리화SDS-PAGE감정,획득순화적중조FlgE단백.각막상피세포용20 μg/ml FlgE처리후,세포내염성상관분자IL-6화IL-8적표체량명현상승,이멸활적FlgE단백칙상실해자격활성.결론 획득순화적중조록농간균편모단백FlgE,차가자격각막상피세포상조염성인자IL-6、IL-8적표체.
Objective To construct the prokaryotic expression and purification protocols for Pseudomonas aeruginosa type E flagellin (FlgE) and to study its bioactivity.Methods With analysis of Pseudomonas aeruginosa flagellin FlgE sequences,the whole-length FlgE gene was amplified from Pseudomonas aeruginosa genomic DNA by using PCR and primers with proper restriction enzyme sites.The amplified FlgE fragment and prokaryotic expression plasmid pET24a were digested with Nde Ⅰ and Hind ⅢⅣ respectively.The target fragment and vector were recovered and ligated to obtain the recombinant plasmid pET24a-FlgE.DNA sequencing of positive clone confirmed that the target gene and the junctions with vectors were all correct.The plasmid pET24a-FlgE was transformed into BL21 bacteria.The culture conditions like temperature,rotation speed,inducer concentrations,time length were optimized to achieve maximal expression of the target recombinant FlgE with 6×His tag at C terminal.FlgE-His proteins were purified using His-Trap affinity chromatography columns and identified by SDS-PAGE.The purified proteins were further subjected to endotoxin elimination with proper kits.The purified recombinant FlgE was added to cultured corneal epithelial cells for 4 h and the expression of several inflammation-related molecules was examined by using real-time quantitative PCR.Results The recombinant plasmid pET24a-FlgE was successfully constructed and high level FlgE expression was achieved in BL21 with rotation at 16℃ and 1 mmol/L isopropyl-β-D-glucopyranosyl-galactosidase induction for 20 h.Purified recombinant FlgE-His was obtained and used for primary bioactivity assay.After treatment of corneal epithelial cells with 20 μg/ml FlgE for 4 h,the expression of inflammatory cytokines IL-6,IL-8 were significantly increased.Inactivation of the FlgE with ethanol abolished its stimulatory activity.Conclusion The prokaryotic expression and purification system for recombinant Pseudomonas aeruginosa flagellin FlgE was set up,and the recombinant FlgE stimulated the expression of inflammatory factors in corneal epithelial cells.