原核表达载体pET28a-EGFP的构建与表达

Construction and Expression of Prokaryotic Expression Vector pET28a-EGFP

以质粒PEGFP-N3中增强型绿色荧光蛋白(Enhanced Green Fluorescent Protein,EGFP)基因片段为模板,利用PCR技术扩增得到EGFP基因片段,并设计引物在其2端引入酶切位点EcoRⅠ和HindⅢ,对引入酶切位点的EGFP片段和pET28a质粒进行双酶切处理后,利用T4连接酶连接得到了重组质粒pET28a-EGFP。利用热击法把得到的重组质粒pET28a-EGFP转化至E.coliBL21(Escherichia coliBL21)感受态细胞中,当大肠埃希菌LB(Luria-Bertani)培养液在600 nm下的光密度值OD600=0.4时,通过添加异丙基硫代β-D-半乳糖苷(IPTG)作为诱导剂诱导EGFP表达。结果表明:重组质粒酶切鉴定及测序结果正确。在自然光下,转化子在LB固体培养基(含1 mmol/L的IPTG和50μg/mL的卡那霉素(Kan))中菌落呈绿色。在荧光显微镜下受蓝光激发,可以清楚观察到发绿色荧光的转化子。成功构建的原核表达载体pET28a-EGFP在E.coliBL21中得到了高效表达,为以后作为荧光标记物标记食源性病原菌提供了一定的理论和技术支持。

An enhanced green fluorescent protein（EGFP） gene fragment in plasmid PEGFP-N3 as a template was used to amplify and obtained the EGFP gene fragment by PCR,and then designed a primer to introduce EcoR I and Hind III sites to its both ends.After treated with double restriction enzyme,the introduced enzymatic site EGFP gene fragment,and pET28a plasmid,a recombinant expression plasmid pET28a-EGFP was obtained using T4 ligase.The pET28a-EGFP was transformed into competence cells of E.coli BL21 with heat shock method.The inducer of isopropyl β-D-1-thiogalactopyranoside（IPTG） was added to induce EGFP expression,as the optical density under 600 nm OD600=0.4 of the E.coli LB（Luria-Bertani） culture medium.The results indicated that the recombinant plasmid enzymatic sites characterization and sequencing was correct.Under the natural light,the transformant colonies assumed green in LB solid medium（contains 1 mmol/L IPTG and 50 μg/mL kanamycin（Kan））.When excited with blue-ray under fluorescence microscope,these recombinants emitting green fluorescence could clearly be observed.The successfully constructed prokaryotic expression vector pET28a-EGFP that expressed effectively in E.coli BL21 will provide certain theoretical and technical supports for marking food borne pathogens as fluorescent markers in the future.