pET15b-EGFP原核表达载体的构建及其表达和纯化

目的:利用中间载体pGEM-T easy vector构建表达型载体pET15b-EGFP,在大肠杆菌中高效表达可溶性蛋白EGFP并将其纯化。方法:以质粒pLEGFP-C1为模板采用聚合酶链反应(PCR)的方法特异性扩增EGFP cD-NA序列,利用Taq DNA聚合酶的非模板依赖性末端转移酶活性催化PCR扩增的EGFP序列和三磷酸脱氧腺苷(dATP)反应,在EGFP序列两3’端加“A”,将纯化后的EGFP序列与中间载体pGEM-T easy vector连接后转化感受态大肠杆菌DH5α,经蓝白筛选,挑取白色单菌落进行扩增、酶切鉴定,正确重组的质粒命名为pGEM-T-EGFP。用XhoI和BamH I分别双酶切pGEM-T-EGFP和pET15b,低熔点琼脂糖回收EGFP cDNA和线性化的pET15b片段后将两片段相连,转化感受态大肠杆菌DH5α并对重组质粒进行酶切鉴定和DNA测序,获取正确重组的表达型质粒并命名为pET15b-EGFP。将正确重组的质粒pET15b?EGFP转化感受态大肠杆菌BL21(DE3),用异丙基β-D-半乳糖苷(IPTG)诱导表达。利用表达蛋白N端的组氨酸“标签”(H is-tag)进行N i2+-树脂柱亲和层析纯化,SDS-PAGE和W estern b lotting鉴定纯化蛋白质。结果:经测序证实重组质粒pET15b-EGFP中的EGFP cDNA序列与从C lontech公司购买的质粒pLEGFP-C1中的EGFP cDNA序列完全一致,从而成功构建了表达型重组质粒pET15b-EGFP。pET15b-EGFP转化感受态大肠杆菌BL21(DE3)并经诱导后得到高效表达,表达量可达66 mg/100 m l菌液,SDS-PAGE和W estern b lotting显示纯化蛋白为目的蛋白EGFP。结论:表达型重组质粒pET15b-EGFP的成功构建和表达、纯化为细胞穿透肽-EGFP融合蛋白穿透细胞能力的研究奠定了基础。

The construction of pET15b-EGFP and cnhanced green fluorescent protein expression and purification within BL21(DE3)

Objective The expression of vector pET15b-EGFP was constructed and the expression and purification of EGFP was performed.Methods Using pLEGFP-C1 plasmid as template,full-length EGFP cDNA was amplified by polymerase chain reaction.Then the amplified EGFP cDNA was added single "A" at its 3' end by using the template-independent terminal transferase activity of Taq DNA polymerase when there was only dATP exsisted.The purified EGFP cDNA being added single "A"at its 3' end was then ligated with pGEM-T easy vector,and the ligated products were transformed into DH5α.After the blue-white selection,a single white clone was picked to be propagated and identified,the correct recombinant plasmid was named after pGEM-T-EGFP.pGEM-T-EGFP and pET15b plasmids were further double-enzyme digested by XhoI and BamHI,respectively,the EGFP cDNA fragment and linearized pET15b fragment were purified by low melting point agarose electrophoresis and was ligated with Ligafast DNA ligation system.The resultant ligation product was transformed into DH5α.The positive clone was propagated and the recombinant plasmid was extracted for further identification and sequencing.The correct recombinat plasmid was named after pET15bEGFP.E.coli BL21(DE_(3)) was transformed with pET15b-EGFP,and induced with IPTG for protein expression.Fusion protein with an N-terminal His-tag could be purified by Ni~(2+)-resin affinity chromatography.Purified protein was identified by SDS-PAGE and Western blotting.Results It was confirmed by DNA sequencing that the EGFP cDNA sequence in recombinant plasmid pET15b-EGFP was in accordance with the EGFP cDNA sequence in plasmid pLEGFP-C1 which was purchased from Clontech company.The recombinant plasmid pET15b-EGFP were constructed successfully.EGFP was expressed efficiently after pET15b-EGFP had been transformed and induced in E.coli BL21(DE_(3)),the yield of the purified protein was approximately 66 mg/100ml bacteria medium.SDS-PAGE and Western blotting demonstrated that the purified protein was EGFP indead.Conclusions Construction of recombinat plasmid pET15b-EGFP and expression and purification of the EGFP provides a basis for the research on the penetrating capability of Cell-penetrating peptide-EGFP fusion protein.