pQE-EGFP原核表达载体的构建及其表达和鉴定

目的:利用分子克隆技术构建原核表达载体pQE-EGFP,在大肠埃希菌中高效表达与鉴定。方法:以质粒pEGFP-N2为模板,采用PCR方法特异性扩增增强型绿色荧光蛋白(EGFP)基因序列,将其克隆于原核表达载体pQE-31,所构建的重组质粒经测序鉴定后转化大肠埃希菌JM109,用异丙基巯基半乳糖(IPTG)诱导表达;十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)和Western blotting鉴定表达蛋白质。结果:PCR扩增得到了EGFP全长结构基因。所构建的pQE-EGFP重组质粒经酶切及测序鉴定结果与设计序列一致;转化大肠埃希菌JM109,经IPTG诱导后,目的蛋白表达率约为25%;SDS-PAGE、Western blotting初步测定目的蛋白的相对分子质量约为28 500,与理论预期值一致。结论:pQE-EGFP重组质粒的成功构建、表达和鉴定为TAT-EGFP融合蛋白穿透细胞能力的研究奠定了基础。

Construction of pQE-EGFP and Enhanced green fluorescent protein expression and identification

Objective: To construct a recombinant plasmid pQE-EGFP and to investigate its expression and identification in E.coli. Methods: With pEGFP-N2 plasmid as the template,the full-length EGFP gene was amplified by polymerase chain reaction and then cloned into the pQE-31 vector.The recombinant plasmid pQE-EGFP was transformed into E.coli JM109 and induced with IPTG for protein expression.The expression of EGFP was identified by SDS-PAGE and Western blotting.Results: The full-length EGFP gene was successfully cloned into pQE-31.The results of SDS-PAGE and Western blotting showed that the molecular weight of the expressed protein was 28 500,with an expression rate of 25%.Conclusion: The successful construction,expression and identification of recombinat plasmid pQE-EGFP have provided a basis for the research on the penetrating capability of the TAT-EGFP fusion protein.