EST3-EGFP融合蛋白重组体的构建和表达

Construction and identification of EST3-EGFP fusion protein recombinant

目的构建EST3-EGFP融合蛋白穿梭载体并检测其表达。方法重叠延伸PCR获取EST3-EGFP融合基因,克隆至pTriEx-4穿梭载体中,重组体转化至细菌Rosetta(De3)pLacI,诱导蛋白的表达,SDS-聚丙烯酰胺凝胶电泳鉴定表达的融合蛋白;免疫印迹法检测重组蛋白的免疫学活性;荧光显微镜观察转化宿主细菌的发光活性。结果成功构建了EST3-EGFP重组体;融合蛋白主要分布于表达菌细胞质中;免疫印迹杂交检测表明重组蛋白具有EST3的免疫原性;诱导后的菌体在荧光显微镜下可见强烈的绿色荧光。结论成功构建的EST3-EGFP穿梭表达载体具有EST3免疫原性和GFP发光特性。

Objective EST3-EGFP fusion protein expressing shuttle vector was constructed. Methods EST3-EGFP fusion gene was obtained by overlapping extension PCR, and was cloned into shuttle vector pTriEx-4. The fusion protein was induced to express by IPTG. SDS-PAGE and western-blot were carried out to identify the fusion protein and fluorescence microscope was used to detect EGFP activity of the fusion protein. Results The recombinant pTriEx-EST3-EGFP was successfully constructed and the fusion protein was expressed. Western blot showed that the recombinant protein is immunocompetent and the IPTG-induced Rosetta （De3）pLacI was observed to emit green fluorescence. Conclusion EST3-EGFP fusion protein expressing shuttle vector was successfully constructed and induction was highly efficient in Rosetta （De3）pLacI. Bioactivity of EST3 and EGFP in this fusion protein is intact.