PTD-Cyclin D1融合蛋白的原核表达及纯化

目的:构建融合重组表达载体并对其进行诱导表达和蛋白纯化以获得大量重组融合蛋白.方法:通过PCR方法扩增出Cyclin D1基因,克隆入pMD-18T载体,进行测序分析,将该基因亚克隆入原核表达载体pET-16b中PTD的下游构建重组质粒pET16b-PTD-CCND1,转化感受态细胞BL21(DE3),经IPTG诱导表达重组融合蛋白,对表达产物进行SDS-PAGE电泳和Western blot检测分析.结果:构建了重组融合表达质粒pET16b-PTD-CCND1,表达的融合蛋白经SDS-PAGE分析,在约Mr 38×103处出现了一条新生的蛋白条带,经灰度扫描检测,表达量约占菌体总蛋白的22%,纯化后得到了目的蛋白.结论:成功地克隆了小鼠的Cyclin D1基因并纯化了融合基因PTD- CCND1的原核表达产物.

Prokaryotic expression and purification of fusion protein PTD-Cyclin D1

AIM: To construct the recombinant expression vector and to obtain large amount of mouse Cyclin D1 protein fused to PTD. METHODS: A 888 bp of mouse Cyclin D1 gene fragment was amplified by PCR method and cloned into pET16b vector immediately downstream of the PTD fragment, an E.coli expression vector, to construct a recombinant plasmid pET16b-PTD-CCND1. The plasmid was transformed into E.coli BL-21 (DE3) and induced to express fusion protein PTD-Cyclin D1 with IPTG. The expression of PTD-Cyclin D1 was detected by SDS-PAGE electrophoresis and Western blot. RESULTS: A novel protein with expected molecular mass was expressed upon induction with IPTG. The expressed product showed good reactivity to anti-His tag antibody, and was mostly in the form of inclusion bodies. CONCLUSION: Our successful cloning and expression of Cyclin D1 gene and purification of Cyclin D1 protein lay a basis for further study on the application of this protein to accelerate cell proliferation in vitro and in vivo.