PTD-hBDNF融合蛋白的表达与生物活性测定

发布时间：2018-09-05 14:45

【摘要】：目的 PTD-hBDNF基因的克隆、表达和表达产物的纯化及生物活性测定。方法 先从人血白细胞中提取基因组总DNA,以基因组总DNA为模板,采用PCR技术扩增人BDNF全长基因,插入到pUC19质粒载体,筛选阳性克隆,经酶切鉴定无误后进行测序;以阳性克隆重组质粒pUC19-hBDNF为模板,再用含PTD编码序列作为PCR反应的5’引物,经PCR反应扩增得PTD-hBDNF融合基因,并插入pUCm-T载体;然后将PTD-hBDNF融合基因克隆至温控表达载体pJW2,转化大肠杆菌DH-5 α,42℃诱导表达。重组蛋白经初步复性纯化后,将其加入海马神经元细胞中进行重组蛋白的生物活性测定。结果PTD-hBDNF在大肠杆菌中的表达主要是以包涵体的形式存在。细菌的菌体蛋白经SDS-PAGE分析,在15kD处有一条明显的蛋白条带,经Western-blot分析表明PTD-hBDNF具有明显的免疫反应。复性纯化后的蛋白质纯度达到了90%以上,且具有明显的生物活性。结论成功构建了含PTD-hBDNF融合基因的表达载体,并在大肠杆菌中获得表达且对表达产物进行了有效的复性和纯化,得到了具有生物活性的融合蛋白。
[Abstract]:Objective to clone, express and purify PTD-hBDNF gene and determine its biological activity. Methods Total genomic DNA, was extracted from human leukocytes and genomic total DNA was used as template. The full-length gene of human BDNF was amplified by PCR technique and inserted into pUC19 plasmid vector to screen positive clones. The fusion gene of PTD-hBDNF was amplified by PCR reaction and inserted into pUCm-T vector using the positive clone recombinant plasmid pUC19-hBDNF as template and PTD coding sequence as 5 'primer for PCR reaction. Then the PTD-hBDNF fusion gene was cloned into the temperature-controlled expression vector pJW2, and transformed into Escherichia coli DH-5 伪 to induce expression at 42 鈩，

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