重组非糖基化尿激酶型纤溶酶原激活剂的研究
发布时间：2018-12-13 23:40
【摘要】：目的 构建非糖基化、抗凝血酶激活的高分子量尿激酶原突变体。 方法 使用PCR扩增的方法,将302位天冬酰胺(Asn302)突变为丙氨酸(Ala302),去掉糖基化位点;将156位精氨酸(Arg156)突变为赖氨酸(Lys156),去掉凝血酶作用位点。 结果 PCR产物经琼脂糖电泳鉴定分子量为1296Da(道尔顿,dalton)。突变体与pUC19克隆载体用HindⅢ、XbaⅠ双酶切。二者连接后,转化入DH5α感受态细胞(大肠杆菌)中。用蓝白斑筛选法识别PCR产物是否与pUC19载体连接。包含PCR产物的克隆呈现白色。将白色克隆提质粒送测序。测序正确的基因与pIPES双表达载体连接后备用。 结论 非糖基化抗凝血酶的高分子量尿激酶原突变体构建成功。
[Abstract]:Objective to construct a non-glycosylated, anti-thrombin-activated high molecular weight urokinase mutant. Methods using PCR amplification, 302-site asparagine (Asn302) was mutated to alanine (Ala302) to remove glycosylation sites, and 156-arginine (Arg156) was mutated to lysine (Lys156) to remove thrombin action sites. Results the molecular weight of PCR product was identified as 1296Da (Dalton, dalton).) by agarose electrophoresis. The mutant and pUC19 clone vector were digested with Hind 鈪，
本文编号：2377464
[Abstract]:Objective to construct a non-glycosylated, anti-thrombin-activated high molecular weight urokinase mutant. Methods using PCR amplification, 302-site asparagine (Asn302) was mutated to alanine (Ala302) to remove glycosylation sites, and 156-arginine (Arg156) was mutated to lysine (Lys156) to remove thrombin action sites. Results the molecular weight of PCR product was identified as 1296Da (Dalton, dalton).) by agarose electrophoresis. The mutant and pUC19 clone vector were digested with Hind 鈪，
本文编号：2377464
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