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重组人瘦素的基因构建、表达及活性鉴定
Authors
吴娜
张长弓
+3 more
王臻
谢莲英
颜江华
Publication date
30 September 2006
Publisher
Abstract
根据NCBI中公布的人瘦素cDNA序列,设计并合成了6个89bp左右的DNA小片段,经重叠延伸PCR扩增获得464bp的rhLep的完整基因片段。构建pET22b(+)/rhLep表达载体,转化大肠杆菌BL21(DE3),IPTG诱导表达。目的蛋白以包涵体的形式表达,表达量占菌体总蛋白的50%以上。表达产物用Ni2+亲和层析柱纯化,SDS-PAGE结果表明,含6×His标签的目的蛋白的相对分子量约16kD。MTT比色法实验显示,当低剂量(10~30ng/mL)时,rhLep具有促进内皮细胞生长的作用;在高剂量(50~225ng/mL)时,rhLep具有杀伤人内皮细胞的活性。其最大杀伤率达到98.8%。吖啶橙荧光染色观察到高剂量rhLep对人内皮细胞有明显的凋亡作用。以上工作为进一步了解瘦素的体内体外生物活性奠定了基础
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Last time updated on 16/06/2016