摘要
微管相关蛋白1轻链3-β(LC3)在哺乳动物细胞中定位于自噬泡上,可作为一种特定的标记用来检测自噬。从SD大鼠脑组织提取总RNA,经RT-PCR扩增得到LC3全长编码基因,并克隆到pMD1 8-T载体。以测序验证正确的重组子为模板扩增LC3基因,并构建pUC18-LC3重组质粒,将其转化到E.coli BL21(DE3)中。SDS-PAGE结果表明,菌体诱导后形成包涵体形式的表达产物,以电泳回收目的蛋白并免疫新西兰白兔,获得大鼠LC3蛋白多克隆抗体。western blot结果显示,制备的多克隆抗体可特异性识别LC3蛋白。
Microtubule-associated protein 1 light chain 3-β （LC3）, which located on autophagosome membranes in mammalian cells, is a distinctive mark to detect autophage. The LC3 gene was amplified by RT-PCR from the template of total RNA extracted from SD rat brain and then cloned into the pMD-18T vector. The correct identified recombinant was used as a template for the amplification of LC3 gene by PCR. The product was inserted into the prokaryotic expression vector pUC18, named pUC18-LC3 and transformed into E. coli BL21 （DE3）. The recombinant protein LC3 was expressed. Following SDS-PAGE, the objective band was reclaimed. Anti-LC3 serum was also prepared in New Zealand rabbit with purified reclaimed protein. The results of western blot showed that the polyclonal antibodies could recognize LC3 specifically.
出处
《中国医药工业杂志》
CAS
CSCD
北大核心
2011年第10期739-741,共3页
Chinese Journal of Pharmaceuticals
基金
教育部高等学校博士学科点专项科研基金(20101301120005)
河北大学自然科学基金青年项目(2008Q49)
河北大学博士基金项目(2009-168)的资助
关键词
微管相关蛋白1轻链3-β
原核表达
多克隆抗体
制备
microtubule-associated protein 1 light chain 3-β （LC3）
prokaryotic expression
polyclonal antibody
preparation
