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OALib Journal期刊
ISSN: 2333-9721
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his标记的小鼠线粒体转录因子a的克隆和表达纯化及其多克隆抗体的制备
, PP. 774-777
Keywords: 线粒体转录因子a,融合蛋白,基因克隆,蛋白表达
Abstract:
目的表达和纯化带his标记的小鼠线粒体转录因子a(mitochondrialtranscriptionfactora,mttfa)融合蛋白,并制备mttfa的特异性多克隆抗体。方法提取正常balb/c小鼠肝脏组织总rna,通过rt-pcr方法扩增出无信号肽的mttfa编码序列,克隆入经kpnⅰ和bamhⅰ酶切后的pet14b。酶切及测序鉴定正确后转化大肠杆菌bl21(de3),经iptg诱导表达后用镍离子亲和树脂(ni2+-ntahis？bindresin)纯化融合蛋白。用纯化的蛋白免疫新西兰大白兔,制备多克隆抗体。结果成功克隆出了mttfa的编码序列,并构建了带his标记的小鼠mttfa融合蛋白的重组表达质粒pet14b/his-mttfa。表达的融合蛋白经亲和树脂纯化后得到大量的高纯度目的蛋白,获得了高特异性兔抗血清。结论获得了his标记的小鼠mttfa原核表达载体,纯化得到高纯度的目标蛋白,并制备出高特异性兔抗血清,对进一步研究其生物学功能及调节细胞核同线粒体之间功能协同性的信号通路有重要意义。
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