摘要
线粒体外膜易位酶是由Tom40基因编码的位于线粒体外膜上的转位酶复合体,能够将外界蛋白质转位运输到线粒体基质中.最近有研究称在一类仅具有简缩进化的线粒体——纺锤剩体的感染家蚕微孢子虫的基因组鉴定到一个NbTom40基因.本文首先通过克隆了长度为839bp的NbTom40基因,通过构建重组载体pET30a(+)-NbTom40,转入E.coli BL21(DE3)感受态细胞中进行诱导表达,经过HIS抗体的免疫印迹对表达的目的蛋白——31～43kDa间存在一条单一条带进行验证,结果显示该条带即为分子量大小正确的带有HIS标签的目的蛋白,然后将其进行Ni-NTA亲和层析以及聚丙烯酰胺凝胶纯化并制备多克隆抗体.NbTom40抗体与家蚕微孢子虫总蛋白的Western Blotting结果显示,在32kD处有明显杂交信号,与理论分子大小一致.最后,通过间接免疫荧光(IFA)对该基因编码的蛋白进行细胞定位,观察到NbTom40以散点状荧光信号分布在孢子内部.本研究为深入了解纺锤剩体的蛋白组成及其膜转运的分子机制奠定了分子生物学基础.
Mitochondrial outer membrane translocase located on the mitochondrial outer membrane. It can is a translocase complex encoded by gene Tom40 and translocate or transport external protein into the mi tocbondrial matrix. Recently, NbTom40 was annotated in the genome of Nosema bombycis, which is the pathogen of silkworm pebrine and belongs to Microsporidia which possess a reduced mitochondrial remnant called mitosome. In this study, we cloned NbTom40 with a whole length of 839 bp and expressed it in E. coli, and then prepared the specific polyelonal antibody, Western blotting results further verified that the specific using the purified fusion protein as the antigen. polyclonal antibody interacted with 32 kD protein of N. bornbycis, which is well consistent with the theoretical molecular weight of NbTom40. Finally, indi rect immunofluorescence assay was used to probe NbTom40 sub-cellular localization, and figured out the asterion signals distributed in the mature spore, which indicated the expression of NbTom40 in this mi crosporidia. These results lay a molecular-biological foundation for the exploration of the composition of protein and the mechanism of its translocation in mitosome in future.
出处
《西南大学学报（自然科学版）》
CAS
CSCD
北大核心
2013年第5期30-36,共7页
Journal of Southwest University(Natural Science Edition)
基金
国家自然科学基金资助项目(30930067
31072089
31272504)
863计划项目(2012AA101301-3)
教育部高校基本业务费重大孵育项目(XDJK2009-A005)
重庆市蚕桑重大专项(CSTC2010AA1003)
关键词
家蚕微孢子虫
线粒体外膜易位酶
原核表达
间接免疫荧光
Nosema bombycis
translocase of the mitochondrial outer membrane 40 kD subunit（Tom40）
prokaryotic expression
indirect immunofluorescence assay（IFA）
