摘要
为进一步研究hhLIM的功能及其与F肌动蛋白(Factin)之间的相互关系,利用PCR扩增hhlim基因并将其克隆到pGEX3X表达质粒上,重组表达质粒转化宿主菌得到稳定表达的可溶性产物,表达产物经GlutathioneSepharose亲和纯化得到纯度达90%的融合蛋白GSThhLIM.进而研究其在F肌动蛋白交联中的作用,以鬼比环肽和细胞松弛素处理C2C12细胞,诱导细胞骨架聚合和解聚.荧光显微镜下观察,hhLIM的分布变化与细胞骨架的形态学变化具有相关性.Western印迹证实,hhLIM主要作为细胞骨架相关蛋白而分布于F肌动蛋白组分中,肌动蛋白交联实验显示,hhLIM蛋白与F肌动蛋白具有较高的亲和力,具有促进F肌动蛋白纤维的交联并将其捆聚成束的作用.结果表明,hhLIM是一种F肌动蛋白交联蛋白,通过将F肌动蛋白纤维捆聚成束而参与骨架重构.
In order to further explore the biological functions of hhLIM and the interaction between hhLIM and actin, full-length cDNA coding hhlim gene obtained by PCR amplication, was cloned into the prokaryotic expression vector pGEX-3X and expressed in soluble form as GST-hhLIM fusion protein. After purification by Glutathione-Sepharose affinity chromatography, the purity of the fusion protein was above 90%. To further explore the effect of hhLIM on cytoskeleton organization, C2C12 cells were treated with cytochalasin B and phalloidin, and hhLIM protein distribution in cytoskeleton was detected. Microscopy images showed that hhLIM protein distributed in the cytoplasm and co-localized with F-actin. Western bolt analysis revealed that hhLIM existed mainly in the extracts of cytoskeleton-associated protein. F-actin cross-linking assay identified that hhLIM induced F-actin to cross-link into bundles. In conclusion, hhLIM may act as an organizer to crosslink F-actin into actin cytoskeleton.
出处
《中国生物化学与分子生物学报》
CAS
CSCD
北大核心
2006年第5期379-383,共5页
Chinese Journal of Biochemistry and Molecular Biology
基金
新世纪优秀人才支持计划项目
国家重点基础研究发展规划(973计划
No.G2000056905)
国家自然科学基金(No.30300132)
河北省自然科学基金(No.2004000644)资助.~~
