【摘要】 目的:探讨SM22αC端功能域肽段与细胞骨架F-actin聚合的关系,明确SM22α在血管平滑肌细胞(VSMC)骨架重构中的作用。方法:构建GST-SM22αC端功能域融合蛋白原核表达质粒pGEX3X-SM22α,诱导E coli高效表达可溶性GST-SM22α融合蛋白,制备抗SM22α抗体,VSMC蛋白分步提取及Western blot检测F-actin/G-actin中SM22α的含量变化,GST-pull down分析和免疫共沉淀检测SM22α与actin的相互作用,细胞免疫双荧光染色观察SM22α和actin在VSMC中的定位关系。结果:所构建的pGEX3X-SM22α原核表达质粒,在0.5mmol/LIPTG,30℃诱导6h条件下,表达可溶性GST-SM22α融合蛋白的水平最高,用纯化的融合蛋白免疫新西兰白兔获得的抗血清效价为1∶16。免疫双荧光染色和蛋白分步提取分析结果表明,在VSMC再分析过程中,SM22α与F-actin共定位,GSTpull down分析和免疫共沉淀结果均显示,SM22α通过C端功能域与F-actin相互作用而参与细胞骨架的重构;但是,SM22α与G-actin的结合能力较弱。结论:本研究重组得到的SM22αC端功能域具有与F-actin结合的活性,SM22α通过该区域与actin相互作用而参与细胞骨架重构。更多还原

【Abstract】 Aim:To investigate the interaction between C-terminal domains of SM22α and cytoskeleton F-actin.Methods:Prokaryotic expression vector containing SM22α cDNA and GST sequence was constructed.The induction conditions were optimized to increase the product of soluble GST-SM22α fusion protein in E coli.Expression products were purified and rabbit anti-GST-SM22α polyclonal antibody was produced by the purified fusion protein.In order to explore the effect of SM22α on cytoskeleton reorganization,VSMCs were treated with serum withdrawal and then serum stimulation to induce contractile/synthetic phenotypic modulation.SM22α protein distribution in F-actin/G-actin fractions was detected by Western blotting.The interaction between SM22α and actin was examined by GST pull down assay and coimmunoprecipitation.Colocalization of endogenous SM22α with F-actin was observed by i-mmunofluorescence.Results:The results showed that the expression of soluble GST-SM22α protein was the highest under condition induced by 30℃,0.5 mmol/L IPTG for 6 h.Immunofluorescence and Western blotting of protein extracts from F-actin/G-actin fractions revealed that SM22α colocalized with F-actin during VSMC redifferentiation.GST pull down assay and coimmunoprecipitation showed that SM22α interacted with F-actin by C-terminal domains to participate in cytoskeleton reorganization.Conclution:The recombinant SM22α C-terminal domains have the ability to bind F-actin,by which SM22α interacts with actin and participates in cytoskeleton reorganization.更多还原