树突状细胞中Rab40b/c相互作用蛋白质的鉴定研究

Identification of Rab40b and Rab40c interacting proteins in dendritic cells

目的改进串联亲和纯化质谱(TAP-MS)技术,鉴定并验证Rab40b和Rab40c在树突状细胞中的相互作用蛋白质。方法用分子克隆技术构建融合蛋白基因e Xact-6×His-EYFP-Rab40b和e Xact-6×His-EYFP-Rab40c的表达载体,采用慢病毒感染建立融合蛋白e Xact-6×His-EYFP-Rab40b和e Xact-6×His-EYFP-Rab40c稳定表达的树突状细胞系。改进串联亲和纯化技术,通过e Xact-6×His标签进行新型串联亲和纯化和q TOF液质检测,运用Comp PASS算法过滤和GO富集分析,鉴定Rab40b和Rab40c的相互作用蛋白,并采用免疫共沉淀验证。结果成功构建e Xact-6×His-EYFP-Rab40b和e Xact-6×His-EYFP-Rab40c融合蛋白稳定表达的树突状细胞系。采用新型串联亲和纯化方法,鉴定获得26个Rab40b高可信相互作用蛋白和16个Rab40c高可信相互作用蛋白。Rab40b、Rab40c分别与Elongin B、Elongin C、Cullin5具有相互作用。结论成功建立新型串联亲和纯化方法。Elongin B、Elongin C、Cullin5是Rab40b、Rab40c的相互作用蛋白。

Objective To identify Rab40 b and Rab40 c interacting proteins in dendritic cells with improved tandem affinity purification strategy. Method Vectors expressing e Xact-6 × His-EYFP-Rab40 b or e Xact-6 × His-EYFP-Rab40 c were constructed by molecular cloning techniques. Stable dendritic cell lines（ DC2. 4） expressing e Xact-6 × His-EYFP-Rab40 b protein and e Xact-6 × His-EYFP-Rab40 c protein were constructed by high-titer lentiviral transduction. Tandem affinity purification strategy was improved to use e Xact-6 × His tag to perform tandem purification and q TOF liquid mass spectrometry. Rab40 b and Rab40 c interacting proteins were screened by unbiased comparative metrics-based algorithm（ Comp PASS） and gene ontology enrichment analysis. The interacting proteins were identified by e Xact pull-down and coimmunoprecipitation. Results Stable dendritic cell lines（ DC2. 4） expressing e Xact-6 × His-Rab40 b protein and e Xact-6 × His-Rab40 c protein were constructed respectively. Twenty-six high-confidence Rab40 b interacting proteins and 16 high-confidence Rab40 c interacting proteins were identified by improved tandem affinity purification. Rab40 b and Rab40 c interacted with Elongin B,Elongin C and Cullin5. Conclusion The tandem affinity purification strategy is improved successfully. Elongin B,Elongin C and Cullin5 are interacting proteins of Rab40 b and Rab40 c.