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目的建立能稳定表达SAV(Salrador Homolog)的人胚胎肾细胞HEK293细胞池并从中纯化出SAV相互作用蛋白复合物。方法从人胚胎肾细胞HEK293中提取RNA,RT-PCR扩增得到SAV基因片断,构建表达质粒pBabe-SBP-FLAG-SAV。将pBabe-SBP-FLAG-SAV质粒和病毒包装质粒共同转染HEK293T细胞来产生病毒,收获病毒后感染HEK293细胞。通过嘌呤霉素筛选直至没有细胞死亡并用Western-blot验证表达情况。用链酶亲和素beads从该稳定细胞池中纯化SAV相互作用蛋白并通过银染检测。结果 pBabe-SBP-FLAG-SAV真核表达载体构建成功,包装病毒感染HEK293后通过Western-blot检测可见SAV蛋白的表达,FLAG beads和链酶亲和素beads免疫沉淀进一步证明了该融合蛋白表达的正确性。链酶亲和素纯化出的SAV相互作用蛋白复合物在银染时可见。结论成功构建了pBabe-SBP-FLAG-SAV真核表达载体及稳定表达细胞池,纯化得到SAV相互作用蛋白复合物。为阐明这些蛋白复合体在Hippo信号通路中的作用提供了可靠信息。
Objective To establish a human embryonic kidney cell HEK293 cell pool that can stably express SAV (Salrador Homolog) and to purify SAV-interacting protein complex from it. Methods RNA was extracted from human embryonic kidney cells HEK293. The SAV gene fragment was amplified by RT-PCR and the expression plasmid pBabe-SBP-FLAG-SAV was constructed. HEK293T cells were co-transfected with pBabe-SBP-FLAG-SAV plasmid and virus packaging plasmids to produce virus, which was then infected into HEK293 cells. Purification by puromycin was performed until no cell death was observed and the expression was verified by Western-blot. SAV interacting proteins were purified from this stable cell pool with streptavidin beads and detected by silver stain. Results The eukaryotic expression vector pBabe-SBP-FLAG-SAV was successfully constructed. The expression of SAV protein was detected by Western-blot after the HEK293 cells were infected with the virus. FLAG beads and streptavidin beads immunoprecipitation further confirmed the expression of the fusion protein Correctness Streptavidin-purified SAV-interacting protein complexes were visible on silver stain. Conclusion The eukaryotic expression vector pBabe-SBP-FLAG-SAV was successfully constructed and the cell pool was stably expressed. The SAV-interacting protein complex was purified. It provides reliable information to elucidate the role of these protein complexes in the Hippo signaling pathway.