Consturction and expression of mouse Cdc25B protein
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摘要: 目的 研究小鼠N末端缺失286氨基酸的野生型Cdc25B原核表达载体的构建和表达.方法 应用Primer5.0软件分析并设计小鼠Cdc25B5′及3′端引物(N末端缺失286氨基酶),化学合成并用聚合酶链反就(PCR)方法扩增,经限制性内切酶酶切后连接原核表达载体PGEX4T-2,构建成PGEX4T-2-Cdc25B融合表达载体,转化大肠埃希菌BL21后,通过异丙基硫代-β-D半乳糖苷(IPTG)诱导进行目的蛋白表达.结果 通过双酶切、电泳分析及测序证明成功构建了Cdc25B融合表达载体,融合蛋白产物经蛋白印迹(Westernblot)显示,相对分子量为53KD的蛋白谱带,与预期一致.结论 成功构建并表达了Cdc25B-N末端缺失的基因,并在原核细胞中获得较高水平的表达,为近一步研究Cdc25B的功能提供基础依据.
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关键词:
- Cdc25B /
- 基因表达 /
- 谷胱甘肽转移酶(GST) /
- 聚合酶链式反应
Abstract: Objective To study construction and expression of mouse cdc25B protein.Methods The Cdc25B gene was amplified by PCR,enzyme digest and linked behind PG EX4T-2.After inducing with I PT G,the targ et gene was primarily expressed.Results T he Cdc25B gene consisted of about 900bp by DN A electrophoresis.The prokaryotic expression vector of PGEX4T-2/Cdc25B was constructed.After induction,a new anticipated Mr 53K D band appear ed in Westen-Blot.Conclusion Fusion proteins of GST/Cdc25B are successfully expressed in BL 21.This study is a basis for the role of Cdc25B during mouse oocyte maturatin.-
Key words:
- Cdc25B /
- gene expression /
- GST /
- PCR
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