GST-Fank1融合蛋白表达载体构建及表达纯化的研究

摘要

目的 研究转录因子Fank1与GST融合蛋白原核表达栽体的构建和表达.方法 根据Fank1基因的DNA序列,经引物设计软件Primer Premier 5.0优化设计出上下游引物(Ⅰ,Ⅱ),同时在5'端引入BamH Ⅰ酶切位点,3'端引入Sal Ⅰ酶切位点.以pdsRED-Fank1质粒为模板进行PCR扩增Fank1目的 片段,经限制性内切酶酶切后连接到原核表达载体pGEX4T-2,构建成pGEX4T-2-Fank1原核表达载体,转化大肠埃希菌BL21(DE3),通过异丙基硫代-β-D半乳糖苷诱导进行目的 蛋白表达.结果 经菌液PCR、电泳分析及测序证明成功构建了Fank1融合表达载体,融合蛋白产物经蛋白印迹显示,相时分子量为43 kDa的蛋白条带,与预期一致.结论 成功构建了pGEX4T-2-Fank1原核表达载体,并在原核细胞中有表达,为进一步研究转录因子Fank1的功能奠定了基础.%Objective To study the construction of GST-Fank1 fusion protein expression vector. Methods According to the DNA sequence which encodes Fank1 protein,the upstream and downstream primers ( Ⅰ , Ⅱ ) of Fank1 gene which contained BamH I and Sal Ⅰ sites were optimum designed by using the primer design software Premier 5.0. The Fank1 target fragment was obtained by PCR amp1ification with the pdsRED-Fank1 as template. After digested by restriction enzyme,the Fank1 target fragment was inserted into pGEX4T-2 to create the prokaryotc expression vector pGEX4T-2-Fank1. The expression vector was transformed into E. coliBL21 (DE3) and the expressive target protein was induced with IPTG. Results We constructed the prokaryotic expression vector of pGEX4T-2- Fank1 ,which was testified by using bacterium PCR,electrophoretic analysis and sequencing. Western blot result showed that the Mr 43 kDa of fusion protein band was consistent with the expected one. Conclusion GST-Fank1 fusion protein expression vector was constructed successfilly,and the expression of the fusion protein was observed in BL21(DE3).