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目的构建人FHL1各LIM结构域的原核表达载体,获得纯化的GST-FHL1-LIM1/2、-LIM1、-LIM2、-LIM3和-LIM4融合蛋白,并对其活性进行检测。方法用PCR法从FHL1真核表达载体中扩增FHL1的LIM1/2、LIM1、LIM2、LIM3和LIM4基因编码序列,将其正确插入pGEX-KG载体,重组质粒转化大肠杆菌Rossate表达后,用GST-Sepharose 4B珠子纯化融合蛋白,并用Western blot法检测融合蛋白表达,通过GST pull-down技术检测纯化蛋白与已知结合蛋白雌激素受体α(ERα)的相互作用。结果构建得到FHL1-LIM1/2、-LIM1、-LIM2、-LIM3和-LIM4基因的原核表达载体,双酶切鉴定得到与预期片段大小相符的外源基因插入片段,经测序与目的序列完全一致;在Rossate菌中诱导表达出与预期大小相符的目的蛋白,经Western blot法检测,融合蛋白成功表达;纯化得到GST-FHL1-LIM1/2、-LIM1、-LIM2、-LIM3和-LIM4五个融合蛋白,GST pull-down证明FHL1全长以及FHL1-LIM1/2、-LIM2蛋白可以和ERα相互作用。结论成功克隆FHL1-LIM1/2、-LIM1、-LIM2、-LIM3和-LIM4基因,并获得了活性良好的GST-FHL1-LIM1/2、-LIM1、-LIM2、-LIM3和-LIM4蛋白,为研究FHL1在乳腺癌中的作用奠定了实验基础。
Objective To construct a prokaryotic expression vector of LIM domains of human FHL1 and obtain the purified GST-FHL1-LIM1 / 2, -LIM1, -LIM2, -LIM3 and -LIM4 fusion proteins, and test their activities. Methods The coding sequences of LIM1 / 2, LIM1, LIM2, LIM3 and LIM4 genes of FHL1 were amplified by PCR from FHL1 eukaryotic expression vector and inserted into pGEX-KG vector. The recombinant plasmids were transformed into E. coli Rossate and expressed in GST Sepharose 4B beads. The fusion protein was detected by Western blot and the interaction between the purified protein and the known binding protein estrogen receptor alpha (ERα) was detected by GST pull-down assay. Results The prokaryotic expression vectors of FHL1-LIM1 / 2, -LIM1, -LIM2, -LIM3 and -LIM4 were constructed and double-digested to obtain the exogenous gene fragment which was consistent with the expected fragment size. The sequence was exactly the same as the target sequence ; The target protein was induced to express in Rossart bacteria with the expected size, and the fusion protein was successfully expressed by Western blot; five GST-FHL1-LIM1 / 2, -LIM1, -LIM2, -LIM3 and -LIM4 The fusion protein, GST pull-down, demonstrated that FHL1 full length and FHL1-LIM1 / 2, -LIM2 protein can interact with ERα. Conclusion The FHL1-LIM1 / 2, -LIM1, -LIM2, -LIM3 and -LIM4 genes were successfully cloned and the GST-FHL1-LIM1 / 2, -LIM1, -LIM2, -LIM3 and -LIM4 proteins with good activity were obtained. Studying the role of FHL1 in breast cancer lays the foundation for the experiment.