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目的:构建人Cdc25C基因原核表达载体,获得纯化的GST-Cdc25C融合蛋白,并对其功能进行初步检测。方法:用PCR方法从乳腺文库中扩增Cdc25C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Ros-sate表达后,用GST-Sepharose 4B珠子纯化融合蛋白,并用SDS-PAGE和Western blot方法检测融合蛋白表达,通过GSTpull-down技术检测纯化蛋白与已知结合蛋白Chk2的相互作用。结果:构建得到Cdc25C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菌中诱导表达出相对分子质量(Mr)约为80 000的目的蛋白,经SDS-PAGE和Western blot检测,融合蛋白成功表达,并纯化得到GST-Cdc25C融合蛋白,GST pull-down检测证实GST-Cdc25C蛋白可以和已知相互作用蛋白Chk2相互作用。结论:成功克隆Cdc25C基因,并获得了活性良好的GST-Cdc25C蛋白,为进一步研究细胞周期蛋白调控机制奠定了实验基础。
OBJECTIVE: To construct the prokaryotic expression vector of human Cdc25C gene and obtain the purified GST-Cdc25C fusion protein, and to test its function. METHODS: The coding sequence of Cdc25C gene was amplified from the mammary gland by PCR and inserted into pGEX-KG vector. After the recombinant plasmid was transformed into E. coli Ros-sate, the fusion protein was purified with GST-Sepharose 4B beads and analyzed by SDS-PAGE Western blot was used to detect the expression of the fusion protein. The interaction between the purified protein and the known binding protein Chk2 was detected by GSTpull-down assay. Results: The prokaryotic expression vector of Cdc25C gene was constructed. The inserted fragment was identified by double enzyme digestion and was consistent with the expected fragment size. The sequence was consistent with the target sequence. The relative molecular mass (Mr) was induced in Rossate strain GST-Cdc25C fusion protein was confirmed by SDS-PAGE and Western blot. GST pull-down assay confirmed that GST-Cdc25C protein can interact with the known interaction protein Chk2 . CONCLUSION: The Cdc25C gene was successfully cloned and the GST-Cdc25C protein with good activity was obtained, which laid the experimental foundation for further study on the regulatory mechanism of the cell cycle proteins.