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目的:构建人14-3-3σ基因的原核表达载体,获得其原核表达产物,并对融合蛋白进行纯化及活性检测。方法:采用PCR技术从人乳腺文库中扩增14-3-3σ基因编码序列,将其克隆到pGEX-KG载体中,重组质粒转化大肠杆菌Rossate后表达重组蛋白,利用GST-Sepharose 4B亲和珠对原核表达产物进行纯化,并通过SDS-PAGE和Western印迹检测融合蛋白的表达,采用GST pull-down技术检测已纯化的蛋白与已知体外相互作用蛋白AKT之间的相互作用。结果:从人乳腺文库中扩增获得约750 bp的DNA片段,并成功克隆至pGEX-KG载体上,经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,测序与目的基因序列完全一致;在Rossate菌株中诱导表达出相对分子质量约52 000的目的蛋白,SDS-PAGE和Western印迹结果表明融合蛋白表达成功,并纯化得到GST-14-3-3σ融合蛋白;通过GST pull-down技术检测证实GST-14-3-3σ融合蛋白可以和AKT在体外结合,并证实其具有生物学活性。结论:获得了原核表达的活性较好的GST-14-3-3σ蛋白,为后续研究细胞周期蛋白调控机制奠定了实验基础。
OBJECTIVE: To construct a prokaryotic expression vector for human 14-3-3σ gene and obtain its prokaryotic expression product. The fusion protein was purified and its activity assayed. Methods: The coding sequence of 14-3-3σ gene was amplified from human breast cDNA library by PCR and cloned into pGEX-KG vector. The recombinant plasmid was transformed into E. coli Rossate to express the recombinant protein. GST-Sepharose 4B affinity beads The prokaryotic expression product was purified and the fusion protein was detected by SDS-PAGE and Western blotting. The interaction between the purified protein and the known in vitro interaction protein AKT was detected by GST pull-down technique. Results: A DNA fragment of about 750 bp was amplified from the human breast cDNA library and cloned into the pGEX-KG vector. The double-digested DNA fragment was inserted into the exogenous gene and sequenced. The results of SDS-PAGE and Western blot showed that the fusion protein was successfully expressed and the GST-14-3-3σ fusion protein was purified. By GST pull- down assay confirmed that GST-14-3-3σ fusion protein can bind with AKT in vitro and confirmed its biological activity. CONCLUSION: GST-14-3-3σ protein with good activity in prokaryotic expression was obtained, which laid the experimental foundation for further study on the regulation of the cell cycle proteins.