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目的构建带PET-28a标签的人ENO1基因原核表达产物,观察其表达并纯化出带PET-28a标签的ENO1融合蛋白,为研究肿瘤糖酵解的相关机制奠定实验基础。方法以人乳腺文库为模板,利用PCR技术扩增出ENO1基因编码片段,将其插入到PET-28a载体中,鉴定正确的重组质粒转化大肠杆菌Rossate,小量诱导表达后,利用PET-28a-琼脂糖凝胶4B亲和珠纯化PET-28a-ENO1融合蛋白,经SDS-PAGE电泳和Western印迹检测,获得纯度较好的PET-28a-ENO1蛋白。结果利用PCR技术从乳腺文库中扩增得到大小约1300 bp的目的基因片段,插入PET-28a载体中,经双酶切鉴定及测序,结果表明PET-28a-ENO1质粒构建成功;转化Rossate菌并小量诱导,表达鉴定成功后纯化得到相对分子质量(Mr)约为57×103的目的蛋白。结论成功获得了人糖酵解相关基因ENO1的原核表达产物,为进一步研究ENO1在糖酵解过程中的作用机制奠定基础。
Objective To construct the prokaryotic expression product of human ENO1 gene with PET-28a tag and observe its expression and purify the ENO1 fusion protein with PET-28a tag, which lays an experimental foundation for the study on the mechanism of tumor glycolytic. Methods Human breast cDNA library was used as a template. The ENO1 gene was amplified by PCR and inserted into PET-28a vector. The correct recombinant plasmid was identified as E. coli Rossate. After induced by small amount, PET-28a- PET-28a-ENO1 fusion protein was purified by agarose gel 4B affinity chromatography and purified by SDS-PAGE electrophoresis and Western blotting. Results The target gene fragment of about 1300 bp was amplified by PCR and inserted into PET-28a vector. The double-restriction enzyme digestion and sequencing showed that the recombinant plasmid PET-28a-ENO1 was successfully constructed. A small amount of induction, the expression was identified after the successful purification of the relative molecular mass (Mr) of about 57 103 of the target protein. Conclusion The prokaryotic expression product of human glycolysis-related gene ENO1 was successfully obtained, which laid the foundation for further study on the mechanism of ENO1 in glycolysis.