人α-磷酸丙酮酸水合酶原核表达载体的构建及蛋白纯化  

Construction of human ENO1 gene prokaryotic expression vector and purification of the ENO1 protein

作　　者：宋烨琼 董倩[2] 梁迎春[2] 李玲[2] 洪甜[2] 晋帅 徐小洁[2] 叶棋浓[2] 吕朝晖[1] 

机构地区：[1]解放军总医院,北京100853 [2]军事医学科学院生物工程研究所,北京100850

出　　处：《军事医学》2016年第5期384-386,391,共4页Military Medical Sciences

基　　金：国家自然科学基金资助项目(81372161;81472589;31100604);北京市科技新星计划资助项目(Z141102001814055);军事医学科学院创新基金转化医学项目(ZHYX003);北京市自然科学基金资助项目(7132155)

摘　　要：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 vector of human ENO1 gene and to express and purify PET-28a-ENO1 fusion protein. Methods Human ENO1 coding region was amplified from human mammary gland c DNA by PCR and inserted into the prokaryotic expression vector of PET-28 a. The recombinant plasmid PET-28a-ENO1 was transformed into E. coli Rossate. The expressed product was purified by PET-28a-Sepharose 4B beads and identified by SDSPAGE and Western blotting. Results The DNA fragment of about 1300 bp was successfully amplified by PCR and inserted into the vector of PET-28 a. The results of double digestion and sequencing indicated that the PET-28a-ENO1 recombinant plasmid was successfully obtained. The PET-28a-ENO1 fusion protein of about 57 × 10^3（ Mr） was successfully induced,and identified by SDS-PAGE and Western blotting. Conclusion The prokaryotic expression vector of PET-28a-ENO1 is constructed successfully,which will facilitate further research.

关 键 词：人ENO1基因 磷酸丙酮酸水合酶 原核表达 纯化 糖酵解 

分 类 号：R346[医药卫生—基础医学]