新克隆的硝基还原酶基因NOR_1的表达及其产物的纯化

Expression of a New Cloned Nitroreductase Gene NOR_1 and Purification of Expressed Product

背景与目的:NOR1是与鼻咽癌密切相关的抑瘤/易感基因候选者之一,本实验旨在构建NOR1基因原核表达载体,在大肠杆菌中表达并纯化NOR1基因所编码的蛋白。方法:抽提人鼻咽正常组织中总RNA,利用RT-PCR扩增NOR1基因全长,经BamHⅠ、XhoⅠ双酶切后插入同样经BamHⅠ、XhoⅠ双酶切后的pGEX-4T-2质粒,构建表达载体pGEX-4T-2/NOR1重组表达质粒;转化大肠杆菌Jm105,用异丙基-β-D硫代半乳糖苷(isopropyl-thiogalactoside,IPTG)诱导pGEX-4T-2/NOR1/Jm105表达GST融合蛋白后,采用Westernblot验证,并用GST琼脂糖亲和层析柱对目的蛋白进行纯化。结果:酶切鉴定和测序验证成功地构建了NOR1基因原核表达载体,经十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(sodiumdodecylsulphatepolyacrylamidegelelectrophoresis,SDS-PAGE)分析在大肠杆菌Jm105中成功诱导表达了一分子质量约为74kDa的融合蛋白;该融合蛋白存在于细菌裂解液的上清和沉淀中,Westernblot证实表达获得了成功;并利用亲和层析法获得了纯化蛋白。结论:成功获得了高效表达NOR1基因的原核表达产物,通过对该融合蛋白的纯化为它的多抗制备奠定了基础。

BACKGROUND & OBJECTIVE:NOR1 is a good candidate of tumor suppressor/susceptibility gene associated with nasopharyngeal carcinoma. This study was designed to construct the prokaryotic expression vector and to investigate the expression of nitroreductase gene NOR1 in Escherichia coli and to purify expressed product. METHODS:Total RNA was subtracted from normal nasopharyngeal carcinoma tissue. The full length of NOR1 gene was amplified by reverse transcription polymerase chain reaction (RT PCR) and digested with BamHⅠand XhoⅠ restriction endonucleases. The plasmid pGEX 4T 2 was also digested with BamHⅠ and XhoⅠ,then the NOR1 gene was inserted into vector pGEX 4T 2. The recombinant expression vector pGEX 4T 2/NOR1 was identified by sequencing and digested with restriction enzymes. E.coli Jm105 transformed with the recombinant plasmid was induced by IPTG to express GST fusion protein. The result was confirmed by Western blot analysis and the purified targeted protein was obtained by affinity chromatography. RESULTS:The 1 25kb NOR1 gene was successfully isolated. After induction, a new anticipated protein of 74 kDa appeared on sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS PAGE). It existed not only in supernatant but also in precipitation of broken bacteria. The result was confirmed by Western blot analysis, and the purified targeted protein was obtained by affinity chromatography. CONCLUSION:The successes in construction of expression vector of NOR1, expression and purification of GST/NOR1 fusion protein make it possible to prepare for the polyantibodies for NOR1.