CAJ | 학술논문

利用GST融合蛋白表达系统表达与纯化人N-Ras蛋白
이용GST융합단백표체계통표체여순화인N-Ras단백
Expression and Purification of Human N-Ras Protein with Fused GST Protein Expression System

万方数据

生物技术通讯生物技術通訊 생물기술통신
LETTERS IN BIOTECHNOLOGY
2015年 4期 497-500 ,共4页

张云静%冯滢滢%徐小洁%梁迎春%张立%王涛%李玲%郭靖%纪贝贝%张蓉%叶棋浓

장운정%풍형형%서소길%량영춘%장립%왕도%리령%곽정%기패패%장용%협기농

人N-ras蛋白%小G蛋白%原核表达%纯化人N-ras蛋白%小G蛋白%原覈錶達%純化
인N-ras단백%소G단백%원핵표체%순화
human N-Ras protein%small G-protein%prokaryotic expression%purification

目的：克隆人N-ras蛋白全长编码区基因，获得其原核表达产物，并对融合蛋白进行纯化。方法：采用PCR技术从人乳腺文库中扩增出人N-ras蛋白全长编码区基因，将其克隆到pGEX-KG载体中，在大肠杆菌Rossate中表达后，利用GST-Sepharose 4B亲和珠对原核表达产物进行纯化，SDS-PAGE鉴定表达与纯化产物。结果：从人乳腺文库中扩增获得约600 bp的DNA片段，并克隆至pGEX-KG载体上，经测序与目的序列完全一致；在大肠杆菌Rossate中诱导表达出相对分子质量约47×103的目的蛋白；纯化后，经鉴定获得了纯度较高的重组蛋白GST-N-Ras。结论：获得了重组蛋白GST-N-ras，为后续深入研究Ras基因与其他癌基因、抑癌基因的相互作用奠定了基础。
목적：극륭인N-ras단백전장편마구기인，획득기원핵표체산물，병대융합단백진행순화。방법：채용PCR기술종인유선문고중확증출인N-ras단백전장편마구기인，장기극륭도pGEX-KG재체중，재대장간균Rossate중표체후，이용GST-Sepharose 4B친화주대원핵표체산물진행순화，SDS-PAGE감정표체여순화산물。결과：종인유선문고중확증획득약600 bp적DNA편단，병극륭지pGEX-KG재체상，경측서여목적서렬완전일치；재대장간균Rossate중유도표체출상대분자질량약47×103적목적단백；순화후，경감정획득료순도교고적중조단백GST-N-Ras。결론：획득료중조단백GST-N-ras，위후속심입연구Ras기인여기타암기인、억암기인적상호작용전정료기출。
Objective: To construct the prokaryotic expression vector of human N-Ras gene, obtain the purified GST-N-Ras protein. Methods: Human N-Ras gene coding region was amplified from human mammary DNA li?brary by the technique of PCR, and was inserted into prokaryotic expression vector pGEX-KG. The recombinant plasmid pGEX-KG-N-Ras was transformed into E.coli Rossate. The expressed product was purified by GST-Sepha?rose 4B beads and identified by SDS-PAGE analysis. Results: The DNA fragment of N-Ras about 600 bp was amplified by PCR, successfully cloned into pGEX-KG and identified by sequencing. The recombinant protein GST-N-Ras with Mr 47 kD was successfully induced, purified well. Conclusion: The recombinant protein of GST-N-Ras is obtained successfully, which lay the foundation for further research between Ras gene and other gene, in?cluding oncogene and tumor suppressor gene.