摘要
目的将X连锁凋亡抑制蛋白(XIAP)插入质粒pTAT-HA,构建pTAT-XIAP原核表达载体,以获得高产量、高纯度的pTAT-XIAP融合蛋白。方法将pTAT-XIAP融合蛋白表达载体转入大肠杆菌BL21plysS,异丙基硫代半乳糖苷(IPTG)诱导融合蛋白表达,产物经镍离子亲和层析柱(Ni-NTA)亲和层析纯化,并用Western blot方法鉴定。结果 pTAT-XIAP融合蛋白在大肠杆菌中获得表达,纯化后蛋白呈单一条带,表达产物经Western blot分析证实目的蛋白表达。结论构建重组融合表达载体,在大肠杆菌中成功表达并纯化pTAT-XIAP融合蛋白。
Objective To construct the prokaryotic expression vector for X-linked inhibitor of apoptosis protein（XIAP） and express it in E.coli,so as to obtain the recombinant transactivator of transcription（pTAT）-XIAP protein with high production and high purity.Methods The XIAP gene was synthesized and cloned into pTAT-HA plasmid.After sequence analysis,they were transducted into E.coli.BL21plysS was induced to express TAT-XIAP fusion protein with isopropy-β-D-thiogalactoside（IPTG）.The recombinant protein was purified by Ni-NTA affinity chromatography under denaturing conditions and then detected by Western blot.Results A band with molecular weight 64 kD was observed after SDS-PAGE and it was equivalent to the expected value of TAT-XIAP fusion protein.Conclusion The pTAT-XIAP vector is successfully constructed and fusion protein is expressed and purified.
出处
《江苏医药》
CAS
CSCD
北大核心
2011年第7期772-775,共4页
Jiangsu Medical Journal
基金
2009桂林医学院青年科研启动基金项目(ky2008001)
桂林医学院博士启动基金项目(BS200603)
