摘要
为了构建pGEX-4T-1-Sam68原核表达载体,表达并鉴定GST-Sam68融合蛋白,采用PCR扩增Sam68基因,插入pGEX-4T-1的Eco R I和Sal I位点,并转化Rosetta(DE3)大肠杆菌,IPTG诱导表达,SDS-PAGE和Western Blot验证蛋白表达,GST pull-down技术验证Sam68的结合活性。酶切和测序结果证实Sam68基因正确插入pGEX-4T-1载体中,载体能够在Rosetta(DE3)细胞中正确表达,且纯化的GST-Sam68蛋白具有与PI3K p85特异结合的活性,说明成功构建了原核表达载体pGEX-4T-1-Sam68。
In order to prokaryotically express and identify the nuclear protein Sam68,Sam68 gene fragment was firstly amplified by PCR.The PCR product was cloned into EcoR I and Sal I site of pGEX-4T-1.The recombinant vector pGEX-4T-1-Sam68 was transformed into Rosetta （DE3）,and competent cells were induced by IPTG to express the fusion protein.GST beads were used for purifying the GST-Sam68 fusion protein.SDS-PAGE and Western blot were used for identification of the fusion expression.GST pull- down was performed for detection of binding activity of GST-Sam68.Restriction endonuclease digestion and sequence analysis confirmed that Sam68 was inserted into pGEX-4T-1 ,and the vector expressed the GST-Sam68 fusion protein correctly.The purified GST-Sam68 fusion protein could bind to PI3K p85,suggesting that prokaryotic expression vector pGEX-4T-1-Sam68 had been constructed successfully.
出处
《黑龙江八一农垦大学学报》
2017年第3期73-76,81,共5页
journal of heilongjiang bayi agricultural university
基金
国家自然科学基金(31300145
31570159)
黑龙江省教育厅科学技术研究面上项目(12541578)
中国博士后科学基金面上项目(2016M590297)
黑龙江省政府博士后资助经费(LBH-Z15188)
黑龙江八一农垦大学博士科研启动基金(XDB2015-16)
关键词
Sam68
原核表达
载体构建
鉴定
Sam68
prokaryotic expression
construction of vector
identification
