摘要
BRD7基因是一个鼻咽癌侯选抑瘤基因 ,为了构建BRD7基因的原核表达载体并使其在大肠杆菌得到表达 ,设计了带有SalⅠ ,NotⅠ酶切位点的引物 ,以已构建好的质粒 pGEM TEasy/BRD7为模板 ,用PCR扩增出BRD7基因的完整阅读框架 ,并用SalⅠ ,NotⅠ酶切PCR产物和原核表达载体PGEX 4T 2 ,然后用T4DNA连接酶将其连接 ,得到重组表达质粒PGEX 4T 2 /BRD7,经双酶切鉴定和测序验证 ,表达载体构建正确 .重组表达质粒转化感受态大肠杆菌Jm10 5后用IPTG诱导 ,成功表达了一分子质量约为 90ku的融合蛋白 ;37℃诱导 4h后 ,SDS 聚丙烯酰胺凝胶 (PAGE)电泳后 ,经扫描分析该融合蛋白产量占菌体蛋白总量 2 8 4 8% ,蛋白质印迹 (Western blot)证实了该融合蛋白的表达获得成功 .这为BRD7基因的蛋白纯化及抗体制备 ,进一步开展其功能研究奠定了基础 .
BRD7 gene is a good candidate tumor suppression gene associated with NPC. In order to construct prokaryotic expression vector of BRD7 and express BRD7 in E.coli , The coding region with Sal Ⅰ and Not Ⅰ restriction sites of BRD7 was obtained from pGEM T Easy/BRD7 plasmid by PCR. PCR product and plasmid PGEX 4T 2 were digested by corresponding restrict endonucleases respectively. The fragments were ligated by T4 DNA ligase to gain recombinant expression vector. Endonuclease digesting and DNA sequencing confirmed that the coding region of BRD7 gene was correctly inserted into the vector. The recombinant plasmid PGEX 4T 2/BRD7 was transferred into competent Jm105 strain. The GST/BRD7 fusion protein was expressed in the bacteria under induction of IPTG. After induction, a new protein band of 90 ku appeared on SDS PAGE. The result was confirmed by Western blot. The recombinant protein of 90 ku amounted to 28 48% of the total bacterial protein after inducing with IPTG for 4 h at 37℃. It existed not only in supernatant but also in precipitation of broken bacteria. The successes in construction of expression vector of BRD7 and expression of BRD7 in E.coli make it possible to study further on its biological function and antibody preparation.
出处
《生物化学与生物物理进展》
SCIE
CAS
CSCD
北大核心
2002年第4期631-634,共4页
Progress In Biochemistry and Biophysics
基金
国家高技术"863"计划资助项目 (10 2 10 0 1 0 5 )
国家重点基础研究发展规划项目 (973 ) (G19980 5 10 0 8)
国家自然科学基金资助项目 (3 970 0 15 8
3 0 0 0 0 0 65 )~~
