医学论文-PTD.docVIP

医学论文-PTD ??????????????????????????????????????? 作者：邱波，马庆久，鲁建国，阴继凯，李金茂，王青 【关键词】? SH2基因;,融合蛋白；表达；纯化；pET 　　Prokaryotic expression and purification of PTDSH2 fusion protein 　　【Abstract】 AIM: To construct the recombinant expression vector containing protein transduction domain （PTD） and Src homology 2 （SH2） fusion gene and express PTDSH2 fusion protein in E. coli and purify the fusion protein. METHODS: A 297 bp of human SH2 gene fragment was amplified by PCR and subcloned into pET16b vector downstream of the PTD fragment, an E.coli expression vector, to construct a recombinant plasmid pET16bPTDSH2. The plasmid was transformed into E.coli BL21 (DE3) and induced to express fusion protein PTDSH2 with IPTG. The expression of PTDSH2 was detected by SDSPAGE and Western blot. The expressed protein was purified by NiNTA affinity chromatographic column. RESULTS: SH2 was identical to what reported by GenBank. A novel protein with expected molecular mass (about Mr 15×103) was expressed under the induction with IPTG. The expressed product showed good reactivity to antiHis tag antibody, and was mostly in the form of inclusion bodies. The expressed proteins could be purified via NiNTA affinity chromatography in denatured condition. CONCLUSION: Our successful construction of recombinant express vector pET16bPTDSH2 and efficient expression of PTDSH2 fusion protein in E. coli and purification of interest protein lay a basis for further study on SH2 functions. 　　【Keywords】 SH2; fusion proteins; expression; purification; pET16b vector 　　【摘要】 目的：构建含SH2与蛋白转导结构域(PTD)融合基因片段的重组表达载体，在大肠杆菌中进行表达并纯化蛋白. 方法: 通过PCR方法扩增出SH2基因，克隆入pMD18T载体，进行测序分析，将该基因亚克隆入原核表达载体pET16b中PTD 的下游构建重组质粒pET16bPTDSH2,转化感受态细胞BL21(DE3)，经IPTG诱导表达重组融合蛋白，对表达产物进行SDSPAGE电泳和Western blot检测分析，将已表达的蛋白质通过NiNTA亲和色谱柱进行纯化. 结果:构建了重组融合表达质粒pET16bPTDSH2, 表达的融合蛋白经SDSPAGE分析，在约Mr为15×103处出现了一条新生的蛋白条带，经灰度扫描检测，表达量约占菌体总蛋白的16%, 可溶性分析发现融合蛋白主要以包涵体形式存在，纯化后得到了目的蛋白. 结论: 成功构建了PTDSH2的重组表达载体，使融合蛋白在E. coli DE3中高效表达，亲和层析后获得了纯化目的蛋白，为后续的SH2结构域的功能研究奠定了基础. 　　【关键词】 SH2