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目的 利用基因重组技术构建人IκBα基因原核表达质粒 ,制备TrxA IκBα融合蛋白 ,以便进一步研究IκBα的生物学功能和制备相应抗体。方法 以重组质粒pGEM T IκBα为模板 ,利用PCR方法扩增出带有BamHⅠ和HindⅢ酶切位点的人IκBα基因cDNA ,经相应酶切后插入原核表达载体pET 32a(+)。重组表达质粒pET32a(+) IκBα转化大肠杆菌BL2 1(DE3) ,经IPTG诱导表达TrxA IκBα融合蛋白。Westernblot试验鉴定表达蛋白。超声波破菌后采用Ni NTA树脂对TrxA IκBα融合蛋白进行纯化。结果 酶切鉴定证实人IκBα基因cDNA已插入原核表达载体pET 32a(+)。重组表达质粒pET32a(+) IκBα在大肠杆菌BL2 1(DE3)中成功地表达了TrxA IκBα融合蛋白 ,其相对分子质量(Mr)约为 5 6× 10 3 ,表达量约占细菌总蛋白的 2 5 %。Westernblot试验显示TrxA IκBα融合蛋白与兔抗IκBα多克隆抗体呈特异性免疫反应。经Ni NTA树脂纯化后 ,TrxA IκBα融合蛋白的纯度可高达 95 %以上。结论 人IκBα基因原核表达质粒的构建及TrxA IκBα融合蛋白的制备为进一步研究IκBα的生物学功能和制备相应抗体奠定了物质基础。
Objective To construct a prokaryotic expression plasmid of human IκBα gene by gene recombination technology and prepare TrxA IκBα fusion protein for further study of biological function of IκBα and preparation of corresponding antibodies. Methods The recombinant plasmid pGEM T IκBα was used as a template to amplify the human IκBα cDNA with BamH Ⅰ and Hind Ⅲ restriction sites by PCR and inserted into the prokaryotic expression vector pET 32a (+). The recombinant plasmid pET32a (+) IκBα was transformed into E. coli BL21 (DE3), and induced by IPTG to express TrxA IκBα fusion protein. Western blot assay identifies the expressed protein. The TrxA IκBα fusion protein was purified by Ni NTA resin after sonication. Results The restriction enzyme digestion confirmed that human IκBα gene cDNA was inserted into prokaryotic expression vector pET 32a (+). The recombinant plasmid pET32a (+) IκBα successfully expressed TrxA IκBα fusion protein in E. coli BL21 (DE3). The relative molecular mass (Mr) was about 56 × 10 3, and its expression level was about 2 5%. Westernblot assay showed that the TrxA IκBα fusion protein was specifically immunoreactive with rabbit anti-IκBα polyclonal antibody. After purification by Ni NTA resin, the purity of the TrxA IκBα fusion protein can be as high as 95% or more. Conclusion The construction of prokaryotic expression plasmid of human IκBα gene and the preparation of TrxA IκBα fusion protein provide the material basis for further study on the biological function of IκBα and preparation of corresponding antibodies.