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目的 :用大肠杆菌分别表达抗角蛋白抗体的轻链和Fd片段 ,体外复性得到Fab抗体。方法 :从已构建的质粒p3MH/Fab中 ,亚克隆抗角蛋白抗体的轻链和Fd片段基因 ,并分别插入载体pET32a中 ,构建重组质粒。以重组质粒分别转化大肠杆菌BL2 1(DE3) ,在IPTG诱导下进行表达。SDS PAGE分析发现 ,在相对分子量 (Mr)为 2 5 0 0 0处有外源蛋白表达。轻链和Fd片段变性后等量混合于折叠液中 ,复性后形成Mr 约为4 5 0 0 0的蛋白。结果 :成功地表达抗角蛋白抗体的轻链和Fd片段 ,ELISA和Westernblot证实 ,复性产物具有与角蛋白结合的能力。结论 :获得了具有活性的抗角蛋白的Fab抗体 ,为其应用研究打下了基础 ,也表明包涵体表达基因工程抗体技术是可行的。
OBJECTIVE: To express Fab antibody in vitro by using the light chain and Fd fragment of anti-keratin antibody respectively expressed in Escherichia coli. Methods: The light chain and Fd fragment of the anti-keratin antibody were subcloned from the constructed plasmid p3MH / Fab and inserted into the vector pET32a respectively to construct a recombinant plasmid. The recombinant plasmids were transformed into E. coli BL21 (DE3) and expressed under the induction of IPTG. SDS PAGE analysis showed that foreign protein was expressed at a relative molecular weight (Mr) of 25000. The light chain and Fd fragments were denatured and mixed in the same amount of the folded solution to form a protein of Mr about 450,000 after annealing. RESULTS: The light and Fd fragments of the anti-keratin antibody were successfully expressed. ELISA and Western blot confirmed that the renaturation product had the ability to bind to keratin. Conclusion: Obtaining an active anti-keratin Fab antibody laid the foundation for its application and also indicated that it is feasible for inclusion bodies to express the genetically engineered antibody technology.