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目的用基因工程技术制备人源性抗角蛋白抗体单链抗体(singlechainFv,ScFv),并对其抗原结合特性等进行鉴定。方法利用基因重组技术对从半合成噬菌体抗体库中克隆的人源性抗角蛋白抗体Fab片段进行改造,获得了人源性抗角蛋白ScFv片段,并进行DNA序列分析。同时用ELISA法鉴定ScFv的抗原结合活性和特异性。结果DNA序列分析结果表明,单链抗体表达载体(pScFv)的Vκ和VH基因序列同Fab片段的Vκ和VH基因序列完全相同,表明在Fab片段改建成ScFv过程中未发生基因突变。可溶性表达的ScFv抗体能特异性地与角蛋白结合,而与其它抗原,如铁蛋白、胃蛋白酶、HBsAg等无关抗原不结合,说明人源性抗角蛋白Fab抗体改建为ScFv后具有较好的特异性,但可溶性表达的ScFv与角蛋白的结合活性低于可溶性Fab片段。结论成功表达并鉴定了人源性抗角蛋白的ScFv可溶性片段,为人源性抗角蛋白抗体工程化、进一步研究该抗体的生物活性并提高其临床应用价值奠定了基础。
OBJECTIVE: To prepare singlechainFv (ScFv) of human anti-keratin antibody by genetic engineering and to identify its antigen binding properties. Methods The human anti-keratin antibody Fab fragment cloned from the semi-synthetic phage antibody library was modified by gene recombination technology to obtain the human anti-keratin protein ScFv fragment and DNA sequence analysis. At the same time, the antigen binding activity and specificity of ScFv were identified by ELISA. Results The results of DNA sequence analysis showed that the Vκ and VH sequences of pScFv were exactly the same as the Vκ and VH genes of Fab fragment, indicating that no gene mutation occurred during the Fab fragment was converted into ScFv. Soluble expressed ScFv antibodies specifically bind to keratin, but do not bind to other antigens such as ferritin, pepsin, HBsAg and other unrelated antigens, indicating that human anti-keratin Fab antibody has better Specific, but soluble expression of ScFv with keratin binding activity than soluble Fab fragments. Conclusion The soluble fragment of human keratin protein ScFv was successfully expressed and identified, which laid the foundation for the further study on the bioactivity of human anti-keratin antibody and its bioactivity and clinical value.