人源性抗角蛋白单链抗体的构建及表达

目的 用基因工程技术制备人源性抗角蛋白抗体单链抗体(single chainFv,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。

Construction and Expression of Human Anti-Keratin ScFv

Objective To construct and express human ScFv against keratin in E.coli,and identify its binding activity with antigens and antigenic specificity.Methods By genetic engineering technology,human anti-keratin ScFv was constructed from Fab fragment selected from established semi-synthetic phage antibody library.The binding activity with antigens and antigenic specificity of expressed products were identified by ELISA.Results Results of DNA sequence analysis showed that nucleotide sequence of Vκand VH of ScFv gene was similar to that of Vκand VH of Fab gene,suggesting that there was no mutation in the construction of ScFv.The soluble anti-keratin ScFv was found to have a good antigenic specificity as well as excellent binding activity with target antigens by ELISA.However,the binding activity of ScFv with keratin was lower than that of Fab.Conclusions The successful expression and identification of human anti-keratin ScFv might lay a solid foundation for further observation of biological activities of anti-keratin autoantibody,manufacture of genetic-engineering anti-keratin products,and promotion of clinical application of genetic-engineering anti-keratin antibodies.