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目的鉴定抗原人干扰素α1b(huIFN-α1b)与1株全人源抗体蛋白AIFNa1IgG1的结合表位。方法从正常人分离纯化淋巴细胞并抽提mRNA进行CDNA合成,以CDNA为模板通过PCR扩增得到huIFN-α1b基因,通过重叠延伸PCR法构建huIFN-α1b蛋白29-35位氨基酸残基缺失突变体及点突变体,野生型和突变基因与载体pET30a连接构建重组表达载体,将以上重组表达载体转化大肠杆菌表达宿主E.coli Rosseta(DE3),诱导表达后的huIFN-α1b蛋白及突变体通过Western blot分析其与全人源抗huIFN-α1b基因工程抗体蛋白的结合活性。结果 huIFN-α1b蛋白在E.coli Rosseta(DE3)细胞中成功表达,Western-blot分析表明野生型huIFN-α1b蛋白与抗体结合,29-35位氨基酸缺失与抗体失去结合活性,其中27位的Ser,33位的Asp,34位的Arg,35的His,36位的Asp突变为Gly后,抗体与huIFN-α1b的结合可减弱到不可检测的水平。结论 huIFN-α1b蛋白29-35位氨基酸形成的表位是与抗体AIFNa1IgG1结合的重要表位。
Objective To identify the binding epitopes of human interferon alb (huIFN-α1b) and a human full-length antibody AIFNa1IgG1. METHODS: Lymphocytes were isolated and purified from normal individuals and mRNA was extracted for CDNA synthesis. The huIFN-α1b gene was amplified by PCR using CDNA as a template. The mutant of amino acid residues 29-35 of huIFN-α1b protein was constructed by overlap extension PCR And point mutants, wild-type and mutant genes were ligated with the vector pET30a to construct a recombinant expression vector. The above recombinant expression vector was transformed into E. coli Rosseta (DE3), and the expressed huIFN-α1b protein and the mutant were induced by Western blot analysis of its binding activity with whole human anti-huIFN-α1b genetically engineered antibody protein. Results The huIFN-α1b protein was successfully expressed in E.coli Rosseta (DE3) cells. Western-blot analysis showed that the wild-type huIFN-α1b protein bound to the antibody and the amino acid at positions 29-35 lost the binding activity to the antibody. Serine 27 , Asp at position 33, Arg at position 34, His at position 35, Asp at position 36 to Gly, the binding of the antibody to huIFN-alb can be reduced to undetectable levels. Conclusion The epitope formed by amino acids 29-35 of huIFN-α1b protein is an important epitope binding to the antibody AIFNa1IgG1.