全人源抗人干扰素α1b基因工程抗体与抗原的结合表位鉴定

The epitope identification of human interferon α1b against a gene engineering human antibody

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。

We aim to identify the epitope of human interferon α1b（huIFN-α1b） against a gene engineering human antibody AIFNa1IgG1.Firstly,the huIFN-α1b gene was amplified by PCR technology using CDNA as template.The huIFN-α1b protein mutant with amino acid residues 29-35 deletion and point mutation was build through overlap extension PCR method.The wild-type and mutant huIFN-α1b genes were ligated with expression vector pET30a and transformed into E.coli Rosseta（DE3）.The expression of huIFN-α1b and its mutant were analyzed by SDS-PAGE Western blot test.Consequently,the antigen huIFN-α1b and its mutant were successfully expressed in DE3.Western blot test showed that wild-type huIFN-α1b could bind with antibody,but huIFN-α1b mutant with 29-35 deletion could not bind with antibody.The huIFN-α1b mutant with single amino acid mutated to Glycine,such as point mutation of site 27,site 33,site 34,site 35,site 36 from Ser,Asp,Arg,His,and Asp to Gly.The result show that huIFN-α1b epitope of amino acid from site 29 to 35 is the binding site of gene engineering human antibody AIFNa1IgG1.