抗TNF-α骆驼化人源sdAbs库的构建及筛选

Construction and selection of camelized human sdAbs library against TNF-α

目的探索有效的抗体稳定性改构策略,筛选制备抗肿瘤坏死因子(tumor necrosis factor alpha,TNF-α)稳定、高亲和力人源抗体。方法基于定点突变技术和核糖体展示技术,对人源抗体片段VH进行骆驼化稳定性改构,构建骆驼化人源单域抗体(single-domain antibodyies,sdAbs)库,以TNF-α作为筛选抗原,筛选特异骆驼化人源sdAbs。结果以前期构建的人源VH/K基因库为模板扩增VH基因;采用定点突变方法,将VH基因FR2中的4个疏水性氨基酸突变为亲水性氨基酸构建人源骆驼化sdAbs基因库;基于核糖体抗体库技术,构建用于核糖体展示的sdAbs基因库;经体外转录翻译后,以重组TNF-α作为筛选抗原,采用亲和富集法淘选特异性抗体基因;将筛选富集mRNA的RT-PCR基因产物克隆,并在原核系统pET-22b(+)/BL21(DE3)中实现可溶性表达,表达产物经ELISA鉴定,确认具有极高ELISA值的阳性克隆12、40,其序列分析表明是全新的抗TNF-α人源sdAbs;抗原特异性结合活性实验显示两者均可以特异性识别TNF-α,而不能与狂犬病毒糖蛋白和BSA结合,表明它们是特异针对TNF-α的人源基因工程抗体,而且均有很好的抗原浓度依赖性。结论该研究将抗体稳定性的定向改造法和进化方法相结合,筛选得到了具有较好抗原特异性和良好稳定性的人源抗体,为稳定性人源抗体的制备提供了理论依据及技术基础。

Objective To prepare single-domain antibodies（sdAbs） against TNF-α with high neutralizing potency.Methods A camelized human sdAbs library was constructed using site-directed mutagenesis technology.The residues of Val-37,Gly-44,Leu-45 and Trp-47 in the framework-2 region were changed into Phe-37,Glu-44,Arg-45 and Gly-47.Specific camelized human sdAbs were selected over five cycles of ribosome display using recombinant TNF-α as screening antigen.The selected sdAbs genes were cloned into pET-22b（＋）/BL2l（DE3）,from which soluble sdAbs were prepared.The expressed products of selected clones were analyzed by ELISA.The clones of No.12 and No.40 with high ELISA signals were sequenced.The assay of specificity characteristics and stability showed that the two distinct clones with new human immunoglobulin genes could recognize TNF-α specifically with good stability.Results A camelized human sdAbs library was constructed,and four hydrophobic residues were changed into hydrophilic residues in FR2 of VH gene by site-directed mutagenesis technology.A camelized human sdAbs ribosome display library was successsfully constructed.After transcription and translation in vitro,the specific sdAbs to TNF-α were selected.Conclusion A human antibody with good stability can be prepared by a combination of camelizing human fragments and ribosome display technology.