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 explore an effective strategy for antibody stability modification and to screen for stable and high-affinity human antibody against tumor necrosis factor alpha (TNF-α). Methods Based on the site-directed mutagenesis and ribosome display technology, the human antibody fragment VH was restructured into camelized stable and the camelized human-derived single domain antibody (sdAbs) library was constructed. TNF-α was used as the screening antigen , Screening specific camelized human sdAbs. Results Human VH / K gene library was used as template to amplify VH gene. The site-directed mutagenesis was performed to mutate four hydrophobic amino acids in VH gene FR2 to hydrophilic amino acids to construct human camelid sdAbs gene library. Based on the ribosomal antibody library technology, a sdAbs gene library for ribosome display was constructed. After in vitro transcription and translation, recombinant TNF-α was used as the screening antigen, and the specific antibody gene was panned by affinity enrichment. The mRNA of RT-PCR was cloned and expressed in the prokaryotic expression system pET-22b (+) / BL21 (DE3). The expressed product was identified by ELISA and confirmed to have the highest ELISA value. Analysis showed that it was a brand new anti-TNF-α human sdAbs. Antigen-specific binding activity assay showed that both could specifically recognize TNF-α, but not to rabies virus glycoprotein and BSA, indicating that they are specific to TNF-α Of human genetically engineered antibodies, and have good antigen concentration-dependent. Conclusion In this study, a combination of directed modification of antibody stability and evolutionary methods was used to screen out human antibodies with good antigen specificity and good stability, which provided a theoretical basis and technical basis for the preparation of stable human antibodies .