抗HER-2纳米抗体的筛选、表达及功能鉴定

Screening,Expression and Functional Identification of Anti-HER-2 Nanobody

目的筛选获取抗HER-2纳米抗体序列,偶联人IgG1 Fc片段,采用原核表达系统表达重组抗HER-2纳米抗体并验证其生物学特征。方法基于天然噬菌体纳米抗体展示库,采用生物素-链霉亲和素液相筛选法,以生物素化HER-2抗原为靶点经三轮淘选,随机挑取单克隆进行ELISA鉴定并测序。获得抗HER-2纳米抗体序列,将其偶联人IgG1 Fc片段,并将重组抗HER-2纳米抗体序列克隆至原核表达载体pET-30a,转化至表达菌Arctic Express,诱导重组抗HER-2纳米抗体表达并纯化,采用SPR、Western blot、细胞免疫荧光对纯化后的重组抗HER-2纳米抗体特征和功能进行验证。结果经淘选获得了一条抗HER-2纳米抗体序列,与人IgG1 Fc片段偶联,成功构建了原核表达载体,并表达纯化获得的重组抗HER-2纳米抗体;SPR显示其亲和力较好,Western blot表明其可与HER-2抗原结合,细胞免疫荧光实验结果提示其可与人乳腺癌细胞SKBR-3结合。结论本实验获得了亲和力、特异性均良好且具有一定生物学功能的重组抗HER-2纳米抗体,可为其后续临床应用提供基础。

Objective To screen and obtain the anti-HER-2 nanobody sequence,couple with human Ig G1 Fc,and use the prokaryotic expression system to express recombinated anti-HER-2 nanobody and verify its biological characteristics. Methods Based on the naive phage nanobody display library,the anti-HER-2 nanobody was selected by biotin-streptavidin liquid-phase screening method. After three rounds of panning,single clones were randomly selected for ELISA identification,then the positive clones were sent for sequencing.The obtained anti-HER-2 nanobody sequence was fused to human Ig G1 Fc fragment,and then recombined into prokaryotic vector p ET-30a and expressed by Arctic Express stain. SPR,Western blot,cellular immunofluorescence were used to verify the characteristics and functions of the purified recombinated anti-HER-2 nanobody.Results An anti-HER-2 nanobody sequence was obtained and coupled with human Ig G1 Fc fragment,and the prokaryotic expression vector was successfully constructed. The recombinated anti-HER2 nanobody was successfully expressed and purified. SPR showed that its affinity behaved well,Western blot revealed that it could bind to HER-2 antigen,and cell immunofluorescence indicated that it could bind to human breast cancer cells SKBR-3. Conclusion The recombinated anti-HER-2 nanobody with high affinity,specificity was obtained,and proved to have biological activity,which could provide the basis for further clinical application.