抗表皮生长因子受体3(HER3)单链抗体的制备及鉴定  

Preparation and identification of single-chain fragment variable against epidermal growth factor receptor 3

作　　者：刘梦苗 何佩彦 温碧燕 邱创楠 李黄金 赵林 LIU Mengmiao;HE Peiyan;WEN Biyan;QIU Chuangnan;LI Huangjin;ZHAO Lin(Guangdong Provincial Key Laboratory for Biotechnology Drug Candidates,School of Life Sciences and Biopharmaceutics,Guangdong Pharmaceutical University,Guangzhou 510006,China)

机构地区：[1]广东药科大学生命科学与生物制药学院,广东省生物技术候选药物研究重点实验室,广东广州510006

出　　处：《细胞与分子免疫学杂志》2019年第10期938-943,共6页Chinese Journal of Cellular and Molecular Immunology

基　　金：国家自然科学基金(81273423);广东省自然科学基金(2015A030310120);广东省科技计划项目(2014A020210027);广东省高校重大科研项目(2016KZDXM042)。

摘　　要：目的表达、纯化并鉴定抗人表皮生长因子受体3(HER3)的单链抗体(scFv).方法从NCBI查找抗HER3单克隆抗体UM716的轻链和重链序列,构建抗HER3-scFv基因;利用毕赤酵母组成性表达载体pGAPZαA构建重组表达载体,电转化毕赤酵母,筛选高表达工程菌;工程菌发酵上清经疏水层析和金属离子亲和层析纯化,Weslem blot法和ELISA鉴定纯化产物.结果成功构建了抗HER3-scFv基因,获得了高表达工程菌;发酵上清经两步层析纯化,获得了纯度达95%以上的抗HER3-scFv,产率为192mg/L;Western blot结果显示抗HER3-scFv得到正确表达,ELISA结果表明抗HER3-scFv可特异识别HER3.结论成功制备了特异的抗HER3-scFv.Objective To express,purify and identify the single-chain fragment variable(scFv)against human epidermal growth factor receptor 3(HER3).Methods We searched NCBI for the light chain sequence and heavy chain sequenee of anti-HER3 mAb LJM716 to construct the gene of scFv against HER3.The recombinant expression vector pGAPZαA-anit-HER3-scFv was constructed using the constitutive expression vector pGAPZαA and then electro-transformed into Pichia Pastoris X-33 to screen the strains with high expression of the protein of interest.After shaking flask fermentation,the supernatant was purified by hydrophobic chromatography and metal ion affinity chromatography.The purified product was identified by Western blotting and ELISA.Results The anti-HER3-scFv gene was successfully constructed and the strains with high expression of anti-HER3-scFv were obtained.The anti-HER3-scFv was purified to a purity of more than 95%by two-step chromatography,and the purified yield was 192 mg/L.Western blotting showed that the anti-HER3-scFv was correctly expressed and ELISA indicated that anti-HER3-scFv could specifically recognize HER3.Conclusion The anti-HER3-scFv has been successfully prepared.

关 键 词：人表皮生长因子受体3(HER3) 单链抗体(scFv) 表达 纯化 鉴定 

分 类 号：Q781[生物学—分子生物学] Q392.1