人源抗ICAM-1单链抗体的制备及其生物学活性鉴定
Preparation and identification of anti-human ICAM-1 scFv
投稿时间:2018-03-12  
DOI:  10.13345/j.cjb.180083
中文关键词:噬菌体展示技术,细胞间黏附分子,ICAM-1,单链抗体,人源化抗体
英文关键词:phage display technology, intercellular adhesion molecule, ICAM-1, scFv, humanized antibody
基金项目:国家自然科学基金 (Nos. 81703546,81272485),安徽省自然科学基金 (No. 1808085QH265),吉林省科技发展计划 (No. 20160520045JH),皖南医学院博士科研启动基金 (No. RCQD201617),国家重点研发计划“宠物病毒性传染病血液生物制品研究与产品创制” (No. 2016YFD0501002) 资助。
作者单位
陈云雨 1 皖南医学院 药学院 新药筛选与评价中心安徽 芜湖 241002
2 军事医学研究院 军事兽医研究所吉林 长春 130122 
孙红 2 军事医学研究院 军事兽医研究所吉林 长春 1301223 黑龙江生物科技职业学院 生物制药系黑龙江 哈尔滨 150025 
刘刚 1 皖南医学院 药学院 新药筛选与评价中心安徽 芜湖 241002 
胡华波 1 皖南医学院 药学院 新药筛选与评价中心安徽 芜湖 241002 
张国利 2 军事医学研究院 军事兽医研究所吉林 长春 130122 
刘晓平 1 皖南医学院 药学院 新药筛选与评价中心安徽 芜湖 241002 
岳玉环 2 军事医学研究院 军事兽医研究所吉林 长春 130122 
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中文摘要:
      利用噬菌体展示技术筛选特异性人源抗ICAM-1单链抗体(Anti-human ICAM-1 scFv)并进行生物学活性鉴定。应用Tomlinson I+J噬菌体抗体库,以P1抗原肽为包被抗原,经过4轮“吸附-洗脱-扩增”进行亲和富集筛选。以PCR反应、ELISA抗原交叉反应和Dot blotting实验进行阳性克隆的鉴定。scFv经原核表达和分离纯化后,以Western blotting实验、竞争ELISA实验和细胞黏附抑制实验对其生物学活性进行初步鉴定。Tomlinson I+J噬菌体抗体库经4轮亲和富集筛选,利用ELISA方法成功筛出4株阳性克隆。通过PCR鉴定反应、ELISA抗原交叉反应和Dot blotting实验,最终获得了1株既能与P1抗原肽特异结合又能与人ICAM-1抗原特异结合的阳性克隆J-A1。对scFv进行原核表达和亲和层析后获得了高纯度的目的蛋白。竞争ELISA实验和细胞黏附抑制实验证实纯化的scFv具有良好的亲和活性和抗细胞黏附活性。文中成功利用噬菌体展示技术筛选到特异性人源抗ICAM-1 scFv,为进一步探索该抗体在炎症相关性疾病治疗中的应用奠定了基础。
英文摘要:
      To screen the specific anti-human intercellular adhesion molecule-1 (ICAM-1) single chain fragment variable (scFv) using phage display library technology and to identify its biological activity. P1 peptide was used as antigen, and the phage antibodies against human ICAM-1 antigen were panned by four binding-eluting-amplifying cycles using Tomlinson I+J phage display library. After four rounds of selective enrichment screening, the positive clones were determined by PCR, enzyme linked immunosorbent assay (ELISA)-based antigenic cross reaction and Dot blotting. Then the binding specificity and biological activity of purified scFv were identified by Western blotting, competitive ELISA and cell adhesion inhibition assay respectively. Furthermore, four positive clones were first panned through P1 peptide coated-ELISA assay, and then J-A1 was obtained and identified by PCR, ELISA-based antigenic cross reaction and Dot blotting, which could show a specific binding between P1 peptide and human ICAM-1 protein antigen. Subsequently, the purified scFv showed a satisfactory specificity and anti-adhesive activity in competitive ELISA and the cell adhesion inhibition assay. The specific anti-human ICAM-1 scFv was prepared successfully from Tomlinson I+J phage display library, which pave the way for further application of anti-human ICAM-1 scFv for inflammation diseases therapeutics.
陈云雨,孙红,刘刚,胡华波,张国利,刘晓平,岳玉环.人源抗ICAM-1单链抗体的制备及其生物学活性鉴定[J].生物工程学报,2018,34(12):2016~2024
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