人源抗ICAM-1单链抗体的制备及其生物学活性鉴定
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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。


Preparation and identification of anti-human ICAM-1 scFv
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National Natural Science Foundation of China (Nos. 81703546, 81272485), Anhui Provincial Natural Science Foundation (No. 1808085QH265), Jilin Province Science and Technology Development Program (No. 20160520045JH), The Doctoral Starting-up Fund of Wannan Medical College (No. RCQD201617), National Key Research and Development Program of China (No. 2016YFD0501002).

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    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。

    Abstract:

    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.

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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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  • 收稿日期:2018-03-12
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  • 在线发布日期: 2018-12-24
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