| 宋丽敏,张维,陈明,林敏,潘家荣.抗微囊藻毒素单链抗体基因的构建、表达及初步鉴定[J].高技术通讯(中文),2010,20(3):327~330 |
| 抗微囊藻毒素单链抗体基因的构建、表达及初步鉴定 |
| Construction, expression and identification of scFv against microcystin LR |
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| DOI： |
| 中文关键词: 微囊藻毒素(MC)， 单链抗体， 原核表达， ELISA |
| 英文关键词: microcystins (MC), single chain variable fragment, prokaryotic expression, ELISA |
| 基金项目:863计划(2006AA10Z449)资助项目 |
| 作者 | 单位 | | 宋丽敏 | 中国农业科学院农产品加工研究所 | | 张维 | 中国农业科学院生物技术研究所 | | 陈明 | 中国农业科学院生物技术研究所 | | 林敏 | 中国农业科学院生物技术研究所 | | 潘家荣 | 中国农业科学院生物技术研究所 |
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| 中文摘要: |
| 利用RT－PCR方法从抗微囊藻毒素 LR(MC－LR)单克隆抗体的杂交瘤细胞中扩增出抗体的VH和VL基因，构建了抗MC LR分子的单链抗体(scFv)基因。SDS PAGE和Western blot分析结果显示，该单链抗体基因在大肠杆菌Origami 2中特异性表达出分子量约为30kD的融合蛋白。通过Ni NTA金属亲和层析法对可溶性表达产物进行纯化，获得的目的蛋白浓度为0115mg/mL。ELISA反应结果表明该单链抗体能与MC LR特异性结合。此研究为制备多价高亲和力抗MC LR抗体奠定了基础。 |
| 英文摘要: |
| The VH and VL genes were amplified by RT PCR from hybridoma cell secreting anti microcystin LR monoclonal antibody, and the scFv gene was spliced by sequence overlap extension PCR with VH and VL SDS PAGE and Western blot analysis confirmed the expression of scFv with the molecular weight of about 30kD. The recombinant protein was purified by metal affinity chromatography using Ni NTA, and the concentration of purified scFv was 0115mg/mL. The ELISA assay revealed that the scFv protein could bind specifically to MC LR. The results provide a basis for the application of genetic engineering methods in the preparation of multivalent antibodies with high affinity. |
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