| Flt-1胞外Ⅲ区蛋白的可溶性表达、纯化及活性测定 |
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| 引用本文： | 谢印良,谷岳,黄蕊,李雪霞,杜雪,王金宏,熊冬生,杨纯正,许元富.Flt-1胞外Ⅲ区蛋白的可溶性表达、纯化及活性测定[J].生物工程学报,2009,25(4):580-586. |
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| 作者姓名： | 谢印良 谷岳 黄蕊 李雪霞 杜雪 王金宏 熊冬生 杨纯正 许元富 |
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| 作者单位： | 1. 中国医学科学院北京协和医学院血液学研究所血液病医院,实验血液学国家重点实验室,天津,300020
2. 天津医科大学总医院,天津,300020 |
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| 基金项目： | 国家自然科学基金(No.30400405);;天津市自然科学基金(No.05YFJZJC01200)资助~~ |
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| 摘 要： | 本研究旨在克隆和表达人血管内皮生长因子受体-1胞外Ⅲ区蛋白,并测定其生物学活性。应用RT-PCR法从人脐静脉内皮细胞中克隆Flt-1胞外Ⅲ区基因片段,经测序鉴定后再克隆到原核表达载体pAYZ中,构建出的表达载体pAYZflt-1Ⅲ转化大肠杆菌16C9后,用低磷培养基诱导表达目的蛋白;采用E-tag亲和层析柱纯化目的蛋白;用SDS-PAGE、Western blotting和BCA法对其进行定性、定量检测鉴定;用ELISA、损伤愈合试验和Transwell法检测靶蛋白生物学活性。克隆的Flt-1胞外Ⅲ区基因经测序鉴定正确。所构建的pAflt-1Ⅲ表达载体经低磷培养基诱导后高表达出可溶性Flt-1胞外Ⅲ区蛋白,产量约为1.1mg/L;ELISA结果显示该蛋白可以结合VEGF165,并表现为剂量依赖性,其与配体结合的解离常数Kd为1.180pmol/L。损伤愈合试验和Transwell结果显示该蛋白可以抑制VEGF165(50ng/ml)和bFGF(100ng/mL)诱导的脐静脉内皮细胞的迁移,并呈剂量依赖性。这将为今后开展人flt-1基因Ⅲ区的功能研究及其单抗研制奠定了实验基础。
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| 关 键 词： | 血管内皮生长因子受体1 血管新生 血管内皮生长因子165 |
| 收稿时间： | 2008/10/28 0:00:00 |
Soluble expression, purification and activity analysis of extracellular domain III of Flt1 |
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| Yinliang Xie,Yue Gu,Rui Huang,Xuexia Li,Xue Du,Jinhong Wang,Dongsheng Xiong,Chunzheng Yang and Yuanfu Xu.Soluble expression, purification and activity analysis of extracellular domain III of Flt1[J].Chinese Journal of Biotechnology,2009,25(4):580-586. |
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| Authors: | Yinliang Xie Yue Gu Rui Huang Xuexia Li Xue Du Jinhong Wang Dongsheng Xiong Chunzheng Yang and Yuanfu Xu |
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| Institution: | The State Key Laboratory of Experimental Hematology, Institute of Hematology, Chinese Academy of Medical Sciences & Peking Union Medical College, Tianjin 300020, China;The State Key Laboratory of Experimental Hematology, Institute of Hematology, Chinese Academy of Medical Sciences & Peking Union Medical College, Tianjin 300020, China;The State Key Laboratory of Experimental Hematology, Institute of Hematology, Chinese Academy of Medical Sciences & Peking Union Medical College, Tianjin 300020, China;The State Key Laboratory of Experimental Hematology, Institute of Hematology, Chinese Academy of Medical Sciences & Peking Union Medical College, Tianjin 300020, China;The General Hospital under Tianjin Medical Sciences University, Tianjin 300020, China;The State Key Laboratory of Experimental Hematology, Institute of Hematology, Chinese Academy of Medical Sciences & Peking Union Medical College, Tianjin 300020, China;The State Key Laboratory of Experimental Hematology, Institute of Hematology, Chinese Academy of Medical Sciences & Peking Union Medical College, Tianjin 300020, China;The State Key Laboratory of Experimental Hematology, Institute of Hematology, Chinese Academy of Medical Sciences & Peking Union Medical College, Tianjin 300020, China;The State Key Laboratory of Experimental Hematology, Institute of Hematology, Chinese Academy of Medical Sciences & Peking Union Medical College, Tianjin 300020, China |
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| Abstract: | To prepare a soluble human extracellular III domain of Flt1 and analyze its biological activity. The gene encoding extracellular domain III of Flt-1 was cloned into the expression vector pAZY by RT-PCR from human umbilical vein endothelial cell (HUVEC),and induced to express in Escherichia coli by low phosphoric medium,the product was purified by E-tag affinity chromatography. SDS-PAGE and Western blotting analysis showed that Flt-1 gene domain III gene was expressed in E. coli and the yield of the soluble ... |
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| Keywords: | Flt-1 angiogenesis VEGF165 |
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