Xie Yinliang, Gu Yue, Huang Rui, Li Xuexia, Du Xue, Wang Jinhong, Xiong Dongsheng, Yang Chunzheng, Xu Yuanfu
The State Key Laboratory of Experimental Hematology, Institute of Hematology, Chinese Academy of Medical Sciences & Peking Union Medical College, Tianjin 300020, China.
Sheng Wu Gong Cheng Xue Bao. 2009 Apr;25(4):580-6.
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 fusion protein was about 1.10 mg/L. Enzyme-Linked ImmunoSorbent Assay (ELISA) revealed that the Flt-1 domain III was able to bind to VEGF165 dose-dependently. Monolayer denudation assay and Transwell assay showed that the fusion protein could inhibit HUVECs migration induced by conditional medium with 50 ng/mL VEGF165 and 100 ng/mL bFGF. In conclusion, Flt-1 gene domain III gene has been successfully cloned and expressed in E. coli, which will be useful in both the research on the function of Flt-1 gene domain III and preparation of anti-Flt-1 monoclonal antibody in the future.
制备可溶性人Flt1细胞外III结构域并分析其生物学活性。通过RT-PCR从人脐静脉内皮细胞（HUVEC）中克隆编码Flt-1细胞外结构域III的基因至表达载体pAZY中，利用低磷培养基在大肠杆菌中诱导表达，产物经E-tag亲和层析纯化。SDS-PAGE和Western印迹分析表明Flt-1基因结构域III基因在大肠杆菌中表达，可溶性融合蛋白产量约为1.10 mg/L。酶联免疫吸附测定（ELISA）显示Flt-1结构域III能够剂量依赖性地结合VEGF165。单层剥脱试验和Transwell试验表明该融合蛋白能够抑制由含有50 ng/mL VEGF165和100 ng/mL bFGF的条件培养基诱导的HUVEC迁移。总之，Flt-1基因结构域III基因已成功在大肠杆菌中克隆并表达，这将对未来Flt-１基因结构域III功能研究及抗Flt-1单克隆抗体的制备具有重要意义。
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