Flt-1胞外Ⅲ区蛋白的可溶性表达、纯化及活性测定

Soluble expression, purification and activity analysis of extracellular domain III of Flt1

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1.1 mg/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1.180 pmol/L。损伤愈合试验和Transwell结果显示该蛋白可以抑制VEGF165(50 ng/ml)和bFGF(100 ng/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。

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 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.