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

作　　者：谢印良[1] 谷岳[1] 黄蕊[1] 李雪霞[1] 杜雪[2] 王金宏[1] 熊冬生[1] 杨纯正[1] 许元富[1] 

机构地区：[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

出　　处：《生物工程学报》2009年第4期580-586,共7页Chinese Journal of Biotechnology

基　　金：国家自然科学基金(No.30400405);天津市自然科学基金(No.05YFJZJC01200)资助~~

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。To prepare a soluble human extracellular Ⅲ domain of Fltl 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 affmity 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 TransweU 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 Fit-1 gene domain Ⅲ and preparation of anti-Fit-1 monoclonal antibody in the future.

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 克隆 单抗 

分 类 号：Q78[生物学—分子生物学]