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利用PCR技术 ,从人胎盘cDNA文库扩增出 4个截短的人血管内皮生长因子受体 (Flt 1)cDNA ,分别含胞外第 2、1 2、2 3、1 3个环 ,应用酵母双杂交系统对Flt 1的配体结合域进行了研究 ,结果表明不仅其胞外 1 3环能与hVEGF165结合 ,比其更小的片段 2 3环也具有与配体结合的能力。进一步构建了重组表达质粒pPIC9K/Flt 1(1 3)和 pPIC9K/Flt 1(2 3) ,转化Pichiapastoris酵母菌GS115 ,经摇瓶培养 ,甲醇诱导表达。SDS PAGE显示 ,表达的Flt 1以可溶性分子形式存在于上清中 ,诱导 4d的表达量占上清总蛋白质的 6 0 %以上。表达产物经CM SepharoseFF阳离子交换层析和SephacrylS 10 0分子筛层析纯化 ,纯度达 90 %以上。生物学活性检测证实表达的Flt 1(2 3)具有与Flt 1(1 3)比较接近的结合hVEGF165的能力和抑制hVEGF165对HUVEC的促增殖功能。
Four truncated human vascular endothelial growth factor receptor (Flt1) cDNAs were amplified from the human placenta cDNA library by PCR. The cDNAs contained the extracellular loops 1, 2, 2, 3, and 3, respectively. Two-hybrid system of Flt 1 ligand binding domain was studied, the results show that not only the extracellular 13 ring with hVEGF165 binding, smaller than its fragment 23 ring also has the ability to bind ligand. The recombinant plasmids pPIC9K / Flt1 (1 3) and pPIC9K / Flt1 (2 3) were further constructed and transformed into Pichia pastoris GS115. The recombinant plasmids were induced by methanol and shake flask culture. SDS PAGE showed that the expressed Flt 1 was present in the supernatant as a soluble molecule, and the expression level of Flt 1 accounted for more than 60% of the total protein in the supernatant after induction for 4 days. The expressed product was purified by CM Sepharose FF cation exchange chromatography and Sephacryl S 10 0 molecular sieve chromatography with a purity of over 90%. The biological activity assay confirmed that the expressed Flt1 (23) has the ability to bind hVEGF165 which is relatively close to Flt1 (13) and inhibits the proliferation-promoting function of hVEGF165 on HUVECs.