CAJ | 학술논문

目的应用酵母双杂交系统筛选人血管内皮生长因子受体Flt-1胞外最小配体结合域.方法采用PCR技术,从人胎盘cDNA文库扩增出4个截短的Flt-1 cDNA,分别含胞外第2、1-2、2-3和1-3个loop,构建酵母双杂交系统融合表达质粒,并将pGBT9/hVEGF165与pGAD424/Flt-ls两两配对转化酵母菌SFY526.采用滤纸法和液体培养法对阳性克隆进行β-半乳糖苷酶活性分析.结果Flt-1胞外loop2-3与loop1-3的配体结合能力相差不大,loop1-2的结合力较弱,单独第2个loop无配体结合能力.结论利用酵母双杂交系统,筛选出了Flt-1胞外最小配体结合域-2-3loop,这对研制分子量更小、安全性更高的可溶性Flt-1片段,开发其在肿瘤、糖尿病性视网膜病变等血管生成相关疾病基因治疗中的应用具有重要的指导意义.
목적응용효모쌍잡교계통사선인혈관내피생장인자수체Flt-1포외최소배체결합역.방법채용PCR기술,종인태반cDNA문고확증출4개절단적Flt-1 cDNA,분별함포외제2、1-2、2-3화1-3개loop,구건효모쌍잡교계통융합표체질립,병장pGBT9/hVEGF165여pGAD424/Flt-ls량량배대전화효모균SFY526.채용려지법화액체배양법대양성극륭진행β-반유당감매활성분석.결과Flt-1포외loop2-3여loop1-3적배체결합능력상차불대,loop1-2적결합력교약,단독제2개loop무배체결합능력.결론이용효모쌍잡교계통,사선출료Flt-1포외최소배체결합역-2-3loop,저대연제분자량경소、안전성경고적가용성Flt-1편단,개발기재종류、당뇨병성시망막병변등혈관생성상관질병기인치료중적응용구유중요적지도의의.
Aim To screen the minimal extracellular ligand- binding domain of vascular endothelial growth factor (VEGF) receptor Flt - 1using yeast two - hybrid system. Methods Four turncated Flt - 1 cDNA fragments containing extracellular domain loops 2, 1 - 2, 2 - 3 and 1 - 3respectively were amplified from human placental cDNA library by PCR and used for constructing fusion expression plasmids in yeast two - hybrid system. Each of the four pGAD424/Flt - 1 plasmids was paired up with pGBT9/hVEGF165 and transformed into yeast SFY526, The 3- galac tosidase activities of positive colonies were analyzed by filter assay and liquid culture assay. Result The binding capacity of loop2 - 3 was close to that of loop1 -3. However, the binding capacity of loop1 -2 was weaker. Loop 2 alone showed no binding capacity. Conclusion The minimal extracellular ligand binding domains of Flt - 1 that were sufficient to achieve VEGF binding to loop2 - 3 were screened out using yeast two -hybrid system, which was of an important guiding significance in developing the more safe sFlt - 1 fragments with smaller molecular weight and studing the their application in the treatment of angiogenesis related diseases, such as tumor and diabetic retinopathy.