人VEGF165基因的克隆、表达和活性鉴定

The clone, expression and activity identify of human vascular endothelial growth factor

目的 克隆人VEGF165基因,构建其真核表达质粒,转染293细胞,并鉴定表达VEGF的生物学活性。方法 采用PCR法从人心脏cDNA文库钓取人VEGF165基因,插入pUC19载体测序后与真核表达质粒pAdTrackCMV连接,阳离子脂质体Lipofectamin介导VEGF基因转染293细胞,Northem blot和Western blot法分别检测VEGFmRNA和蛋白的表达,Miles试验进行活性鉴定。结果(1)琼脂糖凝胶电泳分析得到两个片段,分别为582bp的VEGF165cDNA和2.68kb的pUC19线性质粒,结果与理论值相符,VEGF165测序结果与GENE BANK中VEGF165序列完全一致。(2)转染293细胞后表达绿色荧光蛋白,转染后第3天(1 024pg／ml)和5天(986pg／ml)VEGF表达最高,明显高于转染前(102pg／ml)。(3)Miles试验结果显示,用转染AdTrackCMV-VEGF165细胞培养上清皮下注射后10分钟左右皮丘变为蓝色,成倍连续稀释后,出现蓝斑的时间延长,蓝斑范围也相应缩小。而注射未转染293细胞培养上清和生理盐水组则未见蓝斑出现。结论 成功克隆人VEGF165基因,并表达出具有生物学活性的VEGF蛋白。

Objective To clone, identify and detect the activity of human vascular endothelial growth factor (hVEGF 165). Methods The VEGF165 gene was amplified from the human heart cDNA library and then inserted into plasmid pUC19 and sequenced. The target gene VEGF165 and plasmid pAdTrackCMV were ligated together by T4 ligase and the recombinant eukaryonic expression plasmid AdTrackCMV-VEGF165 was transgered by lipofectamin mediated technique into 293 cell. The results of transfection were detected by northern and western blot. The production of VEGF was assessed by ELISA and the biological activity of VEGF was detected by Miles test. Results 1. The 582bp VEGF165 cDNA fragment was obtained by PCR The sequencing maps showed that phVEGF165 sequence matched well with Genebank completely. 2. The green fluorescence from some 293 cells could be observed on a black background with fluorescence microscopy. 3. The expression of VEGF165 could be detected by Western blot analysis, which protein molecular weight was 22kd. However there was no product in untransfected 293 cells. The results of ELISA revealed that the concentration of VEGF165 was higher at 3rd day and 5th day. There was significant difference between transfected and untransfected 293 cells. The blue dot could be observed where injected with supernatant from transfected 293 cells. Conclusion VEGF165 gene was successfully cloned and its expressing protein was of bioactivity.