绿色荧光蛋白(GFP)融合于人整合蛋白α_(Ⅱb)C端不影响α_(Ⅱb) β_3复合物在CHO细胞正常表达

GFP Fused to the Cytoplasmic Tail of Integrin α_(Ⅱb) Allows the Normal Expression of α_(Ⅱb)β_3 Compound in CHO Cells

本研究选择αⅡb作为靶蛋白观察绿色荧光蛋白(GFP)标记对αⅡbβ,复合物生物合成过程和表达的影响。 从人αⅡb表达载体p3.1-2b中经PCR扩增的αⅡb基因全长cDNA,插入表达载体pEGFP-N1中构建αⅡbGFP融合蛋 白表达载体,测序正确后利用脂质体将重组质粒与表达人整合蛋白β3亚基的真核表达质粒p3.1-3a共转染CHO 细胞,对转染后细胞用Western blot方法鉴定αⅡbGFP基因在CHO细胞中的表达并用激光共聚焦显微镜确定融合 蛋白的细胞内定位。结果表明:经限制性内切酶酶切图谱分析和DNA序列测定证实目的基因已插入重组质粒, Western blot证明转基因CHO细胞有人αⅡbGFP基因的表达,融合蛋白细胞内定位研究显示αⅡbGFP可以由内质网 转运至高尔基体。结论:成功构建了人αⅡbGFP融合蛋白真核表达载体;GFP融合于αⅡbC端不影响αⅡbβ3复合物 在CHO细胞的正常表达。

To investigate the effect of GFP fused to C terminal of integrin αⅡbon the biosynthesis and expression of αⅡb β3 compound, the αⅡbGFP expression plamid, named p αⅡbGFP, the cDNA of αⅡb was constructed from p3. 1-2b and fused to pEGFP-N1 in frame. When the sequence of p αⅡbGFP was confirmed by sequencing it was transferred to Chinese Hamster Ovary (CHO) cells with or without p3. 1-3a expressing integrin β3. Then the expression of αⅡbGFP fusion protein was confirmed by Western blot and then its subcellular localization was determined with laser confocal scanning microscopy. The results showed that the target gene was cloned into recombinant vector by restriction analysis and sequencing. Overex-pression of the fusion protein in the transfected CHO cells was identified with Western blot. Subcellular localization analysis confirmed that αⅡbGFP was expressed in CHO cells and could be transferred from endoplasmic reticulum to Golgi apparatus. It is concluded that the eukaryotic expression plasmid containing αⅡbGFP fusion gene is successfully constructed. GFP fused to the cytoplasmic tail of integrin αⅡballows the normal expression of αⅡbβ3 in CHO cells.