ICAM-1-GFP的构建及其与Molt-4细胞的结合(英文)

Construction of ICAM-1-GFP and Its Binding with Molt-4 Cells

本研究构建人细胞间黏附分子1(ICAM-1)全长基因的真核表达载体pEGFP-C1-ICAM-1,转染中国仓鼠卵巢细胞(CHO-K1)细胞株,并检测其在CHO细胞中的表达及与Molt-4细胞的结合。采用RT-PCR法从健康人外周血中分离单个核细胞,钓取ICAM-1全长基因(1622bp),与pMD18-T载体连接做全自动序列测定。将测序正确的克隆质粒pMD18-T-ICAM-1和表达载体pEGFP-C1分别用HindⅢ和Ⅰ进行双酶切,应用基因重组技术构建ICAM-1全长基因真核表达载体pEGFP-C1-ICAM-1,质粒经HindⅢ和SacII双酶切和PCR电泳鉴定后,采用脂质体转染法转染CHO细胞,并进行G418筛选。用RT-PCR、流式细胞术和荧光显微术检测ICAM-1-GFP的表达及亚细胞的定位,用检测ICAM-1-GFP/CHO细胞与Molt-4细胞的结合能力评价ICAM-1-GFP融合蛋白的功能。结果表明:重组质粒经限制性酶切鉴定得到与ICAM-1全长基因长度一致(1622bp)的酶切产物;测序分析证实,PCR产物与GenBank上登录的ICAM-1基因(NM_000201)序列完全一致,表明成功地完成了ICAM-1的扩增和表达载体的构建;荧光显微镜下可见转染的CHO细胞有绿色荧光蛋白的表达,表达的融合蛋白较均匀地分布于整个细胞;FACS检测ICAM-1-GFP的荧光转染率为(13±5.5)%,表明ICAM-1-GFP基因进入到CHO细胞并获得了有效表达,成功构建了ICAM-1-GFP/CHO细胞,并且ICAM-1-GFP/CHO细胞能够结合PMA处理的Molt-4细胞。结论:成功构建了ICAM-1-GFP真核表达载体,构建的ICAM-1-GFP真核表达载体在CHO细胞内稳定表达,ICAM-1-GFP/CHO细胞能与Molt-4细胞结合,这为进一步研究ICAM-1分子的功能打下基础。

This study was aimed to clone human intercellular adhesion molecule-1 （ ICAM-1 ） gene, to transfect the constructed eukaryotic expression vector ICAM-1-GFP into CHO cells, as well as to detect ICAM-1-GFP expression in CHO cells binding with Molt-4 cells. ICAM-1 cDNA gene was amplified by RT-PCR and inserted in PMD18-T vector. Then ICAM-1 cDNA from pMD 18-ICAM-1 vector was subcloned into eukaryotic expression vector pEGFP-C1 to construct recombinant ICAM-1-pEGFP-C1 vector. Restriction analysis and DNA sequencing were used to confirm the recombinant vector. After stable transfection of CHO-K1 cells with the recombinant vector, the expression and subcellular localization of ICAM-1-GFP were detected by RT-PCR, flow cytometry and fluorescence microscopy. The function of ICAM-1 -GFP fusion protein was assessed by the binding of ICAM-1-GFP/CHO cells to Molt4 cells. The results showed that 1622 bp full-length ICAM-1 cDNA obtained and was successfully ligated with pMD18-T-vector, subcloned to construct recombinant ICAM-1-pEGFP-C1 vector. Restriction analysis and DNA sequencing indicated that recombinant ICAM-1-GFP was successfully constructed and ICAM-1-GFP was expressed stably in CHO cells. ICAM-1-GFP expression was only observed in the cytoplasm of ICAM-1-GFP/CHO cells by fluorescence microscopy. The ICAM-1-GFP/ CHO cells were bound to PMA-treated Molt-4 cells. The expression of MEM-148 was very weak in PMA-treated Molt- 4 cells. It is concluded that the ICAM-1-GFP eukaryotic expression vector has been constructed successfully and expresses stably in CHO cells. PMA can increase the binding of Molt4 cells to 1CAM-1-GFP/CHO cells by inducing specialized form of ICAM-1 clustering.