ICAM-1-GFP的构建及其与Molt-4细胞的结合

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—CI—ICAM—1，转染中国仓鼠卵巢细胞（CHO—K1）细胞株，并检测其在cH0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Hindm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-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-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，／CAM—1—GFP／CHO细胞能与M0lt-4细胞结合，这为进-步研究ICAM-1分子的功能打下基础。

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

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 subeloned into eukaryotic expression vector pEGFP-Cl to construct recombinant ICAM-1-pEGFP-Cl 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 Molt-4 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-Cl 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 microcopy.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 Molt4 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 Molt-4 cells to ICAM-1-GFP/CHO cells by inducing specialized form of ICAM-1 clustering.