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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。
In this study, an eukaryotic expression vector pEGFP-C1-ICAM-1 containing the full-length ICAM-1 gene was constructed and transfected into Chinese hamster ovary cells (CHO-K1) And the binding to Molt-4 cells. The mononuclear cells were isolated from peripheral blood of healthy people by RT-PCR, and the full-length ICAM-1 gene (1622bp) was obtained and ligated with pMD18-T vector. The cloned plasmid pMD18-T-ICAM-1 and the expression vector pEGFP-C1 with the correct sequencing were double-digested with HindIII and Ⅰ, and the full-length ICAM-1 eukaryotic expression vector pEGFP-C1- 1, The plasmid was digested with HindIII and SacII and identified by PCR electrophoresis. The CHO cells were transfected by lipofection and G418 selection. ICAM-1-GFP expression and subcellular localization were detected by RT-PCR, flow cytometry and fluorescence microscopy. The ICAM-1 expression was assessed by measuring the binding ability of ICAM-1-GFP / CHO cells to Molt-4 cells -GFP fusion protein function. The results showed that the restriction endonuclease digestion of the recombinant plasmid was consistent with the full-length gene of ICAM-1 (1622bp). The sequencing analysis confirmed that the PCR product exactly matches the sequence of ICAM-1 gene (NM_000201) registered in GenBank , Indicating that the expansion of ICAM-1 and the construction of the expression vector were successfully completed. Under the fluorescence microscope, transfected CHO cells showed the expression of green fluorescent protein, and the expressed fusion proteins were more evenly distributed throughout the cells. FACS detection of ICAM- The results showed that ICAM-1-GFP gene was successfully transfected into CHO cells and the expression of ICAM-1-GFP / CHO cells was successfully established. ICAM-1- GFP / CHO cells were able to bind to PMA-treated Molt-4 cells. CONCLUSION: The eukaryotic expression vector ICAM-1-GFP was constructed successfully. The constructed eukaryotic expression vector ICAM-1-GFP was stably expressed in CHO cells. ICAM-1-GFP / CHO cells could bind to Molt-4 cells. Lay a foundation for further study on the function of ICAM-1 molecule.