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目的 :观察 p2 7KIP1 绿色荧光蛋白 (GFP)融合蛋白在肝癌细胞系HCC 92 0 4中的表达及定位。方法 :根据p2 7KIP1基因的编码顺序 ,应用PCR技术将 p2 7KIP1基因 3′末端终止密码TAA突变为TGT ,并通过基因重组技术将其与GFP基因融合。再采用脂质体转染法 ,将 p2 7KIP1 GFP融合基因转入HCC 92 0 4细胞中 ,用荧光显微镜观察其表达情况。结果 :DNA序列测定的结果显示 ,p2 7KIP1基因与基因库中已收录的 p2 7KIP1的序列相比较 ,仅有 1个核苷酸的差异。荧光显微镜显示 ,在转染GFP基因的HCC 92 0 4 /GFP细胞中 ,荧光均匀地分布于整个细胞 ;而在转染融合基因 p2 7KIP1 GFP的HCC 92 0 4 /GFP p2 7细胞中 ,绿色荧光主要见于核内。结论 :转染的HCC 92 0 4细胞能高效表达融合基因p2 7KIP1 GFP ,且定位于细胞核内 ,与 p2 7KIP1基因的表达方式相同。
Objective: To observe the expression and localization of p2 7KIP1 green fluorescent protein (GFP) fusion protein in hepatocellular carcinoma cell line HCC 92 0 4. Methods: According to the coding sequence of p2 7KIP1 gene, the termination codon TAA at the 3 ’end of p2 7KIP1 gene was mutated to TGT by PCR and fused with GFP gene by gene recombination technique. Then, the p2 7KIP1 GFP fusion gene was transfected into HCC 92 0 4 cells by lipofection method, and its expression was observed by fluorescence microscope. Results: The result of DNA sequencing showed that there was only one nucleotide difference between the p2 7KIP1 gene and the sequence of p2 7KIP1 in the gene bank. Fluorescence microscopy showed that fluorescence was evenly distributed throughout the cells transfected with GFP gene in HCC 9240 / GFP cells; whereas in HCC 9240 / GFP p27 cells transfected with fusion gene p2 7KIP1 GFP, green fluorescence Mainly seen in the nucleus. CONCLUSION: The transfected HCC 92 0 4 cells can efficiently express the fusion gene p2 7KIP1 GFP and locate in the nucleus in the same manner as the p2 7KIP1 gene.