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Establishment of a eukaryotic expressing vector pcDNA.3.1/myc-His(-)A-HINT1 and its expression in human melanoma cell A375
Author(s): 
Pages: 660-664
Year: Issue:  9
Journal: China journal of Leprosy and Skin Diseases

Keyword:  组氨酸三聚体核苷结合蛋白1黑色素瘤真核表达稳定转染;
Abstract: 目的:构建真核表达载体pcDNA.3.1/myc-His(-)A-HINT1并检测其在人黑素瘤细胞A375中的表达.方法:采用巢式RT-PCR的方法扩增带有Xba I和Kpn I酶切位点的人HINT1编码区基因,经TA克隆技术,将基因测序正确的序列克隆到空白真核表达载体pcDNA.3.1/myc-His(-)A,构建真核表达载体pcDNA.3.1/myc-His(-)A-HINT1.经菌落PCR、双酶切及基因测序鉴定后,用脂质体转染法转染A375细胞,通过G418筛选,获得稳定转染的A375单克隆细胞.用免疫细胞化学法及Western blot(WB)法检测HINT1蛋白在细胞中的表达.结果:pcDNA.3.1/myc-His(-)A-HINT1真核表达载体构建成功,通过脂质体转染,G418筛选,获得稳定转染的A375单克隆细胞,免疫细胞化学及WB检测均显示稳定转染真核表达载体pcDNA.3.1/myc-His(-)A-HINT1的A375单克隆细胞有更高的HINT1蛋白的表达.结论:成功构建了pcDNA.3.1/myc-His(-)A-HINT1真核表达载体,并获得稳定转染且能高表达HINT1的单克隆A375细胞.
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