肝癌抑制基因-1与转运功能相关的451 bp序列区的确定

背景与目的:肝癌抑制基因-1(HCCS1)是一种潜在的肝癌抑制基因,并且在细胞内蛋白分拣运输中也发挥着重要作用,其抑癌作用有可能是通过其蛋白转运的功能而发挥的,本文以寻找HCCS1序列中与转运相关的最小功能序列区域为目的.方法:通过亚克隆技术,构建以pEGFP-C2为载体的含不同长度HCCS1cDNA片段的亚克隆,将构建的亚克隆转染子宫颈癌HeLa细胞,通过免疫荧光共聚焦显微镜观察不同长度HCCS1蛋白的分布,以及与6-磷酸甘露糖受体(M6PR)的共定位.结果:成功构建了以pEGFP-C2为载体的10个含有不同长度HCCS1片段的亚克隆;HCCS1cDNA从3'端向5'端逐渐缺失的片段中:2 100 bp片段至778 bp片段编码的不同长度HCCS1蛋白呈颗粒状分布于核周的胞质内,其中2 100 bp片段至1 571 bp片段编码的不同长度HCCS1蛋白均呈颗粒状、极性分布于核周的胞质内,且与M6PR有共定位;而1 120 bp片段至778 bp片段编码的不同长度HCCS1蛋白虽然也呈颗粒状分布于核周,但极性分布消失,且与M6PR共定位消失;HCCS1cDNA片段678 bp片段至339 bp片段编码的不同长度HCCS1蛋白呈散点状弥散分布于胞质以及胞核内.结论:确定了HCCS1cDNA 1 571 bp片段的3'端451 bp的序列为与HCCS1转运功能相关的最小区域范围.

Establishment of 451 bp fragment that functionally related to transportation in hepatocellular carcinoma supressor gene-1(HCCS1)

Background and purpose:Hepatocellular carcinoma suppressor gene-1(HCCS1)is a potential hepatocellular carcinoma supressor gene,and it also plays an important role in sorting of some cytoplasmic proteins.Its carcinoma suppressor function may be related to its sorting function.So it is important to identify the minimum region of functional sequence in HCCS1 that related to the transportation.Methods:The expression vectors containing various lengths of HCCS1 gene were constructed and transfected into HeLa cells mediated by liposomes.The localizations of different HCCS1 fragments and the colocalizations of M6PR with various truncated HCCS1 were determined by laser scanning confocal microscopy,respectively.Results:10 different expression vectors containing various lengths of HCCS1 gene were successfully constructed,it had been shown that the polar localization of HCCS1 protein and the co-localizations with M6PR disappeared when HCCS1 gene was truncated to 1 120 bp from the 3'end by laser scanning confocal microscopy.Conclusions:We identified the minimum localization of the HCCS1 functional sequence that related to the transportation.