中国肝细胞癌p53基因热点突变R249S的载体构建及其在细胞中的表达

抑癌基因p53突变是人肝细胞癌（Hepatocellular carcinoma，HCC）中常见的现象，中国江苏启东地区的HCC患者中p53基因的第249位密码子突变AGG→AGT/Arg→Ser（R249S）被认为是热点突变。本研究以人肝cDNA为模板，扩增人全长p53基因，将其克隆到pCMV-Myc载体中。利用定点突变引物以pC-MV-p53为模板，构建R249S定点突变体pCMV-R249S，转染p53缺陷型的H1299细胞系。Western Blot检测表明，克隆在pCMV-Myc载体中的p53基因和249位密码子热点突变的p53基因都可以在H1299细胞系中表达。

The Construction of p53 Gene Hotspot Mutation R249S Detecting in Chinese Hepatocelluar Carcinomous and Its Expression in H1299 Cell Line

The tumor suppressor p53 gene mutation is one of the most common abnormalities in human Hepatocellular carcinoma(HCC).The 249th codon of P53 is mutated from AGG to AGT and the amino acid from Arg to Ser(R249S) in more than 50 % HCC patients in Jiangsu Qidong region of China,which is detected as the hotspot mutation.In our study,the total human p53 gene from the human liver cDNA was successfully cloned,and the p53 gene was subcloned into pCMVMyc vector to construct the pCMV-p53.The hotspot mutant pCMV-R249S was constructed taking the pCMV-p53 as template and using the site-directed mutated primers.The pCMV-p53 and pCMV-R249S were respectively transfected into H1299 cell line which is a homozygous deletion of the p53 gene,Western Blot result indicated that both the p53 gene and the mutated p53 gene with the hotspot mutation in the 249th codon could express in H1299 cell line.