人野生型P^(53)与反义mdm_2基因融合体真核表达载体的构建及其在Mc3细胞中的表达

Construction of Eukaryotic Expression Vector of Human Wild-type P^(53) Gene and Antisense mdm_2 Fusion and Its Expression in Mc3 Cell

目的构建P53与反义mdm2cDNA序列真核表达载体,并探讨其在人黏液表皮样癌高转移细胞株Mc3细胞中的表达。方法应用分子克隆技术,将mdm2cDNA序列反向插入到含有P53基因序列pDOR-Neo中的P53基因序列下游,构成含有P53基因与反义mdm2基因融合基因的逆转录病毒表达载体。经酶切分析鉴定后命名为pDOR-Neo-P53-Fmdm2并将重组载体通过脂质体转染Mc3细胞,通过G418筛选转染阳性细胞,并采用Westernblot方法检测P53蛋白表达。结果酶切鉴定证实重组载体含有P53与反义mdm2cDNA片段,实验结果表明:转染后P53蛋白(1.35±0.14)较对照组P53蛋白(6.26±0.11)含量显著减低(P<0.01),转染空载体P53蛋白(6.24±0.12)与对照组比较无统计学差异(P>0.05)。结论P53与反义mdm2基因融合体真核表达载体构建成功,并表达野生型P53蛋白和封闭mdm2基因,为今后进一步研究打下了基础。

Objective To construct the eukaryotic expression vector containing human wild-type p^53 gene and mdm2 anti- sense eDNA sequence,and then discuss its expression in human mucoepidermoid carcinoma high metastatic cell strain Mc3. Methods To insert mdm2 cDNA sequence reversedly into pDOR-Neo gene downstream sequence containing p^53 gene by using the methods of molecular cloning,and the retroviral expression vector containing the fusion gene of p^53 gene and mdm2 antisense gene were constructed. By restriction analysis identification,named the retroviral expression vector pDOR- Neo-p^53-F mdm2,and put the recombinant vector into Mc3 cell by utilizing liposome transfection. Then transfected positive cells could be screen by G418,after that using the western blot method to detect the expression of psa gene. Results The restriction analysis identification confirmed that the retroviral expression vector contains both human wild-type p^53 gene and mdm2 antisense cDNA sequence,the result of the experment showed that the transfected p^53 protein （1.35±0. 14） decreased significantly compared with normal control（6.26±0.11） （P〈0.01） ,the empty vector p^53 protein（6.24±0.12） expression were not significant （P〉0.05）. Conclusion The fusion eukaryotic expression vector containing human wild-type p^53 gene and mdm2 antisense cDNA sequence was constructed successfully,which could express both wild-type p^53 protein and close mdm2 gene. The article makes preparation for the further study.