抗突变型p53 Maxizyme的体外转录和真核表达载体的构建及鉴定

Construction of in vitro Transcription and Eukaryotic Expression Vector of Anti-mutant Type p53 Maxizyme Gene

目的 :构建抗突变型p53Maxizyme(Mz)的体外转录和真核表达载体 ,为进一步探讨抗p53Mz的体外切割作用和在细胞内对突变型 p53的抑制作用打下了基础。 方法 :应用计算机设计针对肝癌细胞突变型 p53(mtp53) 2 4 9位密码子的Mz的基因片断 (MzL和MzR) ,分别克隆入体外转录载体 pBSKU6中 ,命名为 pU6 -MzL和pU6MzR。应用PCR技术使 pU6 -MzR的酶切位点改变 ,亚克隆入 pU6 -MzL中 ,重组质粒命名为pU6 -Mz,同时构建靶基因mtp53的体外转录质粒 .再将嵌U6中的Mz亚克隆入真核表达载体 pEGFP中 ,构建真核重组质粒pEGFP -Mz ,该质粒嵌于U6表达系统中 ,在体外由RNA聚合酶Ⅲ高效转录。结果 :测序结果提示成功构建了Mz的体外转录和真核表达载体。结论 :本研究成功构建了针对突变型p53的Maxizyme的体外转录和真核表达载体 。

Objective To determine the inhibition of mutant-type p53(mtp53) in hepatocellular carcinoma by Maxizyme in both cell-free system and MHCC97 cell lines, the in vitro transcription and eukaryotic expression vector were constructed. Methods The anti-mtp53 maxizyme gene(MzL,MzR) was designed by computer, then coloned into vector respectively pBSKU6,pBSKU6MzL and pBSKU6MzR. After sequencing, the restrictive endonuclease site in pBSKU6MzR were changed by RT-PCR and then MzR was inserted into pBSKU6MzL, the recombinants were named pBSKU6Mz. The pBSKU6Mz was subcoloned into eukaryotic expression vector pEGFPC1. Results Sequencing showed that the vector of pBSKU6Mz and pEGFPCMz were constructed successfully. Conclusion These results lay a good foundation for in vitro cleavage and in vivo study.