MGMT反义RNA真核表达载体的构建与鉴定

Construction and identification of eukaryotic expression vector for MGMT antisense RNA

目的：探讨反义ＲＮＡ逆转胶质瘤细胞耐药的可能性，构建ＭＧＭＴ反义ＲＮＡ的真核表达载体。方法：以ＸｈｏⅠ和ＰｖｕⅡ双酶切质粒ｐＨＭ１４，回收１７８ｂｐ的片段，定向插入以ＨｐａⅠ和ＸｈｏⅠ双酶切ｐＬＸＳＮ所获得的线性化载体，构建针对ＭＧＭＴｍＲＮＡ５＇端的反义表达载体ｐＬａＭＴ５ＳＮ；以ＥｃｏＲⅠ单酶切ｐＨＭ１４，回收７７９ｂｐ的片段，插入以ＥｃｏＲⅠ单酶切ｐＬＸＳＮ并经ＣＩＡＰ去磷酸化的线性载体，构建针对ＭＧＭＴｍＲＮＡ全长序列的反义表达载体ｐＬａＭＴＳＮ。结果：ｐＬａＭＴ５ＳＮ经ＨｉｎｄⅢ酶切可见３７０ｂｐ的片段；ｐＬａＭＴＳＮ经ＰＣＲ扩增证明目的片段反向插入ｐＬＸＳＮ，鉴定结果表明构建成功。结论：成功构建了两个ＭＧＭＴ反义ＲＮＡ的真核表达载体，为研究ＭＧＭＴ反义ＲＮＡ逆转胶质瘤耐药细胞的耐药表型提供了良好的实验材料。

Objective:To construct the eukaryotic expression vectors of MGMT antisense RNA for exploring the effect of antisense RNA reversin g the drug resistance of glioma.Methods:The plasmid pHM14 was d igested with XhoⅠand PvuⅡ, the 178 bp fragment was extracted and cloned into the linear pLXSN vector which was dige sted with HpaⅠand XhoⅠ. The recombinant targets 5'region of MGMT mRNA and desi g nated pLaMT5SN. The pHM14 was digested with EcoRⅠ, the 779bp fragment was extra cted and cloned into the linear pLXSN vector which was digested with EcoRⅠand d ephosphorylated with CIAP. The reverse recombinant targets the whole length of M GMT mRNA and designated pLaMTSN.Results:There was a 370bp band by ag a rose gel electrophoresis after pLaMT5SN was digested with HindⅢ. The reverse or ientation of insert in pLaMTSN was identified with PCR. The identification prove d that the construction was successful.Conclusion:Two eukaryotic exp ression vectors were successfully constructed. These vectors may provide experim ental bases for study of reversing the drug resistance of gliomas with antisens e RNA and provide more information about the mechanisms of antisense RNA.