MGMT基因siRNA质粒载体的构建及鉴定

目的 构建MGMT基因的siRNA（small interfering RNA）质粒载体pRNAT-H1.1/Neo-MGMT siRNA,为进一步研究siRNA对MGMT基因的抑制作用,探讨MGMT在胶质瘤的化疗耐药及逆转胶质瘤细胞耐药表型中的作用奠定基础.方法 针对MGMT的靶序列设计合成二三条shRNA（small hair RNA）的DNA模板单链,同时模板链两端分别设计BamHⅠ和Hind Ⅲ限制酶切位点.退火形成siRNA载体插入片断.用限制性内切酶将pRNAT-H1.1/Neo线性化,T4连接酶将插入片断插入pRNAT-H1.1/Neo中.重组质粒转化大肠杆菌DH5α后培养出阳性克隆,抽提质粒,经酶切、电泳和测序的方法鉴定质粒构建是否成功.结果 设计构建三条针对MGMT基因的siRNA质粒载体,经双酶切,在2%琼脂糖凝胶电泳结果显示：PCR产物大小76bp,产物大小与设计的完全相同.测序结果证明序列正确.结论 成功设计构建了三条针对MGMT基因的siRNA质粒载体,为研究RNA干扰逆转胶质瘤细胞的耐药表型提供了良好的实验材料.

Construction and Identification of MGMT-siRNA Expression Vector

Objective To construct MGMT combinative siRNA vector-pRNAT-H1.1/Neo-MGMT, which may lay the foundation for further investigation on the chemotherapeutic treatment failure in glioma patients,and may conduce the development of strategy of gene therapy targeting MGMT in gliomas. Methods Three shRNAs for blocking MGMT gene was designed.And two single strand DNA templates were designed according to the sequence of each shRNA.When the DNA templates synthesized,two different restriction sites were superimposed, respectively, to the two end of it.The insertion element formed after the DNA templates annealed.Make the blank plasmid linearization by use of restriction enzyme,and then the insertion element was inserted into the blank plasmid by T4 ligase.Enzyme cutting and sequencing was performed to check whether MGMT siRNA plasmid be constructed successfully or not. Result Three siRNA eukaryotic expression vectors--pRNATin-H 1. l/Neo MGMT siRNA were constructed.We gained positive clone after recombinant vector transforming,extracted the vector and gained products by enzyme cutting.And the 2% gel electrophoresis result showed：The product is near 76bp and accord with the design,and the results of DNA sequence proved its preciseness. Conclusion Successfully constructed siRNA eukaryotic expression vector pRNATin-H1.1/Neo MGMT siRNA may provide experimental bases for study of reversing the drug-resistance of gliomas with siRNA and provide more information about the mechanisms of siRNA.