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携带人\AKT2、PDK1、BAD基因的慢病毒表达载体的构建及其在293T细胞中的表达

Construction and Expression of Recombinant Lentiviral Vectors of \AKT2, PDK1 and \BAD

  • 摘要: 目的 构建含人丝氨酸/苏氨酸蛋白激酶B(protein kinase B,PKB/AKT2)、磷酸肌醇依赖激酶-1(phosphoinositide-dependent kinase 1,PDK1)、bcl-2相关性死亡蛋白(bcl-2-associated death protein,BAD)基因的绿色荧光慢病毒载体,鉴定其在293T细胞中的表达。方法 选择病理证实的非小细胞肺癌(NSCLC)组织,采用特异性引物RT-PCR扩增AKT2、PDK1及BAD cDNA。将PCR产物与T载体连接测序,测序正确的目的片段从T载体上酶切下与慢病毒骨架质粒连接,将此重组的慢病毒表达载体质粒及包装系统共转染入293T细胞(人胚肾细胞系),收集病毒上清,Western blot检测AKT2,BAD及PDK1蛋白质表达。结果 凝胶电泳证实AKT2,BAD,PDK1三基因成功转导至以pCDF1-MCS2-EF1-copGFP为骨架质粒的慢病毒包装系统中,磷酸钙转染法72 h后BAD、PDK1及AKT2的转染率分别约为100%、95%、90%。慢病毒包装后测定3种病毒滴度均达6.7×106 PFU/mL,并通过Western blot法检测到AKT2,BAD,PDK1蛋白在293T细胞的表达。结论 成功构建了携带人AKT2、BAD、PDK1原癌基因的慢病毒表达载体,在293T细胞中成功鉴定其表达。

     

    Abstract: 【Abstract】 Lung cancer is the leading cause of cancer-related death worldwide. The topics of this issue include the changes of lung cancer epidemiology and its histological characteristics, the value of low dose CT in early detection of lung cancer and the image characteristics of solitary pulmonary nodule in early diagnosis of lung cancer, the advances in the therapeutic strategies for lung cancer with brain metastases and pulmonary sarcomatoid carcinoma, and new potential targets of molecular targeted therapy in lung cancer. In order to more effectively improve the survival and prognosis of the patients with lung cancer, it is necessary to strengthen basic research on the molecular pathogenesis of lung cancer, and to translate the advances in basic research into the efforts of exploring the specific and sensitive diagnosis tools for lung cancer, and so increasing the accuracy of early screening and diagnosis, as well as classifying the lung cancer by molecular pathology to achieve individualized therapy.

     

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