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目的构建内皮抑素(endostatin,ES)过表达慢病毒载体,并进行鉴定。方法利用载体PUC57-ES构建GV365-ES慢病毒载体,转染293T细胞包装慢病毒,荧光显微镜下观察绿色荧光;Western blot法鉴定表达的目的蛋白;HIV-1 ELISA法检测病毒滴度。结果经PCR及测序鉴定证明慢病毒载体GV365-ES构建正确;转染细胞中荧光显微镜下可见绿色荧光,表达的目的蛋白相对分子质量约23 000;病毒滴度为2.1×10~8 TU/ml。结论已成功构建ES过表达慢病毒载体,为后期该病毒载体治疗实体瘤的观察及其作用机理的研究奠定了基础。
Objective To construct and overexpress lentivirus vector of endostatin (ES). Methods GV365-ES lentiviral vector was constructed by using vector PUC57-ES and transfected into 293T cells. The lentivirus was packaged and the green fluorescence was observed by fluorescence microscopy. The expressed protein was identified by Western blot. The virus titer was detected by ELISA. Results The recombinant lentiviral vector GV365-ES was confirmed by PCR and sequencing. The transfected cells showed green fluorescence under the fluorescence microscope, and the relative molecular mass of the expressed protein was about 23,000. The virus titer was 2.1 × 10-8 TU / ml . Conclusion The ES lentiviral vector has been constructed successfully, which lays the foundation for the observation of the therapeutic effect of this virus vector on solid tumors.