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目的构建过表达miR-19a/19b(miR-19)基因的慢病毒载体pm19H2BmRFP。方法依据miR-19(784 bp)的序列设计引物,以pMIG-miR-19a/19b为模板扩增目的片段miR-19,再经胶回收、酶切并纯化而获连接用目的片段。连接用pHIV-H2BmRFP载体采用内切酶Xba I和Hpa I进行双酶切,获得粘性末端线性化片段并纯化。最后使用T4 DNA连接酶对纯化的线性化载体pHIV-H2BmRFP和miR-19片段进行定向连接,转化,次日挑选单菌落,提取质粒后行PCR和测序鉴定。所构建载体即为pm19H2BmRFP。参考文献方法进行慢病毒包装和确认慢病毒是否成功生产;过表达miR-19和RFP的慢病毒感染293T细胞及肺腺癌细胞株A549-Luc以建立相应病毒感染体系。结果 PCR及测序结果证实成功构建了pm19H2BmRFP,过表达miR-19的慢病毒上清感染A549-Luc和293T细胞48 h后,荧光显微镜下均可观察到红色荧光。结论成功构建过表达miR-19基因的慢病毒载体pm19H2BmRFP,为后续研究奠定基础。
Objective To construct a lentiviral vector pm19H2BmRFP overexpressing miR-19a / 19b (miR-19). Methods According to the sequence of miR-19 (784 bp), primers were designed and miR-19 was amplified using pMIG-miR-19a / 19b as a template. The target fragment was recovered by gel extraction, digestion and purification. The ligation was digested with endonucleases Xba I and Hpa I using the pHIV-H2BmRFP vector to obtain a linearized, sticky-end fragment and purified. Finally, the purified linearized vectors pHIV-H2BmRFP and miR-19 were ligated and transformed using T4 DNA ligase. The single colonies were picked the next day, and the plasmids were extracted and identified by PCR and sequencing. The constructed vector is pm19H2BmRFP. Reference lentivirus packaging and confirm the successful production of lentivirus; Lentiviral overexpression of miR-19 and RFP 293T cells and lung adenocarcinoma cell line A549-Luc infection to establish the corresponding virus infection system. Results PCR and sequencing results confirmed that pm19H2BmRFP was successfully constructed. After transfected with miR-19 over-expressing lentiviral vector for 48 h, A549-Luc and 293T cells were observed under fluorescence microscope. Conclusion The successful construction of lentiviral vector pm19H2BmRFP overexpressing miR-19 gene laid the foundation for further research.