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目的:构建Gαs慢病毒表达载体并检测其表达性能,以便应用过表达技术进一步研究Gαs功能。方法:设计针对人Gαs DNA序列的带酶切位点引物,PCR扩增获得双链DNA后,与酶切后的FUW载体片段进行连接、转化,酶切及DNA测序鉴定重组克隆。提取阳性克隆质粒,转染293T细胞。用293T细胞制备慢病毒颗粒,感染293T细胞后收集细胞全蛋白,用QPCR、Western blot检测慢病毒系统过表达效果。结果:证实人Gαs正确插入慢病毒载体,人Gαs慢病毒过表达质粒及相应病毒颗粒能有效过表达Gαs。结论:人Gαs慢病毒表达系统的成功建立,为应用过表达技术研究人Gαs慢病毒功能打下了基础。
OBJECTIVE: To construct Gαs lentiviral vector and test its expression in order to further study Gαs function by using overexpression technology. Methods: The primers with restriction enzyme sites of human Gαs DNA sequence were designed. After double-stranded DNA was amplified by PCR, the cloned FUW vector fragments were ligated, transformed, digested and sequenced to identify the recombinant clones. Positive clones were extracted and transfected into 293T cells. The 293T cells were used to prepare lentivirus particles, 293T cells were infected after collecting the whole protein, using QPCR, Western blot test lentiviral system overexpression. Results: It was confirmed that Gαs was correctly inserted into the lentiviral vector. The human Gαs lentivirus overexpression plasmid and the corresponding virus particles could effectively overexpress Gαs. Conclusion: The successful establishment of human Gαs lentivirus expression system lays the foundation for the study of human Gαs lentivirus using overexpression technology.