C5orf34基因过表达慢病毒载体的构建及鉴定

Construction and identification of C5orf34 gene overexpression lentiviral vectors

目的构建C5orf34基因的过表达慢病毒载体。方法通过聚合酶链反应(PCR)选择性扩增C5orf34基因目的片段,将目的片段连接至载体,利用基因重组技术构建带有C5orf34基因目的片段的重组载体,将重组载体转化到感受态细胞扩增并对阳性克隆的C5orf34过表达慢病毒载体进行测序,将正确重组的过表达慢病毒载体包装并转染色至293T细胞进行后续的慢病毒转染和滴度测定,采用实时定量PCR法检测C5orf34基因的过表达水平。结果经过测序比对显示,慢病毒载体与设计参考序列一致。重组慢病毒经包装并转染293T细胞后可观察到荧光,显示C5orf34过表达慢病毒载体滴度为1.0×10^(8)TU/mL。实时定量PCR的结果显示,在稳定转染C5orf34基因的细胞中C5orf34基因表达水平明显增高。结论成功构建C5orf34基因过表达慢病毒载体,为进一步研究C5orf34基因在肺癌发生发展过程中的机制奠定了生物学基础。

Objective To construct an overexpression lentiviral vector of C5orf34 gene.Methods The target fragment of the C5orf34 gene was selectively amplified using polymerase chain reaction(PCR).The amplified fragment was then ligated into the vector using gene recombination technology,resulting in a recombinant vector containing the target fragment of the C5orf34 gene.The recombinant vector was transformed into competent cells for amplification,and the positive clones of the C5orf34 overexpression lentiviral vector were sequenced.The correctly recombined overexpression lentiviral vector was packaged and transfected into 293T cells for subsequent lentiviral transduction and titer determination.Real-time quantitative PCR was employed to detect the level of C5orf34 gene overexpression.Results Sequencing analysis revealed that the lentiviral vector was consistent with the designed reference sequence.Fluorescence was observed after the recombinant lentivirus was packaged and transfected into 293T cells,indicating a titer of 1.0×10^(8)TU/mL for the C5orf34 overexpression lentiviral vector.Real-time quantitative PCR results demonstrated a significant increase in C5orf34 gene expression levels in cells stably transfected with the C5orf34 gene.Conclusion The construction of the C5orf34 gene overexpression lentiviral vector was successful,providing a biological basis for further research on the mechanisms of the C5orf34 gene in the occurrence and development of lung cancer.