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目的:克隆小鼠CXCR4基因并建立其慢病毒表达系统。方法:设计合成PCR引物,从小鼠骨髓有核细胞来源的cDNA中扩增并克隆小鼠CXCR4基因的编码区。构建CX-CR4-IRES-GFP表达单元后通过转染细胞,观察GFP的表达证实其表达效能。然后建立慢病毒表达载体,包装慢病毒后感染培养的细胞,用流式细胞术分析其在被感染的细胞中的表达。结果:成功扩增了小鼠CXCR4基因的编码区。克隆入质粒载体后经DNA序列测定证实了其序列。通过转染细胞和流式细胞术以及荧光显微镜观察证实了CXCR4-IRES-GFP的表达效能。成功构建了CXCR4慢病毒表达载体,感染细胞后经流式细胞术分析,证实被感染的细胞可以表达小鼠CX-CR4。结论:成功扩增、克隆了小鼠CXCR4基因,并成功建立起其慢病毒表达系统,为后续的基础和应用研究奠定了基础。
Objective: To clone mouse CXCR4 gene and establish its lentivirus expression system. Methods: PCR primers were designed and synthesized. The coding region of mouse CXCR4 gene was amplified from cDNA of murine bone marrow nucleated cells. After constructing CX-CR4-IRES-GFP expression unit, the expression of GFP was confirmed by transfecting the cells. Then, a lentiviral expression vector was constructed, the cultured cells were infected after packaging the lentivirus, and their expression in the infected cells was analyzed by flow cytometry. Results: The coding region of mouse CXCR4 gene was successfully amplified. After cloning into plasmid vector, its sequence was confirmed by DNA sequencing. The expression of CXCR4-IRES-GFP was confirmed by transfected cells and flow cytometry and fluorescence microscopy. The CXCR4 lentiviral vector was successfully constructed. After infected cells were analyzed by flow cytometry, the infected cells could express CXCR4. Conclusion: The mouse CXCR4 gene was successfully amplified and cloned, and its lentiviral expression system was successfully established, which lays the foundation for the following basic and applied research.