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目的:克隆小鼠的Marf1基因并使其在真核细胞HEK293T中异位表达,以研究MARF1蛋白在细胞中的表达和定位。方法:运用反转录-聚合酶链反应(reverse transcription-polymerase chain reaction,RT-PCR)扩增并获得Marf1基因编码序列,再利用新型分子克隆体系In-Fusion将该目的片段克隆至pCMV6-AC-3DDK和pCMV6-AN-m Kate两个真核表达载体中,由此所得融合质粒经限制性内切酶酶切电泳和测序筛选鉴定,将所得到的阳性质粒转染到HEK293T细胞中进行表达,然后利用DDK和m Kate标签抗体通过Western blot或免疫荧光的方法检测目的蛋白MARF1的表达和定位。结果:DNA测序鉴定证明Marf1基因克隆成功;Western blot检测到MARF1-3DDK和MARF1-m Kate融合蛋白在转染的HEK293T细胞内的表达;免疫荧光显示融合蛋白在转染后的HEK293T细胞质呈颗粒状分布,并与纺锤体共定位。结论:成功克隆了小鼠Marf1基因并实现了其在真核细胞HEK293T中的稳定表达,为进一步研究其功能和作用机制奠定了基础。
OBJECTIVE: To clone the Marf1 gene of mouse and ectopically express it in eukaryotic HEK293T cells to study the expression and localization of MARF1 protein in cells. Methods: The coding sequence of Marf1 gene was amplified by reverse transcription-polymerase chain reaction (RT-PCR). The target fragment was cloned into pCMV6-AC using the new molecular cloning system In-Fusion -3DDK and pCMV6-AN-m Kate, and the resulting fusion plasmid was identified by restriction enzyme digestion electrophoresis and sequencing, and the resulting positive plasmids were transfected into HEK293T cells for expression The expression and localization of the target protein MARF1 were detected by Western blot or immunofluorescence using DDK and mKate tagged antibodies. Results: DNA sequencing confirmed the successful cloning of Marf1 gene. The expression of MARF1-3DDK and MARF1-m Kate fusion protein in HEK293T cells was detected by Western blot. Immunofluorescence showed that the fusion protein was granulated in HEK293T cytoplasm after transfection Distributed and co-localized with the spindle. Conclusion: The Marf1 gene was successfully cloned and its expression was stable in eukaryotic cells HEK293T, which laid the foundation for further study on its function and mechanism.