Cloning of Marf1 gene and its expression in eukaryotic cells
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摘要:
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-mKate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mKate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-mKate 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。
Abstract:
Objective：To clone mouse Marf1 gene and let it ectopically express in HEK293T cells，for studying its expression and mechanism. Methods：Marf1 coding sequence was amplified by reverse transcription-polymerase chain reaction(RT-PCR)and was cloned into the eukaryotic cell-expression vector pCMV6-AC-3DDK and pCMV6-AN-mKate using the advanced In-Fusion cloning system. After verification by restriction digestion followed by electrophoresis and sequencing，the correctly cloned plasmid DNA was transfected into HEK293T cells. The expression of MARF1-3DDK and MARF1-mKate fusion protein in the transfected HEK293T cells was assessed by both Western blot and immunofluorescence using the antibodies against DDK and mKate tag proteins. Results：The correct cloning of Marf1 was confirmed by restriction digestion and sequencing，and the expression of MARF1-3DDK and MARF1-mKate fusion protein was detected by Western blot. MARF1 fusion protein was detected to be localized either in the cytoplasm in a punctate manner or on the mitotic spindles. Conclusion：Mouse Marf1 gene was successfully cloned and expressed in HEK293T cells，which lays solid foundation for further studies on its functions and the related mechanisms.
