Marf1基因的克隆及其在真核细胞中的表达

发布时间：2018-03-27 04:43

本文选题：Marf　切入点：In-Fusion克隆　出处：《南京医科大学学报(自然科学版)》2017年07期

【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-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。
[Abstract]:Objective: to clone mouse Marf1 gene and its expression in eukaryotic cells of ectopic HEK293T, to study the MARF1 protein in the cell expression and localization. Methods: using reverse transcription polymerase chain reaction (reverse transcription-polymerase chain reaction, RT-PCR) were amplified and obtained the Marf1 gene encoding sequence, using novel molecular cloning system In-Fusion the fragment was cloned into pCMV6-AC-3DDK pCMV6-AN-m and Kate two eukaryotic expression vector, the resulting fusion plasmid by restriction endonuclease digestion and sequencing identification, the positive plasmid transfected into HEK293T cells, and then use DDK and m Kate tag antibody through expression and localization of blot or Western immunofluorescence detection of MARF1 protein. Results: DNA sequencing proved that the Marf1 gene was cloned successfully; Western blot detected MARF1-3DDK and MARF The expression of 1-m Kate fusion protein in transfected HEK293T cells; immunofluorescence showed that the fusion protein was granular distribution in the transfected HEK293T cytoplasm and colocalized with spindle. Conclusion: cloned mouse Marf1 gene and its realization in eukaryotic cells in HEK293T stable expression, which lays the foundation for further study the function and mechanism of action.

【作者单位】：南京医科大学生殖医学国家重点实验室;
【基金】：国家重大科学研究计划(2014CB943200,2013CB945500) 国家自然科学基金面上项目(31471351,31271538) 江苏省自然科学基金(BK20140061)
【分类号】：R3416

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