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目的构建示踪细胞内磷脂酸的工具。方法人Raf-1基因磷脂酸结合结构域(Raf-1-PABD)能特异性结合磷脂酸,通过PCR和酶切Raf-1-PABD序列,并连接至带有增强型绿色荧光蛋白(EGFP)标签的载体,测序鉴定正确后,细胞转染pc DNA3.0-mito PLD-HA质粒表达线粒体磷脂酶D(mito PLD)产生大量磷脂酸,同时以Raf-1-PABD-EGFP融合蛋白结合产生的磷脂酸以示踪细胞内磷脂酸的量以及细胞内磷脂酸的改变。结果重组工具载体p EGFP-C1-Raf-1-PABD(磷脂酸结合蛋白)构建并成功表达,可示踪线粒体融合过程中磷脂酸的改变。结论成功构建了示踪细胞内磷脂酸水平的p EGFP-C1-Raf-1-PABD的载体,将其转染NIH3T3细胞进行表达,可用于细胞内线粒体中磷脂酸的示踪。
Objective To construct a tracer to trace intracellular phosphatidic acid. Methods The Raf-1 gene phosphatidic acid-binding domain (Raf-1-PABD) was able to bind specifically to phosphatidic acid. The Raf-1-PABD sequence was cleaved by PCR and ligated to the vector with enhanced green fluorescent protein (EGFP) After the vector was identified by sequencing, the cells were transfected with the pcDNA3.0-mito PLD-HA plasmid to generate a large amount of phosphatidic acid and mitochondrial phospholipidase D (mito PLD), while the Raf-1-PABD-EGFP fusion protein Phosphatidic acid is used to trace the amount of intracellular phosphatidic acid and changes in intracellular phosphatidic acid. Results The recombinant vector p EGFP-C1-Raf-1-PABD (phosphatidic acid-binding protein) was constructed and successfully expressed, which can be used to trace the changes of phosphatidic acid during mitochondrial fusion. Conclusion The vector of pEGFP-C1-Raf-1-PABD which traced intracellular phosphatidic acid levels was successfully constructed and transfected into NIH3T3 cells for expression. It can be used for the tracing of phosphatidic acid in mitochondria of cells.