ω-3多聚不饱和脂肪酸脱氢酶的真核表达及鉴定

Eukaryotic Expression and Characterized of the ω-3 Fatty Acid Desaturase fat1 Gene

目的:构建ω-3多聚不饱和脂肪酸脱氢酶真核表达载体,并在293T细胞(人胚肾细胞)中实现表达。方法:通过RT-PCR法扩增得到ω-3多聚不饱和脂肪酸脱氢酶基因fat1,构建重组真核表达载体pCMV-Myc-fat1,用脂质体法转染293T细胞,Western blot检测fat1的表达,并用间接免疫荧光(IFA)确定其在293T细胞中的定位情况。结果:构建真核表达质粒pCMV-Myc-fat1,转染293T细胞后,可检测到细胞内有fat1的表达并确定其在细胞中的位置。结论:成功构建真核表达质粒pCMV-Myc-fat1,可检测出细胞内有fat1的表达并确定其在细胞膜和细胞质内均有表达,为进行fat1的功能研究奠定了基础。

Object ：To construct eukaryotic recombinant expression vector pCMV-Myc-fatl and express fatl gene inhuman kidney 293T cells. Methods：The cDNA of fatl was amplified by RT-PCR from Caenorhabditis elegans. The vector containing fatl gene were constructed and identified by endonucleases digestion and PCR. Expression plasmids were transfected into 297T cells, the expression of fatl was detected by Western blot, and the distribution of this gene was analyzed by indirect immunofluorescense assay （IFA）. Results： Eukaryotic recombinant expression vector pCMV-Myc-fatl was constructed, fatl gene was effectively expressed in 293T cells and detected successfully by Western blot and laser scanning confocal microscope. Conclusions：The eukaryotic recombinant expression vector pCMV-Myc-fatl was constructed successfully through DNA recombinant technique. The expression of fatl was detected and the expressed fatl was found to distribute in the cell membrane and cytoplasm.