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目的构建F盒蛋白6(FBXO6)基因真核表达载体。方法采用PCR方法合成含有EcoRⅠ和BglⅡ双酶切位点的FBXO6 cDNA全长。分别构建载体pEGFP-C1-FBXO6和pEGFP-C1-anti-FBXO6,采用菌落PCR、双酶切鉴定以及测序证实cDNA片段大小和序列正确。将载体pEGFP-C1-FBXO6和pEGFP-C1-anti-FBXO6分别转染HEK293T细胞,Western blot法检测FBXO6蛋白的表达。结果 pEGFP-C1-FBXO6和pEGFP-C1-anti-FBXO6包含大小、序列正确的FBXO6片段;FBXO6蛋白在转染pEGFP-C1-FBXO6的293T细胞中高表达;在转染pEGFP-C1-anti-FBXO6的HEK293T细胞中表达降低。结论成功构建FBXO6基因正义真核表达载体pEGFP-C1-FBXO6和反义真核表达载体pEGFP-C1-anti-FBXO6。
Objective To construct eukaryotic expression vector of F box protein 6 (FBXO6) gene. Methods The full length cDNA of FBXO6 containing EcoR Ⅰ and Bgl Ⅱ restriction sites was synthesized by PCR. The vector pEGFP-C1-FBXO6 and pEGFP-C1-anti-FBXO6 were constructed respectively. The colony PCR, restriction enzyme digestion and sequencing confirmed the correct size and sequence of the cDNA fragments. The vector pEGFP-C1-FBXO6 and pEGFP-C1-anti-FBXO6 were transfected into HEK293T cells respectively. The expression of FBXO6 protein was detected by Western blot. Results pEGFP-C1-FBXO6 and pEGFP-C1-anti-FBXO6 contained the correct size and sequence of FBXO6 fragments. FBXO6 protein was highly expressed in 293T cells transfected with pEGFP-C1-FBXO6. Decreased expression in HEK293T cells. Conclusion The eukaryotic expression vector pEGFP-C1-FBXO6 of FBXO6 gene and antisense eukaryotic expression vector pEGFP-C1-anti-FBXO6 were successfully constructed.