SYAP1真核表达载体的构建及蛋白表达定位

摘要

Aim: To construct eukaryotic expression vector pcDNA 3.1( -)-Myc-SYAP1.Methods: SYAP1 se-quence was synthesized , joined with Myc-tag sequence and EcoRⅠ,HindⅢrestriction sites, and then connected with lin-earized pcDNA3.1(-) .After the identification by the results of colony PCR , restriction analysis and sequencing ,the plas-mid was transfected into HEK 293 cells by lipid transfection , Western blot and indirect immunofluorescence were applied to observe the expression level and position of SYAP 1 protein in cells .Results:The results of colony PCR , sequencing and restriction enzyme digestion showed that the target nucleotide sequences were successfully inserted into the expected sites of the vector.Western blot showed that SYAP1 protein was successfully expressed in transfected HEK 293 cells,and lind indi-rect immunofluorescence result showed that SYAP 1 protein located in cytoplasm .Conclusion:Recombinant vector of pcD-NA3.1(-)-Myc-SYAP1 has been successfully constructed .%目的：构建pcDNA3．1（-）-Myc-突触相关蛋白1（SYAP1）真核表达载体。方法：合成SYAP1基因序列，加入Myc标签序列、EcoR Ⅰ和Hind Ⅲ酶切位点，并与线性化pcDNA3．1（-）连接，得重组质粒pcDNA3．1（-）-Myc-SYAP1。重组质粒经菌落PCR、酶切及测序鉴定正确后，脂质体转染HEK293细胞，采用Western blot和间接免疫荧光法检测转染细胞中SYAP1蛋白的表达及定位情况。结果：Myc-SYAP1成功插入 pcDNA3．1（-）载体， SYAP1蛋白成功表达且定位于胞浆。结论：pcDNA3．1（-）-Myc-SYAP1载体构建成功。