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目的:构建pNTAP-PRAK真核表达质粒,并建立其稳定表达的HEK293细胞系。方法:将人的PRAK亚克隆至串联亲和纯化(tandem affinity purification,TAP)载体pNTAP质粒上,构建成重组质粒pNTAP-PRAK,转化该重组质粒至感受态大肠杆菌DH5α,阳性克隆进行PCR、酶切及DNA测序验证正确后,利用PolyFect脂质体介导将其转染至HEK293细胞中,再通过G418筛选建立稳定表达TAP tag-PRAK融合蛋白的HEK293细胞系;利用Western blotting和细胞免疫荧光标记法检测融合蛋白TAP tag-PRAK的表达及细胞内定位情况。结果:重组真核表达载体构建正确,该重组质粒能在HEK293细胞中稳定表达,表达产物TAP tag-PRAK主要分布在核内。结论:成功构建pNTAP-PRAK真核表达载体并建立了其稳定表达的HEK293细胞系,TAP标签未对PRAK定位产生明显影响。
Objective: To construct eukaryotic expression plasmid pNTAP-PRAK and establish HEK293 cell line stably expressing it. Methods: Human PRAK was subcloned into pNTAP vector of tandem affinity purification (TAP) vector and constructed into recombinant plasmid pNTAP-PRAK. The recombinant plasmid was transformed into competent E. coli DH5α and the positive clones were subjected to PCR. The enzyme After sequencing and DNA sequencing, the HEK293 cells were transfected with PolyFect liposome and then HEK293 cell line stably expressing TAP tag-PRAK fusion protein was selected by G418 screening. Western blotting and immunofluorescence staining Method to detect the expression of fusion protein TAP tag-PRAK and intracellular localization. Results: The recombinant eukaryotic expression vector was constructed correctly. The recombinant plasmid was stably expressed in HEK293 cells. The expression product TAP tag-PRAK mainly distributed in the nucleus. CONCLUSION: The eukaryotic expression vector pNTAP-PRAK was successfully constructed and its stable expression was established in HEK293 cell line. TAP tag did not affect PRAK localization significantly.