真核表达质粒pcDNA3.1(+)-Vasostatin的构建及其在293T细胞中的表达

Construction of Eukaryotic Expression Vector pcDNA3.1(+)-Vasostatin and Expression of Vasostatin in 293T Cells

目的构建真核表达质粒pcDNA3.1(+)-Vasostatin并检测其在真核细胞内的表达水平。方法将带有信号肽的Vasostatin基因片段克隆至pcDNA3.1(+)真核表达载体上,经酶切鉴定及测序分析证明构建成功后,以脂质体介导法转染293T细胞,通过Westernblot法,检测其在293T细胞内的表达水平。结果所构建的真核表达质粒pcDNA3.1(+)-Va-sostatin转染293T细胞后,在其裂解的上清液中,检测到目的基因的表达。结论已成功构建了pcDNA3.1(+)-Vasostatin表达载体,并在真核细胞中表达了目的蛋白。

Objective To construct eukaryotic expression vector pcDNA3.1（＋）-Vasostatin and express Vasostatin in 293T ceils. Methods Clone the Vasostatin gene fragment containing a signal peptide into eukaryotic expression vector pcDNA3.1 （＋）. Identify the constructed recombinant plasmid pcDNA3.1 （ ＋ ）-Vasostatin by restriction analysis and sequencing, then transfect 293T cells in the mediation of liposome. Identify the expressed product by Western blot. Results A eukaryotic expression vector pcDNA3.1 （ ＋ ）-Vasostatin was successfully constructed. Western blot proved the expression of Vasostatin in the supernatant of lysate of 293T cells transfected with the recombinant plasmid. Conclusion The constructed recombinant plasmid pcDNA3.1 （ ＋ ）-Vasostatin could be used for the expression of Vasostatin in eukaryotic cells.