Src真核表达载体的构建与表达

构建酪氨酸蛋白激酶Src真核表达载体,并检测其在HEK293T细胞中的表达.根据Genebank（GI：4574718）提供的大鼠Src的cDNA序列设计特异性引物,以大鼠海马总RNA为模板,采用RT-PCR方法扩增Src的目的基因,然后克隆至真核表达载体pcDNA3.1.将筛选的阳性重组子转染入HEK293T细胞系,免疫印迹检测Src的蛋白表达水平,经限制性内切酶双酶切及测序分析,扩增的Src的cDNA成功连接入pcDNA3.1,并且序列与Genebank中一致.免疫印迹结果表明,构建的Src-pcDNA3.1可在HEK293T细胞中表达.成功构建了Src-pcDNA3.1真核表达载体,并且可在HEK293T细胞中高效表达.

Construction and Expression of Eukaryotic Expression Vector for Src

To construct eukaryotic expression vector for Src tyrosine protein kinase, and to detect its expression in HEK293T cells were studied in this paper. According to the Src cDNA sequence （Genebank, G1：4574718）, the Src cDNAs by RT- PCR from extracted rat RNA were obtained, and cloned the fragment into the eukaryotic expression vector pcDNA3. 1. The positive recombinants were then transfected into HEK293T cells, and western blot was sent to analyze the protein expression of Src. Results Restriction endonuclease double digestion and sequencing analysis indicate that the amplified Src cDNAs were successfully cloned into pcDNA3.1, and were in consistent with the sequence in Genebank. Western blot analysis showed that the Src - pcDNA3.1 construct were expressed in HEK293T cells. Conclusions Src - pcDNA3.1 eukaryotic expression vector was successfully constructed and was highly expressed in HEK293T cells.