一种新的融合表达载体的构建

Construction of a Novel Fusion Expression Vector

利用 L-ans B基因片段构建了融合表达载体 p EC,其中 L-ans B基因中编码唯一酸水解位点的序列已通过定点突变消除 ,并在它的 3′端引入了新的酸水解位点编码序列和方便外源基因插入的克隆位点 Bam H 和 Hind ,外源基因可从克隆位点 Bam H 和 Hind 处插入 p EC中 ,表达的融合蛋白用酸水解法加工后仅被切割成一大一小两个片段 ,不会导致产物混杂。 h GRF对它进行验证的结果显示 h GRF基因不仅能按正确的读码框插入 ,而且表达的融合蛋白与预期大小一致 ,说明 p

Based on the cloned L asparaginase ( L ansB) gene, a novel fusion expression vector pEC was constructed in which the codon of asp ala replaced that of the original acid sensitive site(asp pro) in L ansB gene, and the codon of a new acid sensitive site and the recognized sites of BamHⅠ, HindⅢ were introduced into the 3′ terminus of L ansB gene. The foreign gene can be inserted into pEC at BamHⅠ and HindⅢ, and the expressed fusion protein can be cleaved at the unique acid sensitive site between the fusion partner and the foreign peptide by acid. The human growth hormone releasing factor (hGRF) was taken as an example to test pEC. The hGRF gene was inserted into pEC with a correct reading frame and its fusion protein was also expressed successfully. The results indicated that pEC was effective to express fusion protein.