论文部分内容阅读
目的：利用重叠延伸PCR技术，构建肿瘤抑素核心活性部位T3肽。方法根据T3肽cDNA序列，人工设计合成三段引物，采用重叠延伸PCR技术扩增目的基因片段T3，非变性聚丙烯酰胺凝胶电泳对其进行分离后，纯化回收扩增产物。将纯化回收的T3片段克隆至T载体，转化大肠杆菌D H5α，选取阳性克隆，提取质粒，双酶切电泳鉴定后进行DNA序列测定。结果琼脂糖凝胶电泳结果显示PCR扩增获得预期大小的目的基因。质粒测序显示目的基因序列与GenBank中记载的cDNA序列完全一致。结论成功构建T3肽基因序列。“,”Objective To construct the core activity site T3 peptide of tumstatin by employing the overlap extension PCR Methods According to the cDNA sequence of T3 peptide, designed and synthesized three primers to amplify the target gene fragment T3 using overlap-extension PCR technique.The amplification products were separated and purified by means of non denaturing polyacrylamide gel electrophoresis. The purified T3 fragment was inserted into T vector, then transformed it into Escherichia coli DH5α.The plasmid was extracted from positive clones, and then identified by double digestion.This recombinant plasmid containing the target gene was sequenced. Results Agarose gel electrophoresis showed that the expected size of gene fragment was amplified. Plasmid DNA sequencing showed that the target gene sequence was the same as the cDNA sequence recorded in GenBank. Conclusion The gene sequence of T3 peptide was successful constructed.