重叠延伸PCR法构建肿瘤抑素核心活性部位T3肽.docVIP

精品论文 参考文献 重叠延伸PCR法构建肿瘤抑素核心活性部位T3肽 顾丽1 张君2 高静3 苏燕4(通讯作者) 　　（1包头医学院病原生物学教研室 内蒙古包头 014060） 　　（2民航总医院检验科 北京 100123） 　　（3包头医学院病原生物学实验室 内蒙古包头 014060） 　　(4包头医学院生物化学与分子生物学教研室 内蒙古包头 014060) 　　【摘要】 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DH5alpha;，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。 　　【关键词】肿瘤抑素 T3肽 重叠延伸PCR 　　【中图分类号】R318 【文献标识码】A 【文章编号】2095-1752（2014）03-0020-02 　　Overlap-extension PCR to construct the core activity site T3 peptide of tumstatin 　　GU Li1,ZHANG Jun2,GAO jing3,SU Yan3* (1.Department of pathogen Biology,Baotou Medical College,Inner Mongolia Baotou 014060;2.Laboratory of General Hospital of Civil Aviation, Beijing 100123;3.Pathogen Biology Laboratory of Baotou Medical College, Inner Mongolia Baotou 014060; 4.Department of biochemistry and molecular biology,Baotou Medical College, Inner Mongolia Baotou 014060, China) 　　【Abstract】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 DH5alpha;.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 success