摘要：研究背景 前期研究发现线粒体转录因子A（mitochondrial transcription factor A,mtTFA）在甲基化放线菌素D诱导肝癌细胞凋亡的过程中表达下调，提示mtTFA可能在肿瘤的发生发展过程中起作用。目的 构建含有mtTFA基因的PEGFP-C1-mtTFA重组质粒并对重组质粒进行鉴定，用于筛选稳定表达的肿瘤细胞株，为进一步研究mtTFA的功能奠定基础。方法 用Trizol试剂提取人胚肾细胞的RNA，反转录为cDNA，PCR扩增得到mtTFA基因，上下游分别加入Xhol I和Hind III酶切位点，双酶切后将其插入PEGFP-C1质粒中，构建PEGFP-C1-mtTFA重组质粒，将PEGFP-C1-mtTFA转化DH5α大肠杆菌进行克隆，质粒提取，酶切和测序鉴定。结果 重组质粒经限制性内切酶Xhol I和Hind III双酶切鉴定及DNA测序鉴定均证实mtTFA基因已正确克隆到PEGFP-C1载体中。结论 成功并正确构建PEGFP-C1-mtTFA重组质粒。51857

毕业论文关键词：mtTFA、PEGFP-C1、重组质粒

Construction and identification of mitochondrial transcription factor A over-expression vector PEGFP-C1-mtTFA

Abstract: Background Previous study shows that mtTFA was downregulated in HepG2 cells treated by mActD. It was suggested that mtTFA may activiated in the development of cancer. Objective To construct PEGFP-C1-mtTFA recombinant plasmid and identify it, for the further screening the tumor cell lines of stable expression. Methods Total RNA was extracted from AD-293 cells using Trizol reagent and was reversely transcribed into cDNA. mtTFA gene was amplified from the cDNA by polymerase chain reaction. Upsteam and downstream promers were introduced into Xhol I and Hind III restriction sites, then the fragments were digested by double enzyme and then inserted into PEGFP-C1 plasmid to construct PEGFP-C1-mtTFA recombinant plasmid. The recombinant plasmid were transformed into E.coli DH5α for cloning, then the plasmind was extracted and identified by restriction enzyme digestion and sequencing.  Results Xhol I and Hind III double digestion and DNA sequencing results confirmed that mtTFA gene had been correctly cloned into PEGFP-C1 vector. Conclusion PEGFP-C1-mtTFA recombinant plasmid was successfully and correctly constructed. 

Key words: mtTFA; PEGFP-C1; recombinant plasmid

 目 录

前言 1

1 材料与仪器 2

1.1 主要试剂 2

1.2 主要仪器 2

1.3 试验方法 2

1.3.1 RNA提取 2

1.3.2反转录（RNA-DNA） 3

1.3.3 PCR检测扩增mtTFA基因 3

1.3.3.1引物的设计 3

1.3.3.2 PCR扩增 3

1.3.3.3琼脂糖凝胶电泳 4

1.4 胶回收 4

1.5 酶切连接 4

1.5.1 Xhol I和Hind III双酶切质粒PEGF-C1和mtTFA基因 5

1.5.2纯化的双酶切产物进行连接 5

1.5.3 连接产物转化DH5.感受态细胞 5

1.5.3.1感受态细胞的制备 5

1.5.3.2连接质粒转化 5

1.6重组质粒的鉴定 6

1.6.1 PCR鉴定 6

1.6.2 酶切鉴定 6

1.6.3测序鉴定 6

2 结果与分析