【作者】 饶泽昌；

【导师】 李文林；

【作者基本信息】 南昌大学 ， 细胞生物学， 2008， 硕士

【摘要】 目的:研究PNP基因的克隆、真核表达载体的构建与鉴定,为肿瘤的基因治疗与基因工程的应用提供实验和理论依据。方法:①大肠埃希氏杆菌K12 PNP基因的PCR扩增;②重组质粒pMSCV-PNP的构建;③用氯化钙法制备感受态大肠杆菌(XL1-Blue);④转化感受态菌:用携带目的基因的重组质粒pMSCV-PNP转化感受态XL1-Blue大肠杆菌,同时设立阳性对照组和阴性对照组。转移适量转化菌于平皿培养,筛选氨苄青霉素抗性转化菌落;⑤提取质粒和酶切重组质粒:提取重组质粒pMSCV-PNP,限制性内切酶EcoRⅠ和BglⅡ双酶切重组质粒,电泳鉴定;⑥PCR鉴定、测序鉴定重组质粒pMSCV-PNP;⑦提取质粒和酶切质粒:限制性内切酶BspHⅠ、BglⅡ、AflⅡ分别酶切pVSV-G,pGAG-POL,pMSCV,电泳鉴定质粒。结果:①PNP基因PCR扩增出特异条带;②重组质粒pMSCV-PNP电泳结果与预期一致;③重组质粒pMSCV-PNP转化结果:实验组平皿上可见近百个菌落,阳性对照组有数百个菌落生长,而阴性对照组则未见菌落生长;④阳性克隆质粒pMSCV-PNP PCR鉴定、酶切鉴定,电泳条带与预期一致;测序结果正常;⑤克隆质粒pVSV-G,pGAG-POL,pMSCV酶切鉴定与预期一致。结论:①从大肠埃希氏杆菌K12中克隆到PNP基因;②构建了表达载体pMSCV-PNP;③抽提了三种载体质粒pMSCV、pVSV-G、pGAG-POL。更多还原

【Abstract】 Objective: To construct and identify the recombinant vector pMSCV-PNP carrying E.coli K12 PNP which can express in eukaryote cells and which will provide the basis for gene therapy.Methods:①PNP were amplified from E.coli K12 bacteria by polymerase chain reaction (PCR).②To construct the recombinant vector pMSCV-PNP by means of T4-DNA ligase.③Competent E. coli (strain XL1-Blue) was prepared using calcium chloroid.④Transformation of the competent bacteria: We transformed the competent bacteria with the plasmids pVSV-G, pGAG-POL, pMSCV and pMSCV-PNP containing PNP gene. The appropriate volume of the transformed bacteria was transfered and spreaded onto agar LB medium to select the ampicillin-resistant colonies.⑤Extraction and restriction endonuclease analysis of the plasmids: The plasmids pVSV-G, pGAG-POL, pMSCV, pMSCV-PNP were extracted from the transformed bacteria with plasmid purification kit, and they were cut with restriction endonucleases BspHⅠ、BglⅡ、AflⅡ, EcoRⅠand BglП, respectively.Results:①Ampicillin resistant tranformed bacteria colonies growth: After transformation with pMSCV , pMSCV-PNP, pGAG-POL and pVSV-G, about 100 colonies were observed on the plate containing ampicillin, and the colonies were not found in the negative control groups.②The normal size of the fragments from the plasmids cut by endonucleases: The results of restriction endonuclease analysis and agar electrophoresis indicated that size of plasmids pMSCV, pMSCV-PNP, pGAG-POL and pVSV-G was normal.Conclusion: E.coli K12 PNP can be successfully cloned and inserted into expression vector. The newly constructed vector may serve as the potential tool to conduct further comprehensive experiments in future on PNP function and on gene therapy.更多还原