The Clone Of PNP Gene And The Construction & Identification Of Its Expressive Vector

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.