婴儿双歧杆菌/胞嘧啶脱氨酶肿瘤靶向性基因治疗系统的构建

Objective： To construct Bifidobacterium Infantis/CD targeting gene therapy system. Methods： CD gene was amplified from E. Coli K12λ using PCR method, pGEX-1LamdaT plasmid and CD gene were digested with dual restriction endonucleas of EcoR Ⅰ and BamH Ⅰ and two segments of 4.9 kb and 1.3 kb were obtained. T4 DNA ligase was added to these two segments to make a recombinant CD/pGEX-1LamdaT plasmid. Then the recombinant plasmid was transfected into Bifidobacterium Infantis by electroporation. The recombinant plasmid was extracted from the positively transfected Bifidobacterium Infantis and digested with dual restriction endonucleases. Then the size of digested fragments was detected and sequencing of the gene segment inserted in extracted recombinant plasmid was performed according to the method of Sanger dideoxynucleotide triphosphate chain termination. Results： 6.2 kb recombinant plasmid was obtained from the positively transfected bacterial colony of Bifidobacterium Infantis. After being digested with dual restriction endonucleases, two segments of approximate 4.9 kb and 1.3 kb were gained from the extracted recombinant plasmid, which were equal to the size of pGEX-1LamdaT plasmid and CD gene, respectively. The full length and sequence of nucleotide acid of the inserted gene in extracted recombinant plasmid was completely identical to the CD gene. Conclusion： The foreign gene, CD gene was correctly inserted into pGEX-1LambdaT plasmid and transferred into Bifidobacterium Infantis. Bifidobacterium Infantis/CD targeting gene therapy system was successfully constructed.

Construction of Bifidobacterium Infantis/CD Targeting GeneTherapy System

Objective: To construct Bifidobacterium Infantis/CD targeting gene therapy system. Methods:CD gene was amplified from E. Coli K12λ using PCR method. pGEX-1LamdaT plasmid and CD gene weredigested with dual restriction endonucleas of EcoR I and BamH I and two segments of 4.9 kb and 1.3 kb wereobtained. T4 DNA ligase was added to these two segments to make a recombinant CD/pGEX-1LamdaTplasmid. Then the recombinant plasmid was transfected into Bifidobacterium Infantis by electroporation.The recombinant plasmid was extracted from the positively transfected Bifidobacterium Infantis and digestedwith dual restriction endonucleases. Then the size of digested fragments was detected and sequencingof the gene segment inserted in extracted recombinant plasmid was performed according to the methodof Sanger dideoxynucleotide triphosphate chain termination. Results: 6.2 kb recombinant plasmid wasobtained from the positively transfected bacterial colony of Bifidobacterium Infantis. After being digestedwith dual restriction endonucleases, two segments of approximate 4.9 kb and 1.3 kb were gained from theextracted recombinant plasmid, which were equal to the size of pGEX-1LamdaT plasmid and CD gene,respectively. The full length and sequence of nucleotide acid of the inserted gene in extracted recombinantplasmid was completely identical to the CD gene. Conclusion: The foreign gene, CD gene was correctlyinserted into pGEX-1LambdaT plasmid and transferred into Bifidobacterium Infantis. BifidobacteriumInfantis/CD targeting gene therapy system was successfully constructed.