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.
Bifidobacterium Infantis - cytosine deaminase - gene therapy