Construction And Identification Of Bifidobacterium As A Delivery System Of IL-12 And IP-10

Objective: Construction and identification of Bifidobacterium as a debvery system of IL-12 and IP-10.These suggest that Bifidobacterium could be used as a new highly specific delivery vector for anticancer gene therapy.Methods: A enhanced selective medium for bifidobacteria ( signature BL) has been developed on basis of the bifidobacteria selective medium such as MRS.A bifidobacterium strain with a resident plasmid was isolate.An expression vector for Bifidobacterium longium was constructed with the E.coli plasmid pBAD-A and pBS-T as the start-piont.The BPP,ara and GFP gene were introduced into a shuttle vector of pBS-BPP-ara-GFP.IL-12 gene cloned from plasmid pORF-hIL-12 by PCR was introduced into the shuttle vector of pBS-BPP-ara-GFPIP-10 gene cloned from plasmid pBLAST49-hIP-10 by PCR was introduced into the shuttle vector of pBS-BPP-ara-GFP.The shuttle vector was transformed into B.longium by electroporation and introduce to expression by L-arabinose.The protein expression of the GFP was observed by using laser scanning conocal microscopy (LSCM).The protein expression of IL-12 and IP-10 was detected by using western blot. Results: (l)We isolated the BPP and ara gene of a Bifidobacterium strains by PCR. The BPP and ara gene has been sequenced and confirmed as DNA.The relative molecular weight of BPP gene is 1963 bp.The relative molecular weight of ara gene is 429 bp.Analysis of the nucleotide sequence showed a high alignment with the same gene in GenBank(96%).The Bifidobacterium strain was identified as B.longium according to its carbohydrate fermentation trials.(2) Green fluorescence was observed by using laser scanning conocal microscopy (LSCM) in the GFP engineered Bifidobacterium.(3)A 58.38 Kilo-Dolton protein band was detected by western blot in the IL-12 engineered Bifidobacterium.(4) A 8.47 Kilo-Dolton protein band was detected by western blot in the IL-12 engineered Bifidobacterium. Conclusion: (1) The BPP gene and ara gene was isolated by PCR in a B.longium strain.(2) The result of Western blot revealed that the active GFP was expressed in B.longium.(3) The result of Western blot revealed that the active IL-12 was expressed...