A new expression plasmid in Bifidobacterium longum as a delivery system of endostatin for cancer gene therapy

Abstract

To utilize Bifidobacterium longum (B. longum) as a safe and stable delivery system for endostatin in cancer gene therapy, we constructed pBV22210 vector combining a chloramphenicol-resistance gene (Cm r ) from pBCSK( þ ) plasmid, a cryptic plasmid pMB1 from B. longum strain with pBV222. Endostatin was cloned directly downstream of an N terminal His6-tag sequence in the pBV22210, so that the endostatin protein expressed in B. longum could be purified with Ni-binding resin. The results indicated that the plasmid electroporated into B. longum was maintained stably in the absence of selective antibiotics and did not significantly affect biological characteristics of B. longum. In addition, the plasmid in B. longum showed a strong inhibitory effect on the growth of mouse solid liver tumor in vivo. These results suggested that this new plasmid may be a stable vector in B. longum for transporting anti-cancer genes in cancer gene therapy.

Gene therapy for cancer: bacteria-mediated anti-angiogenesis therapy

“…In another study, Bifidobacterium longum was shown to efficiently deliver the anti-angiogenic protein endostatin to murine liver tumors and induce anti-tumor activity. 46,47 Furthermore, the anti-tumor effect was enhanced by co-administration of the same bacterial strain expressing tumor necrosis factor-related apoptosis inducing ligand. 48 These findings emphasize the need for combination therapy, in which multiple anti-tumor pathways are inhibited.…”

Bifidobacterium longum as a delivery system of TRAIL and endostatin cooperates with chemotherapeutic drugs to inhibit hypoxic tumor growth

“…26 Fu et al 28 used transformed B. longum carrying shuttle vector pBV220 (Amp þ ) encoding human endostatin as a delivery system and succeeded in selective localization within solid tumors and inhibiting growth of liver tumor xenografts in mice. However, ampicillin is considered to be harmful to bacterial cytoderm synthesis after electroporation, Xu et al 30 constructed a new vector pBV22210-endostatin combining a chloramphenicol resistance gene and a cryptic plasmid pMB1 from WT B. longum strain that made the transformed B. longum more stable, and the expressed recombinant protein from B. longumpBV22210-endostatin exhibited stronger suppression of tumor growth in xenografts models than that from B. longum-pBV220-endostatin in a previous study. Based on the previous work, we further constructed a new plasmid B. longum-pBV22210-TRAIL that encoded the extracellular domain of TRAIL ( Figure 2) and expressed recombinant human TRAIL in B. longum successfully (data not shown) in this study.…”

“…The ligation solution was transferred into competent cells of DH5a and pBV22210-TRAIL was detected on LB agar plate with 10 mg ml À1 chloramphenicol ( Figure 1). 30 Electroporation Electrocompetent cells of B. longum were prepared according to Rossi et al 34 Bacteria were resuspended in about 1/100 of the original culture volume of ice-cold 0.5 M sucrose plus 1 mM ammonium citrate (pH 6.0); 1.0 mg of purified pBV22210-TRAIL plasmid was added in bacteria suspension and incubated at 4 1C for 2-3 h. This mixture was added in a precooled sterile Gene Pulser disposable cuvette (interelectrode distance 0.2 cm; Bio-Rad, Hercules, CA). The pBV22210-TRAIL plasmids were transfected directly into B. longum by electroporation in a Bio-Rad Gene-Pulser apparatus at 25 mF and 2.5 kV with the pulse controller set at 200 O.…”

“…B. longum transfected with pBV22210-endostatin showed a clear inhibitory effect on the growth of mouse solid liver tumor in vivo. 30 Taking advantage of the stability of pBV22210 in B. longum, the tumor sitespecific localization of B. longum and the induction of apoptosis in cancer cells by TRAIL, we reported here the cloning of the extracellular domain of TRAIL (amino acid 114-281) to the downstream of His-tag of pBV22210, the transfection of B. longum, and the characterization of the transfectants. Antitumor activity of B. longumpBV22210-TRAIL combining with chemotherapeutic drugs or B. longum-pBV22210-endostatin in xenograft models of S180 osteosarcoma was examined.…”

“…Though some Bifidobacterium-Escherichia coli shuttle vectors have been constructed in recent years, only some of them expressed foreign genes successfully. [23][24][25][26][27][28][29][30][31][32][33] In our previous studies, we developed a shuttle vector (pBV22210), which could replicate stably and expressed recombinant human endostatin in both E. coli and B. longum. B. longum transfected with pBV22210-endostatin showed a clear inhibitory effect on the growth of mouse solid liver tumor in vivo.…”