Construction of Escherichia coli-Bifidobacterium longum shuttle vector and expression of tumor suppressor gene PTEN in B. longum
大肠杆菌-长双歧杆菌穿梭载体的构建及PTEN在长双歧杆菌中的表达

It was reported that Bifidobacterium longum accumulated specifically in hypoxic solid tumors, therefore could be used as a delivery system for cancer-specific gene therapy. Furthermore, construction of E.coli-B. longum shuttle vectors was proved by other research to be an efficient way for stable gene expression in B. longum. To obtain a shuttle vector and analyze the inhibition on mice solid tumors by genetically engineered B. longum, 48 primers with mutual overlaps were designed, assisted by software package Oligo 6.0. By PCR with the above primers, a linear plasmid was synthesized, which consists of pMB1 and HU gene promoter, both from B. longum. pMB-HU was constructed by cloning the synthesized linear plasmid into E.coli vector pMD 18-T, and was proved to be stably replicated in both E.coli DH5alpha and B. longum L17. By inserting PTEN cDNA into pMB-HU, expression vector pMB-HU-PTEN was obtained, in which PTEN gene was reported as a major tumor suppressor gene encoding a dual-specificity phosphatase. pMB-HU-PTEN was then transferred into B. longum L17 by electroporation. After transformation, an effective expression of PTEN in B. longum L17 was confirmed by Western blot, and significant inhibition on growth of mice solid tumors was also observed with the above genetically engineered B. longum. Those obtained results have laid foundation for tumor-targeting gene therapy with B. longum.