Adenovirus 5 type E1A as a tumor suppressor gene can inhibit tumor growth and enhance the sensitivity of chemotherapy and radiotherapy. E1A have the ability to integrate into the host genome, resulting in long-time ex...Adenovirus 5 type E1A as a tumor suppressor gene can inhibit tumor growth and enhance the sensitivity of chemotherapy and radiotherapy. E1A have the ability to integrate into the host genome, resulting in long-time expres-sion that induces Rb gene inactivation and animal cells im-mortalization. This prompted us to select the E1A protein for treatment of cancer in order to overcome the limitations of E1A gene therapy. Thus, we firstly constructed E1A eu-caryotic expression vector (pPIC9/E1A), transformated the pichia pastoris yeast cells (GS115) and screened the high-expressing recombinant strains. The positive yeast strains were cultured in the shake flask, and induced for 3 d. The crude E1A protein was purified using two steps of col-umn chromatography on HiTrap Q and HiTrap SP. The pu-rified E1A protein was identified by SDS-PAGE and Western blot. E1A protein was mostly located at cellular nuclear when Chariot delivered E1A protein into cells. The analysis in vitro indicated that the E1A protein arrested LN686 cell cycle at G2/M phase, and significantly inhibited the growth of LN686 tumor cells. The current studies firstly provided an experimental basis to further develop E1A protein for tumor treatment.展开更多
The transcription factor nuclear factor kB (NF-kB) plays a key role in the delayed xenograft rejection (DXR). One of the important objects in the field is how to inhibit the NF-kB activity at optimal level. Thus, a mo...The transcription factor nuclear factor kB (NF-kB) plays a key role in the delayed xenograft rejection (DXR). One of the important objects in the field is how to inhibit the NF-kB activity at optimal level. Thus, a modified E1A gene (E1AD) containing function domain (1—80 aa) and nuclear localization domain (139—243 aa) was used and cloned into an eucaryotic expression vector pcDNA3 to transfect the porcine aortic endothelial cells (PAEC). The stable transfectants were screened with G418. E1AD gene was able to be stably expressed in the PAEC and could not affect the growth of PAEC as analyzed by RT-PCR and cell growth rate. Reporter gene assay demonstrated that E1AD was capable of inhibiting NF-kB activity in the PAEC in-duced by TNF-a without sensitizing to apoptosis, and the rate of inhibition was 53%. Furthermore, E1AD inhibited the expression of a NF-kBdependent inflammatory gene E-selectin in the cells, and the rate of inhibition was 63%. In summary, the usage of E1AD gene may be a new strategy to overcome DXR in the xenotransplantation.展开更多
文摘Adenovirus 5 type E1A as a tumor suppressor gene can inhibit tumor growth and enhance the sensitivity of chemotherapy and radiotherapy. E1A have the ability to integrate into the host genome, resulting in long-time expres-sion that induces Rb gene inactivation and animal cells im-mortalization. This prompted us to select the E1A protein for treatment of cancer in order to overcome the limitations of E1A gene therapy. Thus, we firstly constructed E1A eu-caryotic expression vector (pPIC9/E1A), transformated the pichia pastoris yeast cells (GS115) and screened the high-expressing recombinant strains. The positive yeast strains were cultured in the shake flask, and induced for 3 d. The crude E1A protein was purified using two steps of col-umn chromatography on HiTrap Q and HiTrap SP. The pu-rified E1A protein was identified by SDS-PAGE and Western blot. E1A protein was mostly located at cellular nuclear when Chariot delivered E1A protein into cells. The analysis in vitro indicated that the E1A protein arrested LN686 cell cycle at G2/M phase, and significantly inhibited the growth of LN686 tumor cells. The current studies firstly provided an experimental basis to further develop E1A protein for tumor treatment.
基金This work was supported by Beijing "248"High-Tech Program China.
文摘The transcription factor nuclear factor kB (NF-kB) plays a key role in the delayed xenograft rejection (DXR). One of the important objects in the field is how to inhibit the NF-kB activity at optimal level. Thus, a modified E1A gene (E1AD) containing function domain (1—80 aa) and nuclear localization domain (139—243 aa) was used and cloned into an eucaryotic expression vector pcDNA3 to transfect the porcine aortic endothelial cells (PAEC). The stable transfectants were screened with G418. E1AD gene was able to be stably expressed in the PAEC and could not affect the growth of PAEC as analyzed by RT-PCR and cell growth rate. Reporter gene assay demonstrated that E1AD was capable of inhibiting NF-kB activity in the PAEC in-duced by TNF-a without sensitizing to apoptosis, and the rate of inhibition was 53%. Furthermore, E1AD inhibited the expression of a NF-kBdependent inflammatory gene E-selectin in the cells, and the rate of inhibition was 63%. In summary, the usage of E1AD gene may be a new strategy to overcome DXR in the xenotransplantation.
