The Construction and Expression of Recombination Adenovirus Vector of G156A O6-methylguanine-DNA-methyltransferase.

Abstract

OBJECTIVE: To transfect G156A mutant MGMT gene into peripheral blood mononuclear cells(PBMCs), peripheral blood CD34+ cells, umbilical cord blood CD34+ cells and K562 cells through recombinant adenovirus vector and detect the expression of ΔMGMT in these cells, to study the protective effect of the ΔMGMT gene for bone marrow cells against alkylating agent. METHODS: We obtained a full-length cDNA fragment encoding human O6-methylguanine-DNA-methytransferase (MGMT) gene from human liver tissue by RT-PCR. ΔMGMT cDNA was made by site-directed mutagenesis. Positive clones were identified by diagnostic restriction analysis and sequenced to confirm the presence of the desired mutation. ΔMGMT cDNA was released by digestion with XhoI and BamHI, purified and then ligated into adenovirus expression vector pAd5CMV-NpA. The recombinant adenovirus vectors pAd5CMV-ΔMGMT and pAd5CMV-EGFP were constructed and transfected into the packing cell lines HEK293 cells by liposome encapsulation method. The recombination adenvirus were propagated, purified, and titrated to 1x1012pfu/mL. PBMCs, peripheral blood CD34+ cells, umbilical cord blood CD34+ cells and K562 cells were incubated with a mixture containing adenovirus. Fluorescence microscopy was employed to test the transfection of recombinant adenovirus vectors.The expression of ΔMGMT gene was detected by Real-time PCR and Western Blot and contrasted to the cells which were transfected into the recombinant adenovirus including EGFP. 3-(4,5-dimethyhhiazol-2-yl)-2,5-dipheyhetrazolium bromide (MTT) and CFU-assays were used to detect the drug-resistance of the ΔMGMT transfected cells. RESULTS: MGMT gene was cloned successfully and the mutant G156A MGMT genes were obtained. High titer recombinant adenovirus had been made. Real time-PCR showed that mutant drug resistance gene expressed strongly. Western-Blot verified that cells infected with Adv-EGFP displayed EGFP immunoreactivity and basal levels of MGMT immunoreactivity; in contrast, cells infected with Adv-ΔMGMT showed a strong ΔMGMT immunoreactivity. Fluorescence microscopy showed EGFP positive in control group. The mutant drug resistance gene target cells conferred 12.4 ∼ 18.6-fold stronger resistances to BCNU and O6-benzylguanine (BG) in MTT test than control group. CFU-assays showed the colony-forming units was more detected in transfected group compared with vacant vector group (P<0.05). CONCLUSIONS: The results suggested that transduction of the mutant G156A MGMT into hematopoietic progenitor cells mediated by recombinant adenovirus vectors may lead to their selective resistance to the combined use of O6-benzylguanine and alkylating agents, and this study provided an experimental foundation for improving combination chemotherapy tolerance in clinical practice.