| Oltvai et al identified an associated 21 kD protein partner, Bax, that has extensive amino acid homology with Bcl-2 in 1993. Bax homodimerizes and forms heterodimers with Bcl-2 in vivo. Overexpressed Bax accelerates apoptotic death and also counters the death represser activity of Bcl-2. The ratio of Bcl-2 to Bax determines survival or death following an apoptotic stimulus. There are high expression of Bcl-2 and relatively low expression of Bax in glioma. There is relation between unbalance of ratio of Bcl-2 to Bax and histological grade of glioma, capacity of metastasis of glioma, growth rate of glioma, relapse of glioma as well as response of glioma to chemotherapy and radiotherapy. If we can enhance the expression of bax gene in glioma, we'll promote the apoptosis of glioma and enhance the response of glioma to chemotherapy and radiotherapy. Human adenovirus is a kind of double strains DNA (dsDNA) virus. The adenoviral genome exists as an episome in the host cell's neucleus, dose not integrate into host cell's genome, and rarely causes host's gene insert mutation. As a vector, Adenovirus has the advantages of easy operation, easy propagation, wide variety of host cell spectrum, noncell-cycle dependent infectious ability, capacity of ligating large foreign DNA to adenoviral vector. Replication-incompetent(AEl/AE3) human adenovirus is one of the best vector at present because it neither replicate nor actively transcribe in non-package cell science the cell lacks the necessary transcription factors-the El genes. We made replication-incompetentadenovirus as a vector, human cytomegalovirus immediate early promoter as a mammalian expression cassette promoter, Baxa gene as therapeutic gene, to recombine a Bax a -pShuttle-Adeno recombinant adenoviral expression vector. We demonstrated that the recombinant adenoviral expression vector has a high activity of transcription and expression of gene inserted. We also found that the expression of Bax precede the replication and package of adenovirus and overexpression of Bax is cytotoxic in HEK293 cell line, which would impede the propagation of recombinant adenoviral expression vector in sensitive packaging cells-HEK293 by killing the HEK293 cells. This implies that we can replace a controllable promoter to MCV IE promoter, which do not express downstream Bax gene or lowly express in HEK293 cells in order to avoiding its toxicity to HEK293 cell. This make recombinant Bax-spshuttle-adeno expression vector propagate itself enough to proceed the gene therapy to glioma.Experimental materialsFull sequence of Baxa gene was donated by Pro. Jie Pan, College of Life Sciences, Zhejiang University. E coli-PGl was obtained from Biochemical Institute of College of Life Sciences, Zhejiang University. Adeno-X鈩?expression system was bought from BD Biosciences Company, America. HEK293 cell line was bought from Institute of Biochemistry and Cell Biology, SIBS, CAS, China. DMEM, 1640 Medium, and FBS were bought form Introgen Co., America. DOSPER Liposomal Transfection Reagent was bought from Roche Co., Switzeland. MTT and X-gal reagent were bought from Amresco Inc., America. Western Blotting Luminal Reagent was bought from Santa Cruz Biotechnology Co., America. Peroxidase-Conjugated Goat Anti-Mouse IgG was bought from Beijing Zhongshan Biotechnology Co. Ltd., China. Pac I was bought from New England Biotechnology Co., America. Xba I , Kpn I , BamH I , Pst I , DNA Marker DL2000 and DNA Marker DL15000 were bought from TaKaRa Biotechnology(Dalian) Co.Ltd.,China.Experimental MethodsCulture HEK293 cells in high glucose DMEM supplemented with 100 units/ml penicillin G sodium, 100 g/ml streptomycin, and 10% fetal bovine serum in plastic flasks under optimum growth conditions(37 ,5% CO2). Split every 3 days when they reach about 80% confluency. Double-digest pShuttle plasmid DNA with Xba I and Kpn I . Combine the Xba I / Kpn I -digested pShuttle plasmid DNA with the Baxa gene donated by Pro. Jei Pan. Identify putative recombinant Baxa-pShuttle DNA by BamH I or Pst I restrict... |
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