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Construction of conditionally replicative adenovirus vector mediated by dual specific promoters

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Author:
No author available
Journal Title:
Chinese Journal of Biomedical Engineering
Issue:
2
DOI:
10.3760/cma.j.issn.1674-1927.2013.02.010
Key Word:
条件复制腺病毒;人钠碘转染体基因;人端粒酶反转录酶启动子;胶质纤维酸性蛋白启动子;腺病毒E1A基因;Conditionally replicative adenovirus;Human sodium iodide symporter gene;Human telomerase reverse transcriptase promoter;Glial fibrillary acidic protein promoter;AdenovirusE1A gene

Abstract: Objective To construct and identify the conditionally replicative adenovirus vectors which may induce the human sodium iodide symporter (hNIS) expression in the early region 1A (E1A) gene of the human telomerase reverse transcriptase (hTERT) and the glial fibrillary acidic protein (GFAP)promoter regions.Methods Immunoblotting assay was employed to detect the variation in GFAP and telomerase protein expression prior to and following viral infection of the cerebral stellar glioblastoma cells (U87),neuroglioma cells (U251) and human embryonic lung fibroblasts (MRC-5) cells.The hTERT and GFAP promoters and the hNIS genes were amplified by polymerase chain reaction for synthesis of adenoviral E1A genes.The recombinant plasmids containing hTERT and GFAP gene promoters were adopted for transfection into MRC-5,U251 and U87,which entailed assessment of the hTERT and GFAP promoter activity via fluorescent analysis after 24 h.This was followed by ligation with the E1A and hNIS genes and subsequent cloning into the plasmid pDC311 for construction of the recombinant plasmid pDC311-Tp-E1A-Gp-NIS that was identified by double enzyme digestion (EcoR Ⅰ and Sal Ⅰ) and gene sequencing.This recombinant plasmid was co-transfected with adenoviral genomic plasmid pBHGlox△E1-3Cre into the human embryonic kidney 293 cells forming the conditionally replicative recombinant adenovirus Ad-Tp-E1A-Gp-NIS.This Ad-Tp-E1A-Gp-NIS was employed to transfect the 293,MRC-5,U87 and U251 cells,whose conditional replicability was measured by plaque forming assay.The recombinant virus Ad-Tp-E1A-Gp-NIS and Ad-CMV-EGFP controls were used to transfect the U251,U87 and MRC-5 cells for detection of the capacity of 125I uptake by using a γ-ray counter.Results The expression of the 120 000 telomerase and 49 000 GFAP protein could be found in U87 and U251 cells,but not in MRC-5 cells.Glioma target gene expression could be induced by the GFAP and hTERT promoters,the efficiency of which was (62.10±6.26)% and(49.24± 1.73)% in U87 cells,and (59.75±34.36)% and (37.31± 16.86)% in U251 cells.The plasmid pDC311-Tp-E1A-Gp-NIS was digested to 3252 bp and 5500 bp fragments by restriction endonuclease and identified as having the identical sequence before design.The successfully constructed Ad-Tp-E1A-Gp-NIS,a conditionally replicative adenovirus vector,satisfactorily generated considerable viral particles in U87 and U251 cells,could also be achieved replication in 293 cells,but not in MRC-5 cells despite that replication proceeded.Following transfection with Ad-Tp-E1A-Gp-NIS,the U87 and U251 cells showed approximately 93.4 and 107.1 times of 125I uptake of the control cells [(60 679.42±635.61) cpm/100 mg vs (656.67±9.25)cpm/100 mg,and (69 593.10± 1842.89) cpm/100 mg vs (660.63± 16.01) cpm/100 mg,both P<0.05].However,this was not seen in MRC-5 cells [(649.67±13.66) cpm/100 mg vs (649.67±13.66) cpm/100 mg,P>0.05].Conclusion The constructed conditionally replicative adenovirus Ad-Tp-E1A-Gp-NIS has a preferable capacity of selective replication and may uptake the 125I that iodine radiotherapy entails.

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