Construction and target gene expression of recombinant adenovirus vectors containing vascular endothelial growth factor 165 and angiopoietin-1
- VernacularTitle:携带人VEGF_(165)和ANG-1双基因的腺病毒载体的构建及目的基因的表达
- Author:
Jinkang ZHANG
;
Guolin MENG
;
Jian LIU
- Publication Type:Journal Article
- Keywords:
vascular endothelial growth factor 165;
angiopoietin-1;
adenovirus vector;
gene transfect
- From:
Orthopedic Journal of China
2006;0(04):-
- CountryChina
- Language:Chinese
-
Abstract:
[Objective]To construct the recombinant adenovirus vector co-expressing human vascular endothelial growth factor 165(VEGF165) and angiogenin-1(Ang-1),and to observe the expression of target gene after transfection.[Method]Molecular biologic techniques were used to clone the genes of VEGF165 and Ang-1,and PCR was used to amplify the DNA sequence of IRES(internal ribozyme entry site) from pIRES2-EGFP.The genes of VEGF165,IRES and Ang-1 were subcloned to pTrack-CMV one by one to get the plasmid named pTrack-CMV-VIA,which contained all the three genes.The linearized shuttle plasmid was cotransformed into BJ5183 bacteria with backbone vector AdEasy-1.Further the recombinant plasmid was packaged and amplified in QBI-293A cells after Pac I digestion to get adenovirus vector pAd-VIA.The expression of the gene of interests was evaluated by fluorescence microscopic analysis of GFP expression and enzyme linked immunosorbent assay(ELISA).[Result]The recombinant adenovirus plasmid was consistent with that shown by sequencing and restriction endonucleases digestion.The recombinant pAd-VIA vector was packaged and amplified successfully in QBI-293A cells.The titer was 2?1010 PFU/ml after amplifying.High positive GFP was expressed in the field through fluorescence microscopy after being transfected by recombinant pAd-VIA.ELISA indicated the expression of the target genes.The difference between the tansfected and non-transfected group was significant(P
