Construction and identification of recombinant adenovirus containing human vascular endothelial growth factor 165 gene
- VernacularTitle:血管内皮细胞生长因子165基因重组腺病毒的构建及其鉴定
- Author:
Yanfu HAN
;
Jianxing SONG
;
Jun LIU
- Publication Type:Journal Article
- From:
Chinese Journal of Tissue Engineering Research
2007;0(24):-
- CountryChina
- Language:Chinese
-
Abstract:
AIM: Recombinant adenovirus possesses high transfection efficiency and wide host range. This study was designed to construct the recombinant adenovirus vector containing human vascular endothelial growth factor 165 (VEGF165), so as to lay a foundation for the subsequent gene transfection, microencapsulated genetically engineered cells and animal experiments. METHODS: The experiment was conducted in the Laboratory of Cardiothoracic Surgery (the National Key Laboratory), Changhai Hospital of The Second Military Medical University of Chinese PLA from January to May in 2007. Experiment materials: pAxCAwt.VEGF165 was provided by Institute of Cardiothoracic Surgery of Changhai Hospital. pAxCAwt.VEGF165 and DNA-TPC were cotransfected into human embryonic kidney 293 cells by lipofection method. Being propagated, recombinant replication-deficient adenovirus named Ad.VEGF165 was obtained. The target gene of recombinant adenovirus was identified by polymerase chain reaction (PCR) and restriction enzyme digestion. The titer of virus was detected by 50% tissue culture infective dose method. RESULTS: Construction of recombinant adenovirus Ad.VEGF165: The pAxCAwt.VEGF165 and DNA-TPC were successfully cotransfected into human embryonic kidney 293 cells by lipofection method, and replication-deficient adenovirus vectors coding for VEGF165 gene were generated. Identification of recombinant adenovirus Ad.VEGF165: Two fragments of PCR products (597 bp and 146 bp) were obtained by NcoI restriction enzyme. The result was consistent with that calculated with Gene Tool software. The virus titers was 2.2?1015 pfu/L. CONCLUSION: DNA-TPC and pAxCAwt.VEGF165 can be used to construct replication-deficient recombinant adenovirus Ad.VEGF165 in a high titer, low toxicity, high efficiency and safe transfection in vitro.
