Efficient generation of human angiopoietin-1 recombinant adenovirus by COS/TPC homologous recombination
- VernacularTitle:人血管形成素-1重组腺病毒高效制备
- Author:
Shilin CHEN
;
Shengdong HUANG
;
Zhiyun XU
;
Ju MEI
;
Jialin ZHU
;
Baoren ZHANG
- Publication Type:Journal Article
- Keywords:
Adenoviruses, human;
Genes, viral;
Myocardium
- From:
Chinese Journal of Pathophysiology
2000;0(12):-
- CountryChina
- Language:Chinese
-
Abstract:
AIM: To generate the angiopoietin-1 recombinant adenovirus for the further studies about adenoviral mediated angiopoietin-1 gene transfer in ischemic myocardium and its effect on the development of neoangiogenesis.METHODS: Angiopoietin-1-PAxCAwt cosmid DNA and adenoviral DNA-TPC were cotransfected to 293 cells by the use of calcium phosphate precipitation method. All recombinant adenoviruses were propagated and titrated in 293 cells, purified by cesium chloride density purification, dialyzed, and stored at-70 ℃. RESULTS: The result shows that the direction of the insert in the cosmid is correct and the replication-deficient angiopoietin-1 recombinant adenovirus was generated efficiently by COS/TPC homologous recombination, with the titers of 5.6?10 11 pfu/L. The viral stocks were demonstrated to be free of replication-competent wild type adenoviruses. The virus stocks in high titer were harvested in our experiment. CONCLUSION: The COS/TPC is an efficient method to prepare recombinant adenovirus and the angiopoietin-1 recombinant adenovirus could be further used in gene therapy.
