Construction and expression of recombinant adenovirus carrying human endostatin gene
- VernacularTitle:携带人内皮抑素基因的重组腺病毒构建及表达
- Author:
Ying MA
;
Yuanli HE
- Publication Type:Journal Article
- From:
Chinese Journal of Tissue Engineering Research
2007;0(07):-
- CountryChina
- Language:Chinese
-
Abstract:
AIM:So far,endostatin is found playing the strongest effect to inhibit endothelial cells.Because endostatin protein is instable,it is difficult to prepare and apply.Gene therapy is used to assist endostatin to inhibit angiogenesis.This study constructed recombinant adenovirus of human endostatin gene using AdEasy-1 system to investigate the feasibility of expressing endostatin protein in ECV304 cells(human umbilical vein endothelial cells).METHODS:The experiment was performed at College of Life Science,Sun Yat-set University from March to October 2006.Endostatin was amplified from Pshuttle-Endostatin plasmid with PCR and subcloned into the pAdTrack-CMV shuttle vector after sequencing and identification.The resultant plasmid was co-transduced into E.coli BJ 5183 cells with pAdEasy-1 plasmid for homologous recombination.The recombinant plasmid was detected by restriction analysis and DNA sequence analysis.The linearized recombinant plasmid was transfected into AAV 293 cells with LipofectAMINE2000.The recombinant adenovirus was detected by GFP,PCR and restriction analysis.The virus was used to infect ECV304,and the expression of endostatin in vitro was detected by the techniques of RT-PCR and Western blot.RESULTS:The positive clones of the recombinants were constructed and confirmed by restriction endonuclease analysis and DNA sequence analysis.AAV293 cells were transfected.The titer of viral liquid was 2.06?1010 pfu/mL.The transcription of endostatin mRNA in ECV304 cells was checked by RT-PCR and endostatin protein could be detected in the culture by Western blot analysis.CONCLUSION:The recombinant adenoviral vector pAd-Endo carrying human endostatin is successfully constructed,and effective expression is found in ECV304 cells in vitro.
