Construction of recombinant adenovirus vector containing human survivin gene and expression in fibroblast
- VernacularTitle:人Survivin基因重组腺病毒载体的构建及其表达
- Author:
Hong LI
;
Yousheng LIU
;
Changson WANG
- Publication Type:Journal Article
- Keywords:
adenovirus vector;
gene therapy;
gene, Survivin;
dendritic cells
- From:
Medical Journal of Chinese People's Liberation Army
1981;0(06):-
- CountryChina
- Language:Chinese
-
Abstract:
Objective To construct the recombinant adenovirus vector which contained human survivin, then being transfected into dendritic cells. Methods Full length survivin cDNA was obtained from recombinant plasmid pCITE survivin by PCR, the PCR product was double digested with restrictive endonucleases Kpn Ⅰ and Xhol Ⅰ, then being inserted directionally into pAdTrack CMV. The plasmid of pAdTrack survivin was lined with PmeⅠ, the fragment containing survivin was reclaimed and transfected into E coli BJ5183. After having been screened by selected media, the extracted plasmid of positive bacteria was transfected into HEK293 cells with liposome and was identified by observing the green fluorescence protein (GFP) and by PCR method. At the time, the culture media containing virus was transfected into fibroblast, and the expression of survivin was proved by observing the GFP and the Western blot method. Results The recombined adenovirus survivin was constructed successfully and the titer was about 1 65?10 8 pfu/ml. A band was observed by Western bolt and its relative molecular mass was about 16 5kD. Conclusion A recombinant adenovirus vector containing human survivin was constructed successfully, and the survivin protein was expressed by fibroblast. It lays the foundation for further study on survivin as a target antigen for gene therapy
