[Construction and identification of recombinant adenovirus vector pAdEasy-GFP-GITRL].
OBJECTIVE
To construct recombinant adenovirus vector pAdEasy-GFP-GITRL and detect the viral titer.


METHODS
GITRL gene was obtained by double digestion using Bgl II and Sal I, and cloned into the baculovirus transfer vector(pAdtrack-CMV), then the recombinant adenovirus vector (pAdtrack-CMV-GITRL) was digested by restrictive endoenzyme Pme I. The linear recombinant adenorirus vector and pAdEasy-1 were cotransfected into HEK293 cells by co-precipitate of calcium phosphate. Recombinant adenovirus was packaged and purified in HEK293A cells.


RESULTS
Recombinant adenovirus vector pAdEasy-GFP-GITRL was constructed successfully and high titer of recombinant adenovirus was obtained (2.0 x 10⁹ pfu/mL). Western blotting analysis also revealed the expression of GITRL by recombinant adenovirus vector.


CONCLUSIONS
The construction of recombinant adenovirus vector pAdEasy-GFP-GITRL and recombinant adenovirus will facilitate the potential GITRL gene therapy.
Department of Immunology, School of Medical Science and Laboratory Medicine, Jiangsu University, Zhengjiang 212013, China.
Jie
J
Ma
Jie
J
Tian
Yan
Y
Liu
Qing-ling
QL
Liu
Zhao-ming
ZM
Mao
Zhi-jun
ZJ
Jiao
Hua-xi
HX
Xu
Sheng-jun
SJ
Wang
D000256:Adenoviridae / Q000235:genetics; D015153:Blotting, Western; D049452:Green Fluorescent Proteins / Q000235:genetics*; D006801:Humans; D012333:RNA, Messenger / Q000032:analysis; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis*; D048069:Tumor Necrosis Factors / Q000235:genetics*
D012333:RNA, Messenger; D011993:Recombinant Fusion Proteins; C478966:Tnfsf18 protein, mouse; D048069:Tumor Necrosis Factors; D049452:Green Fluorescent Proteins