Construction and identification of recombinant adenoviral vector encoding B7-H4 gene.
- Author:
Pei-yi YAN
1
;
Hang-ping YAO
;
Yun QIAN
;
Li-juan GU
;
Lan TAO
;
Li-huang ZHANG
Author Information
- Publication Type:Journal Article
- MeSH: Adenoviridae; genetics; Animals; B7-1 Antigen; biosynthesis; genetics; Cloning, Molecular; Escherichia coli; genetics; metabolism; Genetic Vectors; Mice; Mice, Inbred C57BL; Plasmids; genetics; Recombinant Fusion Proteins; biosynthesis; genetics; immunology; T-Lymphocytes; immunology; Transfection; V-Set Domain-Containing T-Cell Activation Inhibitor 1
- From: Journal of Zhejiang University. Medical sciences 2008;37(5):499-505
- CountryChina
- Language:Chinese
-
Abstract:
OBJECTIVETo construct the recombinant adenovirus containing B7-H4 gene with AdEasy XL system and to identify its biological activities.
METHODSThe full-length mouse B7-H4 gene was amplified by RT-PCR from C57 mouse lung and put into T vector, then verified by sequencing. Digested with Xhol I and EcoR V the B7-H4 gene was inserted into pshuttle-CMW(PSC). Pme I linearized shuttle plasmid was transformed into E.coli BJ5183-AD-1 to obtain the recombinant adenoviral plasmid pAd-mB7-H4 by efficient homologous recombination. Then the recombinant adenovirus-mB7-H4/Ad was obtained by packaging Pac I linearized in D-293 cells. The mRNA and protein expression of B7-H4 in mB7-H4/Ad infected AD-293 cells were detected by RT-PCR and Western blot, respectively. mB7-H4/Ad was used to infect L929 cells, the bioactivity of expressed B7-H4 in stimulation of T lymphocytes proliferation and cytokine production were tested.
RESULTSThe full-length of mB7-H4 was cloned from mouse lung tissue cDNA and verified by sequencing. The recombinant plasmid pAd-m B7-H4 was successfully generated by homologous recombination, and the primary mB7-H4/Ad was obtained by packaging pAd-B7-H4 in AD-293 cells. Compared with the negative control, L929 cells infected with mB7-H4/Ad effectively inhibited the proliferation of T lymphocytes and cytokines production.
CONCLUSIONThe bioactive recombinant adenovirus mB7-H4/Ad has been successfully constructed.
