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Journal of Zhejiang University. Medical sciences ; (6): 499-505, 2008.
Article in Chinese | WPRIM | ID: wpr-310423

ABSTRACT

<p><b>OBJECTIVE</b>To construct the recombinant adenovirus containing B7-H4 gene with AdEasy XL system and to identify its biological activities.</p><p><b>METHODS</b>The 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.</p><p><b>RESULTS</b>The 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.</p><p><b>CONCLUSION</b>The bioactive recombinant adenovirus mB7-H4/Ad has been successfully constructed.</p>


Subject(s)
Animals , Mice , Adenoviridae , Genetics , B7-1 Antigen , Genetics , Cloning, Molecular , Escherichia coli , Genetics , Metabolism , Genetic Vectors , Mice, Inbred C57BL , Plasmids , Genetics , Recombinant Fusion Proteins , Genetics , Allergy and Immunology , T-Lymphocytes , Allergy and Immunology , Transfection , V-Set Domain-Containing T-Cell Activation Inhibitor 1
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