In vitro study on inhibition of expression of MHC II molecules by adenovirus-mediated gene transfer of CIITA mutant gene / 中国免疫学杂志

ABSTRACT

Objective:To construct adenovirus containing murine MHC class II transactivator (CIITA) mutant gene and to observe expression of this gene mediated by adenovirus as well as function of the expression product in vitro.Methods:The type IV CIITAcDNA gene was cloned from peritoneal macrophage of BALB/c mouse by conventional way of molecular biology, and then it was cloned into expression vector pIRES. CIITA mutant gene was constructed by the way of overlap extension by PCR (OE-PCR), and then it was also cloned into expression vector pIRES. By using of pAdEasy-1 system we acquired the deficient recombination adenovirus Ad-CIITAm, containing CIITA mutant gene, which had the ability of infection and the no-loaded control adenovirus Ad-GFP.Then the two adenoviruses strains were processed for a great deal amplification,purification and titer determining. The Ad-CIITAm and Ad-GFP were infected into HeLa cells and Raji cells.Inducible or constitutive expression of HLA-DR molecule and the following changes were observed by the use of flow cytometry.Results:The murine CIITA gene was cloned successfully and the recombinantant adenovirus Ad-CIITAm containing murine CIITA mutant gene was also constructed successfully. It was proved by flow cytometry that expression of HLA-DR molecules on the surface of HeLa cells and Raji cells infected by Ad-CIITAm were all decreased remarkably in comparison with those of control cells infected by Ad-GFP.Conclusion:This experiment proves that expressed murine CIITA mutant mediated by adenovirus could inhibit the expression of MHC II molecules efficiently.