[Construction of mutants of MHC class II molecule transactivator gene capable of suppressing HLA-DR/DQ expression and exploration on their mechanism].
OBJECTIVE
To construct MHC class II transactivator (CIITA) gene mutants which could suppress the expression of MHC class II molecule and explore its mechanism.


METHODS
Restrictive enzymes digestion, PCR and synthesized oligonucleotide strands were used to construct three mutants including pcDNA3mCIITA2, pcDNA3mCIITA3 and pcDNA3mCIITA4. These mutants and pcDNA3 vector were transfected into Hela and Raji cell lines by lipofectamine. RT-PCR and flow cytometry were used to determine the changes of the inducible/constitutive expression of MHC class II-molecule. The mCIITA4 mutant was transferred to the tetracycline dose-dependent plasmid pUHD10-3. By adjusting the concentration of Dox in cultural circumstances, and regulating expressed level of external mutant C II TA, the relationship between the expression level of mC II TA4 mutant and that of MHC class II molecule was observed.


RESULTS
pcDNA3mCIITA3 and pcDNA3mCIITA4 could significantly suppress the expression of HLA-DR/DQ gene in Hela and Raji cells, while pcDNA3 empty vector and pcDNA3mCIITA2 had no effect on the MHC class II expression. The inhibition rate of MHC class II molecule expression was directly proportional to the quantity of transfected CII TA mutant.


CONCLUSIONS
The mutants pcDNA3 mCII TA3 and pcDNA3mCIITA4 can suppress the expression of MHC class II gene. Above date confirmed primarily that mutant C II TA suppressed the expression of MHC class II molecule by competitive binding with transactivator for the normal endogenous wild type C II TA molecule.
Ou
Qi-shui
QS
Shanghai Institute of Immunology, Shanghai Second Medical University, Shanghai 200025, China.
Lin
Lin
L
Huang
Li-dong
LD
Lu
Pei-hua
PH
Zhou
Guan-yan
GY
D005786:Gene Expression Regulation; D006683:HLA-DQ Antigens / Q000235:genetics*; D006684:HLA-DR Antigens / Q000235:genetics*; D006367:HeLa Cells; D006801:Humans; D007371:Interferon-gamma / Q000096:biosynthesis; D009154:Mutation; D009687:Nuclear Proteins / Q000235:genetics*; D015534:Trans-Activators / Q000235:genetics*; D014162:Transfection
D006683:HLA-DQ Antigens; D006684:HLA-DR Antigens; C083728:MHC class II transactivator protein; D009687:Nuclear Proteins; D015534:Trans-Activators; D007371:Interferon-gamma