构建MHCⅡ类分子反式激活因子基因突变体抑制转染细胞HLAⅡ类分子的表达  

Suppression of HLA class Ⅱ expression of cell lines transfected with the mutated CⅡTA constructs

作　　者：欧启水[1] 林琳[1] 黄立东[1] 陆佩华[1] 周光炎[1] 

机构地区：[1]上海第二医科大学免疫学研究所

出　　处：《中华微生物学和免疫学杂志》2002年第3期312-315,共4页Chinese Journal of Microbiology and Immunology

基　　金：上海市科技发展基金;福建省自然科研基金资助项目(2 0 0 0A0 1 4 );福建省教委青年科研基金资助项目 (99A0 55)

摘　　要：目的　探讨MHCⅡ类分子反式激活因子 (CⅡTA)突变体抑制HLAⅡ类分子表达的可能性。方法　构建不含起始密码子的pcDNA3mCⅡTA2、含起始密码子的pcDNA3mCⅡTA3和含起始密码子及NLS(nuclearlocalizationsignal)的pcDNA3mCⅡTA4突变体。用脂质体转染法将上述 3种突变体及pcDNA3空载体转入HeLa细胞和Raji细胞。用流式细胞术和RT PCR法观察它们对HeLa/Raji细胞HLA DR/DQ分子的诱导性和组成性表达的影响。结果　在细胞和基因水平证实pcDNA3mCⅡTA3和pcDNA3mCⅡTA4对HeLa/Raji细胞HLA DR/DQ的表达起明显抑制作用 ,而pcDNA3mCⅡTA2和pcDNA3空载体无此作用。Objective To explore the depression of MHC class Ⅱ molecules by transfecting the mutated CⅡTA (MHC class Ⅱ transactivator) constructs. Methods Restriction enzymes, PCR technique and synthesized oligonucleotide strand were used to construct three mutants as pcDNA3mCⅡTA2, pcDNA3mCⅡTA3 and pcDNA3mCⅡTA4. All 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 MHC class Ⅱ inducible/constitutive expression. Results Mutants pcDNA3mCⅡTA3 and pcDNA3mCⅡTA4 suppressed the inducible/constitutive expression of MHC class Ⅱ genes. However, pcDNA3 vector and pcDNA3mCⅡTA2 in which the initiation codon ATG was not contained had no effect on the MHC class Ⅱ expression. Conclusion The constructed mutants pcDNA3mCⅡTA3 and pcDNA3mCⅡTA4 suppressed the expression of MHC class Ⅱ genes. [

关 键 词：MHCⅡ类分子反式激活因子 突变体构建 HLA-DR/DQ 基因转染 流式细胞术 RT-PCR法 

分 类 号：R392[医药卫生—免疫学]