构建可抑制HLA-DR/DQ表达的MHC-Ⅱ类分子反式激活因子基因的突变体及其机制  

Construction of mutants of MHC class Ⅱ molecule transactivator gene capable of suppressing HLA-DR/DQ expression and exploration on their mechanism

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]上海第二医科大学上海市免疫学研究所

出　　处：《细胞与分子免疫学杂志》2003年第5期417-420,共4页Chinese Journal of Cellular and Molecular Immunology

基　　金：上海市科技发展基金资助项目(No .99JC1 4 0 33);福建省自然科学基金资助项目(No.C0 0 1 0 0 11 );福建省高等学校科研基金资助项目(No .K990 4 9)

摘　　要：目的 :构建能抑制MHC Ⅱ类分子表达的MHC Ⅱ类分子反式激活因子 (MHCclassⅡtransactivator,CⅡTA)基因的突变体 ,并探讨其抑制MHC Ⅱ类分子表达的机制。方法 :用PCR、酶切及连接技术 ,构建不含起始密码子的pcDNA3mCⅡTA2 ,含起始密码子的pcDNA3mCⅡTA3以及含起始密码子及NLS(nu clearlocalizationsignal)的pcDNA3mCⅡTA4突变体。用脂质体转染法 ,将上述 3种突变体及空载体pcDNA3转入Hela细胞和Raji细胞中。用流式细胞术和RT PCR法 ,观察他们对Hela/Raji细胞HLA DR/DQ分子的诱导性和组成性表达的影响。将mCⅡTA4转移到对四环素浓度依赖的质粒pUHD10 3上 ,通过改变培养环境中四环素的浓度 ,调节外源CⅡTA突变体的表达量 ,观察突变体的表达量与MHC Ⅱ类分子受抑率的关系。结果 :细胞和基因水平证实 ,pcDNA3mCⅡTA3和pcD NA3mCⅡTA4对Hela/Raji细胞HLA DR/DQ的表达均有明显的抑制作用 ;而pcDNA3mCⅡTA2和空载体pcDNA3无此作用。MHC Ⅱ类分子被抑制的程度与外源转入CⅡTA突变体(pUHD10 3mCⅡTA4 )的量明显相关。结论 :成功地构建pcD NA3mCⅡTA3和pcDNA3mCⅡTA4 ,并能抑制HLA Ⅱ类分子的表达。初步证实CⅡTA突变体是通过与胞内的野生型CⅡTA竞争性结合反式激活蛋白 ,来抑制MHC Ⅱ类分子的转录和表达。AIM: 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 CⅡTA, the relationship between the expression level of mCⅡTA4 mutant and that of MHC class Ⅱ molecule was observed. RESULTS: pcDNA3mCⅡTA3 and pcDNA3mCⅡTA4 could significantly suppress the expressions of HLA DR/DQ gene in Hela and Raji cell, 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. CONCLUSION: The mutants pcDNA3 mCⅡ TA3 and pcDNA3mCⅡTA4 can suppress the expression of MHC class Ⅱ gene. Above date confirmed primarily that mutant CⅡTA suppressed the expression of MHC class Ⅱ molecule by competitive binding with transactivator for the normal endogenous wild type CⅡTA molecule.

关 键 词：CⅡTA 突变体 HLA-DR/DQ 基因转染 

分 类 号：Q782[生物学—分子生物学]