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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。
OBJECTIVE: To construct mutants of MHC class Ⅱ transactivator (CⅡTA) gene that can inhibit the expression of MHC class Ⅱ molecules and to explore the mechanism of its inhibitory effect on the expression of MHC class Ⅱ molecules. Methods: pcDNA3mCⅡTA2 without start codon, pcDNA3mCⅡTA3 with start codon and pcDNA3mCⅡTA4 mutant with start codon and NLS (nu clelocalization signal) were constructed by PCR, enzyme digestion and ligation. Three kinds of mutants and empty vector pcDNA3 were transfected into Hela cells and Raji cells by lipofection. Flow cytometry and RT PCR were used to observe their effects on the induction and constitutive expression of HLA DR / DQ in Hela / Raji cells. The mCⅡTA4 was transferred to plasmid pUHD10 3, which is dependent on the concentration of tetracycline. The concentration of tetracycline in the culture environment was adjusted to regulate the expression level of the exogenous CⅡTA mutant. The relationship between the expression level of the mutant and the inhibition rate of MHC class Ⅱ molecules was observed. Results: The cell and gene levels demonstrated that pcDNA3mCⅡTA3 and pcD NA3mCⅡTA4 significantly inhibited the HLA DR / DQ expression in Hela / Raji cells. However, pcDNA3mCⅡTA2 and empty vector pcDNA3 had no effect. The degree of inhibition of MHC class II molecules is significantly correlated with the amount of exogenously transfected C IITA mutant (pUHD10 3mC IITA4). CONCLUSION: The constructed pcD NA3mCⅡTA3 and pcDNA3mCⅡTA4 can inhibit the expression of HLA class II molecules. Preliminary evidence shows that CⅡTA mutants inhibit the transcription and expression of MHC class Ⅱ molecules by competitively binding the transactivator to intracellular wild-type CⅡTA.