一种新的CIITA显性负向突变体的构建和功能研究  

Construction of a novel CIITA dominant negative mutant and its function study

作　　者：路丽明[1] 丁庆[1] 江阳[1] 焦志军[1] 辛利军[1] 周芸[1] 周晓荣[1] 周光炎[1] 

机构地区：[1]上海交通大学医学院上海市免疫学研究所,上海200025

出　　处：《现代免疫学》2006年第2期116-120,共5页Current Immunology

基　　金：863项目(2003AA205009);上海市科技发展基金资助项目(03JC14085);国家自然科学基金重点资助项目(30532690)

摘　　要：II类反式激活因子(CIITA)参与MHC II类基因转录表达的调控,本研究采用RT-PCR等分子克隆技术构建含起始密码子和NLS序列,但是删除N和P/S/T结构域的CIITA突变体质粒pCDNA3.1(+)mCIITA,用脂质体转染法将上述突变体及pCDNA3.1(+)空载体转入来源于BALB/c小鼠的1B4.B6细胞株,用流式细胞术和RT-PCR观察I-A分子表达的影响,并通过混合淋巴细胞反应观察C3H小鼠CD4+T细胞对转入突变体及空载体的1B4.B6细胞的免疫应答强度。结果在细胞和分子水平证实pCDNA3.1(+)mCIITA对1B4.B6细胞I-A的表达起明显抑制作用,强阳性克隆抑制率为90%以上。细胞已基本丧失了对受者CD4+T细胞的刺激能力。以上结果为减低同种异体/异种器官细胞性排斥提供了新的思路和手段。Class Ⅱ transactivator（CⅡTA） is a critical factor for transcription activation of MHC class Ⅱ genes, In the present study, a novel CⅡTA dominant negative mutant（mCⅡTA） plasmid pcDNA3. 1 （＋）mCⅡTA with the N-terminal deleted and P/S/T domain truncated was constructed by using the molecular cloning techniques, such as RT-PCR, and this mutant with the vacant vector pcDNA3.1（＋） was transfected to the 1B4. B6 cell line derived from BALB/c mouse through liposome. The expression of the class Ⅱ molecule I-A was assayed by semi quantitative RT-PCR and flow eytometry analysis, and the intensity of immune responses of the CD4^＋ T cells from C57BL/6J strain of mice to the mCⅡTA-transfeeted 1B4. B6 cells was determined by one way mixed lymphocyte reaction（MLR）, in which the irradiated IB4. B6 cells were to be used as the stimulators. Our experimental results both from molecular and cellular levels demonstrated that the expression of MHC class Ⅱ I-A genes was significantly inhibited in the mCⅡTA-transfeeted cells with a suppression rate of 90%, and these cells failed to stimulate the proliferation of the allogeneie CD4^＋ T cells. It is possible that the alloreaction suppressed by mCⅡTA and the lower expression of the MHC class Ⅱ molecules might be strategically significant for development of the transplantation tolerance.

关 键 词：CⅡTA显性负向突变体 I-A 同种异基因应答 

分 类 号：R392.11[医药卫生—免疫学]