MHCⅡ类分子转录激活因子锤头状核酶的生物学活性  

Biological activity of MHCⅡ transactivator hammerhead ribozyme

作　　者：郭荣[1] 蒋海玉 张敏[3] 邹萍[3] 杜欣[1] 陆泽生[1] 林伟[1] 黄梓伦[1] 

机构地区：[1]广东省人民医院血液科,广州510080 [2]新疆哈密红星医院内一科,新疆哈密839000 [3]华中科技大学同济医学院附属协和医院血液研究所,武汉430022

出　　处：《免疫学杂志》2006年第3期294-297,共4页Immunological Journal

基　　金：广东省自然科学基金(04003959);广东省人民医院科学技术研究基金(Y2004087)资助项目

摘　　要：目的探讨MHCⅡ类分子转录激活因子(CⅡTA)锤头状核酶抑制细胞表面MHCⅡ类分子的表达。方法设计并克隆针对CⅡTA第464位点的锤头状核酶(Rz464)及其相应的CⅡTA靶基因,分别插入pGEM-T载体,进行细胞外切割活性鉴定,进一步将Rz464亚克隆入真核表达载体pIRES2-EGFP(pIRES2-EGFP-Rz464,pRz464),并稳定转染Raji细胞株,流式细胞术检测经典的MHCⅡ(HLA-DR-、DP-、DQ)类抗原表达,RT-PCR分析CⅡTA mRNA水平。结果细胞外活性鉴定表明,Rz464具有明显的切割活性。细胞内切割实验显示:pRz464阳性Raji细胞表面HLA-DR、DP、DQ抗原表达分别降低了75.93%、64.14%、78.32%;同时CⅡTA的mRNA含量降低(P<0.05)。结论抗CⅡTA锤头状核酶Rz464可有效抑制MHCⅡ类抗原的表达。Objective To investigate the inhibiting effects of MHC Ⅱ transactivator （ C Ⅱ TA） hammerhead ribozyme on expression of class Ⅱ major histocompafibility complex （ MHC Ⅱ ） on cell surface. Methods The hammerhead ribozyme recognizing C Ⅱ TA at 464 sites （Rz464） and the CⅡTA target RNA were constructed, and then cloned into the pGEM-T vector,respectively. The recombinant ribozyme and its target RNA were incubated in cell-free conditions. The Rz464 was cloned into the pIRES2-EGFP vector for intracellular analyses （ pIRKS2-EGFP-Rz464, pRz464）, and then transfected into Raji cell. The expressions of classical MHC Ⅱ （HLA-DR,-DP,-DQ） were detected by flow cytometry. The mRNA of C Ⅱ TA was analyzed by RT-PCR. Results Rz464 could cleave target RNA exclusively and the expressions of HLADR, -DP, -DQ on pRz464 positive Raji cells were almost totally inhibited. The contents of CⅡ TA mRNA was decreased significantly （ P 〈 0.05）. Conclusion Rz464 can inhibit the expression of MHCⅡ molecules.

关 键 词：MHCⅡ类分子转录激活因子 核酶 自身免疫性疾病 基因疗法 

分 类 号：R392[医药卫生—免疫学]