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.
出处
《免疫学杂志》
CAS
CSCD
北大核心
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
