CIITA锤头状核酶的克隆及其体外活性

Cloning of anti-CIITA hammerhead ribozyme and its biological activity in vitro

通过CIITA核酶抑制HeLa细胞表面MHCⅡ类分子的表达。设计并合成针对人类CIITA的核酶Rz464,通过体外转录和切割实验鉴定其活性。将Rz464亚克隆到真核表达载体pIRES2-EGFP(pRz464),并稳定转染HeLa细胞株,流式细胞术检测MHCⅡ类抗原表达,RT-PCR检测CIITA mRNA水平。结果表明,Rz464与CIITA靶序列体外切割产物电泳见预期切割条带。pRz464^+HeLa细胞与对照组比较,HLA-DR、DP、DQ抗原诱导型表达分别降低了79.21%、90.31%及48.30%;同时CIITA的诱导型mRNA含量明显减少。Rz464通过切割CIITA mRNA,进而阻止了后者调控的MHCⅡ类分子的表达。

The aim of this study was attempted to investigate the feasibility to use a hammerhead ribozyme against CIITA, a major regulator of MHC II antigen to regress the expression of MHC II molecules in HeLa cells. A hammerhead ribozyme （Rz464） specific to 463-465 GUC triplet of CIITA and its target gene were transcribed , then mixed and incubated in vitro. The cleavage products were analysed by PAGE and silver staining Rz464 was then subcloned into eukaryotic expression vector plRES2-EDPF （pRz464） and stably transfected to HeLa cells. The expression of MHC Ⅱ antigens was detected by flow cytometry and the level of CIITA mRNA was determined by RT-PCR. It was demonstrated that Rz464 could exclusively cleave CI- ITA RNA. While induced with IFN-γ, the expressions of HLA-DR, -DP, and -DQ on pRz464^＋ HeLa cells decreased, so did for the mRNA contents of CIITA. It is concluded that through cleavage of CIITA mRNA, Rz464 inhibit the expression of family of MHC II molecules regulated by CIITA.