主要组织相容性复合物Ⅱ类分子转录激活因子核酶的克隆及其活性

目的探讨主要组织相容性复合物(MHC)Ⅱ类分子转录激活因子(CⅡTA)核酶抑制MHCⅡ类分子的表达。方法设计并合成针对人类CⅡTA的一组核酶,通过体外制备和活性鉴定,筛选出活性较高的核酶一Rz464。将Rz464克隆到真核表达载体pIRES2-EGFP,简称为pRz464,并稳定转染Daudi细胞株(pRzA64一D),流式细胞术检测经典的MHCⅡ(HLA-DR、-DP、-DQ)类抗原表达,RT—PCR检测CⅡTA mRNA水平。结果 pRz464-D与无关核酶组比较,HLA—DR、-DP、-DQ抗原表达分别降低了88．4％、83．5％、93．4％;同时CⅡTA的mRNA含量明显减少。结论提示抗CⅡTA锤头状核酶抑制了自身mRNA含量,从而阻止了其调控的MHCⅡ类分子的相应表达。

Cloning and biological activities of riboenzymes against major histocompatibility complex class II transactivator

OBJECTIVE: To investigate the cloning and biological activities of riboenzymes against major histocompatibility complex (MHC) class II transactivator (CIITA) so as to explore the feasibility of using hammerhead riboenzyme to create immune tolerance. METHODS: Three riboenzymes against MHC CIITA were synthesized and their activities were evaluated in vitro. Rz464, the riboenzyme with a better digestion effect, was inserted into the vector pIRES2-EGFP (then called pRz464). Human B lymphoma cells of Daudi line were cultured and transfected with pRz464 (then called pRz464-D). Cultured Daudi cells transfected with riboenzyme without the ability against CIITA (Rz-D) were used as control group. The expressions of classic MHC CIITA (HLA-DR, DP, and DQ) on the surface of Daudi cells before and after transfection were examined by flow cytometry. RESULTS: The expressions of HLA-DR, DP, and DQ on the surface of cultured Daudi cells (Rz-D) were 97.9 +/- 0.8%, 94.3 +/- 1.1%, and 54.4 +/- 3.1% respectively. The expressions of HLA-DR, DP, and DQ were inhibited by 88.4%, 83.5%, and 93.4% respectively on the surface of pRz464-D cells. CONCLUSION: The hammerhead riboenzyme Rz464 inhibits the expression of CIITA mRNAs, thus inhibiting the expression of MHC II molecules. It may be used in antigen-specific tolerance induction in allo-transplantation.