抗CⅡTA RNaseP(M1-3408-GS)的生物学活性  

The biological activity of anti-CⅡTA RNaseP(M1-3408-GS)

作　　者：郭荣[1] 蒋海玉 邹萍[3] 陆华中[4] 范华骅[4] 高峰[4] 商庆新[5] 曹谊林[5] 张敏[3] 

机构地区：[1]广东省人民医院血液科 [2]新疆哈密红星医院内一科 [3]华中科技大学同济医学院附属协和医院血研所 [4]上海市血液中心血液工程研究室 [5]上海第二医科大学组织工程重点实验室

出　　处：《中华微生物学和免疫学杂志》2004年第6期451-455,共5页Chinese Journal of Microbiology and Immunology

基　　金：上海市科技发展基金资助项目 ( 0 14 3nm0 68) ;上海市科技发展基金 重大项目子课题资助项目 ( 0 0DJ14 0 0 1 8)

摘　　要：目的　通过抗CⅡTARNaseP抑制HeLa细胞表面MHCⅡ分子的表达。方法　M1 RNA是RNaseP的催化活性单位 ,从包含M1 RNA的pTK117质粒克隆抗CⅡTARNaseP(M1 340 8 GS) ,定向插入腺相关病毒载体psNAV(psNAV M1 340 8 GS) ;以RT PCR从Raji细胞株克隆靶基因模板 ,插入pGEM 7zf(+)载体 (pGEM 3176 )。psNAV M1 340 8 GS和pGEM 3176分别进行体外转录和体外切割实验。通过纳米载体介导psNAV M1 340 8 GS质粒稳定转染HeLa细胞 ,应用流式细胞术检测其表面HLA DR、DP、DQ抗原表达 ,RT PCR检测其CⅡTAmRNA水平。结果　M1 340 8 GS与pGEM 3176体外切割产物电泳见预期切割条带。M1 340 8 GS+ HeLa细胞与对照组比较 ,其表面HLA DR、DP、DQ抗原诱导型表达分别降低了 79.2 1%、90 .31%及 4 8.30 % ;同时诱导型CⅡTAmRNA含量减少。结论　抗CⅡTA核糖核酸酶P(M1 340 8 GS)有可能发展为新一代抑制自身免疫疾病的核酸药物。Objective To investigate the cleavage activity of RNaseP against CⅡTA mRNA, a major regulator of MHCⅡ expression, on the expression of MHCⅡ molecules on HeLa cell. Methods M1-RNA was constructed with guide sequences recognizing the LRR of CⅡTA at 3408 point(M1-3408-GS) by PCR from pTK117 plasmid, then cloned into the vector psNAV(psNAV -M1-3408-GS). CⅡTA target gene was obtained from Raji cell by RT-PCR and then inserted into the pGEM-7zf(+) plasmid(pGEM-3176). The recombinant plasmids were screened out by sequence analysis. psNAV-M1-3408-GS and pGEM-3176 were transcribed and then mixed up and incubated in vitro. The cleavage products were analyzed by PAGE and silver-staining. Stable transfectants of HeLa cells with psNAV-M1-3408-GS were tested for class Ⅱ MHC induction by recombinant human interferon-gamma(IFN-γ). mRNA abundance of CⅡTA was measured by RT-PCR. Results It was shown that M1-3408-GS acted as a sequence-specific endonucleases and exclusively cleaved pGEM-3176. When induced by IFN-γ, the expression of HLA-DR, -DP, -DQ on psNAV-M1-3408-GS + HeLa were reduced, and the content of CⅡTA mRNA decreased. Conclusion The ribonuclease P effectively induced antigen-specific tolerance through cleaving CⅡTA mRNA. These results provided an insight into the future application of M1-3408-GS as a new nucleic acid drug against auto-immune diseases.

关 键 词：抗CⅡTARNaseP 生物学活性 CⅡTA 核糖核酸酶P HELA细胞 自身免疫性疾病 

分 类 号：R392[医药卫生—免疫学]