抗DEN-2基因核酶的克隆及其体外切割活性的研究

目的：探讨核酶抗登革病毒活性。方法：根据DEN－2基因结构的特点，按照锤头状核酶的结构模型和作用模式，设计，合成针对172－174GUC位点的核酶基因；定向插入质粒pGEM-3Zf( )的Sac I和Sal I位点之间；核酶基因克隆和DEN－2靶基因克隆分别进行体外转录，生成核酶的靶RNA，体外进行切割反应。结果：核酶与登革病毒靶序列体外产物电泳 见预期切割条带。结论：该核酶具有切割相应靶RNA的活性，可进一步用于细胞内核酶抗登革病毒的研究。     

抗乙型肝炎病毒HDV核酶的研究新进展

乙型肝炎病毒(HBV)是严重威胁人类健康的一种病毒,特别是在我国约有1.2亿人为HBV携事业者,占世界的1/3.对已感染HBV者,常用的化学及免疫疗法通常无效.今年来,研究者试图利用反义技术,通过在基轩表达水平上抑制HBV的复制,来达到治疗HBV感染的目的.核酶是具有RNA裂解活性的RNA分子,能特异地结合并切割靶RNA分子.HDV核酶唯一在人体细胞天然具有裂解活性的核酶类型,研究将HDV核酶作为HBV的反义抑制剂可能有着其独特的优势.本文就HDV核酶靶位点的筛选、病毒载体导入系统、靶向性分子的构建等热点问题的研究新进展予以综述.     

抗乙型肝炎病毒HDV核酶的研究新进展

乙型肝炎病毒(HBV)是严重威胁人类健康的一种病毒，特别是在我国约有1．2亿人为HBV携带者，占世界的1／3。对已感染HBV者，常用的化学及免疫疗法通常无效。今年来，研究者试图利用反义技术，通过在基因表达水平上抑制HBV的复制，来达到治疗HBV感染的目的。核酶是具有RNA裂解活性的RNA分子，能特异地结合并切割靶RNA分子。HDV核酶是唯一在人体细胞天然具有裂解活性的核酶类型，研究将HDV核酶作为HBV的反义抑制剂可能有着其独特的优势。本文就HDV核酶靶位点的筛选、病毒载体导入系统、靶向性分子的构建等热点问题的研究新进展予以综述。     

丁型肝炎病毒核酶反式切割HBV mRNA片段的实验研究

目的　探讨反式作用丁型肝炎病毒 (HDV)核酶体外切割乙型肝炎病毒 (HBV)mRNA片段的可行性。方法　将化学合成的核酶cDNA克隆到含有T7启动子的载体PGEM 4Z中。利用体外转录技术转录出核酶及底物 ,研究其体外切割活性。利用E H作图法进行核酶的酶促动力学研究。结果　在体外实验中显示两酶均能成功的将底物切割 ,37℃温浴 90min的切割百分率为 5 0 %和5 1%。利用E H作图法进行的酶促动力学研究中求得Rc1、Rc2的Km值分别为 0 6 1μmol L、0 5 8μmol L,Kcat值分别为 :0 6 4·min 1 、0 6 0·min 1 。结论　反式作用HDV核酶对非HDV底物 HBVmRNA片段的成功切割为寻找新的HBV的反义抑制手段开辟了途径。     

人工核酶M1GS结构与功能相互关系的研究

目的：研究核酶M₁GS其二级结构与体外切 割活性之间的关系。方法：以人巨细胞病毒（HCMV）DNA聚合酶基因UL54 为靶基因，构建了人工核酶M₁GS-T₇。通过软件RNA structure对M1GS在3个具有相对 稳定结构的温度（20 ℃、37 ℃、55 ℃）的空间构象进行模拟,然后通过体外切割实验来 检测不同温度下核酶M1GS体外切割活性的变化。为一步研究核酶M₁GS二级结构与体外切 割活性之间的关系，参照温度变化实验结果及RNA二级结构的模拟结果，引入突变位点，构 建了在37 ℃与55 ℃时M₁GS-T₇具有相同二级结构的突变型核酶mM₁GS-T₇，并通 过体外切割实验对两者的活性进行比较。结果：在温度变化实验中，55 ℃核酶的体外切割活性最高。而在突变实验中，37 ℃ mM₁GS-T₇比M₁GS-T₇的活 性略高。结论：具有某种特定二级结构的M₁GS-T₇有相对较高的体外 切割活性，核酶的结构与其功能之间存在一定的对应关系。     

针对乙型肝炎病毒前S2基因核酶的设计及体外切割 …

目的 体外研究核酶睦乙型肝炎病毒（ＨＢＶ）前Ｓ２基因的作用。方法 设计合成一针对ＨＢＶａｙｗＤＮＡ前Ｓ２区第１１０，１２２和１３２位碱基的三靶位串联锤头状核酶基因，体外转录核酶基因和靶基因并进行切割实验；将核酶基因与逆转录病毒重组，转导２．２．１５细胞观察转导细胞聚人血清白蛋白受体的表达水平。结果 核酶在体外可有产切割靶原因的转录物，转入２．２．１５细胞后４周内对ＰＨＳＡ－Ｒ表达抑制率为５１．４５     

一种HCV靶向性M1GS核酶的构建及其体外活性

目的构建对HCV基因组具有特异切割作用的新型靶向性核酶-M1GS。方法针对HCV基因组的保守区(5′UTR)设计并合成一段引导序列,通过PCR扩增直接将该引导序列连接于大肠杆菌核糖核酸酶P的催化亚基(M1 RNA)的3′末端,从而构建一类靶向性M1GS核酶。结果体外切割实验表明,所构建的核酶(M1GS-HCV/C67)对HCV5′UTR具有明显的切割作用,切割的位点在靶序列67~68 nt之间,属于特异性切割。结论构建了一种对HCV5′UTR具有靶向切割活性的M1GS核酶,为胞内反义效应及动物模型内抗病毒效应的评价提供了实验材料,为新型抗HCV药物的研究奠定了基础。     

RNase P对人巨细胞病毒mRNA的体外靶定切割研究

目的探讨棱酶P对HCMV UL97 mRNA的体外切割能力.方法以人巨细胞病毒(human cytomegalovirus,HCMV)磷酸转移酶mRNA序列为靶设计extemal guide sequences(EGS),共价结合到大肠杆菌来源M1 RNA中,构建成M1GS-T5核酶.对其进行UL97基因亚克隆片段转录产物的体外切割实验.结果该核酶在一定离子浓度下具对UL 97mRNA片段产生特异切割能力.结论新构建得到的核酶MIGS-T5具特异性切割活性,可发展为一种抗病毒试剂.     

抗转化生长因子β1的U1 snRNA嵌合型核酶体外剪切作用

目的： 研究抗转化生长因子β₁ U1snRNA嵌合型锤头状核酶的细胞外切割活性。方法： 通过计算机设计针对TGFβ₁的锤头状核酶，然后把合成的核酶片段克隆入含有U1 snRNA启动子/增强子和终止子的U1 snRNA核酶载体中。通过RT-PCR扩增获得TGFβ₁的部分基因片段，将其克隆入T载体中T7启动子的下游，体外转录获得核酶和靶RNA，转录过程中掺入同位素，通过变性的聚丙烯酰胺凝胶电泳纯化回收。［³²P］标记的核酶与靶RNA在不同条件下进行切割反应，变性PAGE电泳，放射自显影，分析反应结果。结果： 活性的U1 snRNA嵌合型核酶（U1Rz803）在生理温度下具有良好特异的切割活性； 而点突变型核酶U1Rz803m没有切割活性，因此这些结果显示U1Rz803设计是正确的。结论： 本研究中制备的U1Rz803具有良好的特异催化切割活性。U1 snRNA嵌合型核酶U1Rz803有望在胞内抑制TGFβ₁的表达，为研究转化生长因子（TGF）β₁在造血调控中的作用机制提供有效工具。     

CIITA锤头状核酶的克隆及其体外活性

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。     

Efficient inhibition of hepatitis B virus replication by hammerhead ribozymes delivered by hepatitis delta virus

Although it has been suggested that hepatitis delta virus (HDV) can be used as a vector to deliver biologically active RNAs into hepatocytes, modified HDV as a specific transporting and replicating vector in anti-viral research has not been investigated. In this study, we focused on the development of HDV as a replicative vector to deliver hammerhead ribozyme into hepatocytes and the study of the roles of delivered hammerhead ribozyme on the replication of hepatitis B virus (HBV). To investigate the effects of ribozyme delivered by HDV on HBV replication, we designed two hammerhead ribozymes that specifically target the hepatitis B virus genome. These two ribozymes were then inserted into the genome of hepatitis delta virus. Results showed that transfection of cells with tandem modified HDV cDNA resulted in the production of monomer form of sense and anti-sense genomic RNA indicating the recombinant HDV-ribozyme could replicate effectively. Our data also indicated that ribozymes delivered by the modified HDV had higher level of inhibition activity against HBV replication than that of ribozyme alone. This system provides a new approach for the study of mechanisms of HBV replication as well as for the potential treatment of HBV infection.     

通过受体介导法将核酶特异导入肝细胞以阻断HBV蛋白表达的初步研究

应用半乳糖末端糖蛋白受体（ＡＳＧＰ－Ｒ）介导的内吞作用，将外源基因导入真核细胞，与脂质体介导的转染和细胞表面转铁蛋白受体（Ｔｆ－Ｒ）介导的内吞作用相比，虽然三种方式均能有效介导外源基因的转移，但ＡＳＧＰ－Ｒ法具有肝细胞特异性，而脂质体法和Ｔｆ－Ｒ法不具此特性。将克隆于真核表达载体的针对乙型肝炎病毒（ＨＢＶ）ｍＲＮＡＰｒｅＣ／Ｃ区的核酶质粒ｐＣＭＶ－Ｒｉｐｃ特异性导入肝细胞并发挥作用，通过酶联免疫吸附法（ＥＬＩＳＡ）检测细胞培养液中的乙型肝炎表面抗原（ＨＢｓＡｇ）和ｅ抗原（ＨＢｅＡｇ），评价核酶在细胞水平对ＨＢＶ抗原表达的阻断作用。结果表明当核酶质粒ｐＣＭＶ－Ｒｉｐｃ与ＨＢＶ抗原表达质粒ｐＵＣ－２ＨＢＶ共转染ＨｅｐＧ２细胞时，核酶对ＨＢｓＡｇ和ＨＢｅＡｇ表达的抑制率分别为５５．２９％和６８．７３％。     

通过受体介导法将核酶特异导入肝细胞以阻断HBV…

应用半乳糖末端糖蛋白受体介导的内吞作用，将外源基因导入真核细胞，与脂质体介导的传染和细胞表面转锪蛋白受介导的内吴作用相比，虽然三种方式均能有效介导外源基因的转移，但ＡＳＧＰ－Ｒ法具有肝细胞特异性，而脂质体法和Ｔｒ－Ｒ法不具此特性。将克隆于真核表达载体的针对乙型肝炎病毒ｍＲＮＡＰｒｅＣ／Ｃ区的核酶质粒ｐＣＭＶ－Ｒｉｐｃ特异性导入肝细胞 并发挥作用，通过酶联免疫吸附法检测细胞培养液中的乙型肝炎表现抗原     

Ribozyme mediated destruction of influenza A virus in vitro and in vivo

Short catalytic RNAs with inherent, specific endoribonuclease activity, called ribozymes, have recently been shown to exist in nature. According to the structural models artificial ribozymes have been designed that can potentially hydrolyse any chosen target RNA sequence in trans at a specific site. We have constructed and characterized in vitro hammerhead and hairpin ribozymes designed to cleave viral RNA segment 5 of influenza A virus. Both ribozymes were functional under optimal in vitro conditions, but quantitative measurements indicate that the hammerhead ribozyme is considerably more efficient at this target site than the hairpin ribozyme. Mg²⁺ dependent hammerhead ribozyme-mediated cleavage reactions were enhanced at higher temperature and in presence of spermidine, but catalytic activities were retained also in cellular extract S-100 or nuclear extracts at physiological temperatures. Recombinant plasmids derived from transfection vector pSV2-neo were engineered to allow the expression of specific ribozymes under the control of SV40 early promoter or SV40 early+ late promoters. These plasmids were introduced by transfection into COS cells, and their expression and enzymatic activities were analyzed in stable cell lines after selection of neomycin-re-sistance. Several permanent ribozyme-express-ing clones were established and characterized: ribozyme coding DNA sequences and synthesis of ribozyme RNA molecules in the transfected cells were determined and monitored by polymerase chain reactions. It was found that the highest levels (up to 70-80%) of resistance to influenza A virus strain X-31 super-infection was observed in COS cells transfected with plasmids containing SV40 early or SV40 early+late promoters coinciding with relatively high and constitutive rates of ribozyme expression. These results suggest the feasibility of developing ribozymes designed against influenza virus to achieve therapeutic value. © 1994 Wiley-Liss, inc.     

核酶抑制内皮素-1mRNA的表达

目的:从设计针对人内皮素-1mRNA的核酶DNA片段中,筛选能显著抑制ET-1mRNA表达的核酶。方法:应用锤头状核酶设计原理,将计算机设计人工合成的核酶片段亚克隆到真核表达载体pEGFP中,经脂质体转染入ECV304细胞后,用RT-PCR方法测定转染核酶的细胞内ET-1mRNA的相对含量,并用空载体作对照。结果:设计的核酶R145降低ECV304细胞ET-1mRNA的表达。结论:与对照组相比,核酶R145能显著抑制ECV304细胞ET-1mRNA的表达。     

Delivery of a hammerhead ribozyme specifically down-regulates the production of fibrillin-1 by cultured dermal fibroblasts

The hammerhead ribozyme is a small catalytic RNA molecule. Potential hammerhead ribozymes that possess a catalytic domain and flanking sequence complementary to a target mRNA can cleave in trans at a putative cleavage site within the target molecule. We have investigated the potential of hammerhead ribozymes to down-regulate the product of the fibrillin-1 gene (FBN1). Fibrillin is a 347 kDa glycoprotein that is a major constituent of the elastin-associated microfibrils. Mutations in the FBN1 gene are responsible for Marfan syndrome (MFS), a common systemic disorder of the connective tissue. Many FBN1 mutations responsible for MFS appear to act in a dominant-negative fashion, raising the possibility that reduction of the amount of product from the mutant FBN1 allele might be a valid therapeutic approach for MFS. A trans-acting hammerhead ribozyme (FBN1-RZ1) targeted to the 5' end of the human FBN1 mRNA has been designed and synthesized, and shown to cleave its target efficiently in vitro. FBN1-RZ1 cleavage is magnesium dependent and efficient at both 37 and 50 degrees C. Delivery of the FBN1-RZ1 ribozyme into cultured dermal fibroblasts, by receptor- mediated endocytosis of a ribozyme-transferrin-polylysine complex, specifically reduces both cellular FBN1 mRNA and the deposition of fibrillin in the extracellular matrix. These results suggest that the use of hammerhead ribozymes is a valid approach to the study of fibrillin gene expression and possibly to the development of a therapeutic approach to MFS.