抗半胱氨酸蛋白酶-7核酶的克隆、体外表达与切割活性的研究

目的 设计并合成抗小鼠半胱氯酸蛋白酶-7(Caspaso7)的锤头状核酶,并对它们的体外表与体外切割活性进行研究。方法 采用计算机软件对锤头状核酶和小鼠Caspase 7基因的二级结构进行分析,核酶的DNA序列在体外由自动合成仪合成,小鼠Caspase-7日的基因通过RTPCR扩增获得,将核酶基因和caspase-7基因分别克隆入载体pBSKneoU6’和pGEM-T中,通过体外转录得到核酶和caspase-7 mRNA,通过体外切割筛选出具有切割活性的核酶。结果 针对目的基因的333和394位点设计合成了两个核酶：Rz333和Rz394。成功获得caspase-7 mRNA的表达载体PC7及含有核酶的重组质粒PU6Rz333和pU6Rz394,通过体外转录表达出含有caspase7mRNA的987nt的靶mRNA及完整的核酶Rz333(47nt)和Rz394(50nt)。体外切割实验证实Rz333能够定点切割caspase-7mRNA,产生243nt和744nt的切割产物,切割效率为67．98％。Rz394不能切割靶基因。结论 核酶Rz333能够位点特异性的切割caspase-7 mRNA。     

丙型肝炎病毒特异性锤头样核酶体外转录及切割活性的初步研究

目的　研究针对丙型肝炎病毒 (HCV)特异性锤头样核酶 (Rz)的体外转录及切割活性。方法　设计 3种锤头样核酶 :Rz1、Rz2 分别作用于HCVRNA 5′ 非编码区 (5′ NCR)上核酸序列 136～16 0、313～ 337,Rz3 作用于核心 (C)区上核酸序列 373～ 388。为区别于反义核酸的封闭作用 ,设计在Rz3 的催化环上存在点变异 (G取代A)的变异核酶Rzm用作对照。体外转录出靶HCVRNA和核酶RNA ,在一定条件下 ,将3 2 P标记的靶HCVRNA与核酶RNA按不同浓度比例进行切割反应 ,电泳后放射自显影 ,通过同位素扫描成像分析仪分析来评价核酶切割效率。结果　除Rzm外 ,在生理温度下 ,3种核酶均有活性 ,并随核酶浓度增加而提高 ;核酶切割靶位点距HCV起始密码子近切割效率高。结论　设计并观察到体外具有HCV特异性切割活性的锤头样核酶 ,为进行核酶的细胞内应用研究奠定基础     

腺病毒介导核酶对细胞内HBV基因表达的抑制作用

目的观察针对HBV(乙型肝炎病毒)C区双位点核酶在细胞内阻断C区基因表达的作用。方法采用亚克隆技术,从pGEM—Rz123(含针对HBV C区双位点核酶)上切下双位点核酶的片段,定向克隆于腺病毒表达载体pAd-CMV中。利用Lipofectamine2000介导,将重组病毒pAdCMV—Rz及pAdCMV—laz感染2.2.15细胞中,采用打点杂交技术观察核酶的表达,采用ELISA(酶联免疫)、免疫荧光、共聚焦定量、图象分析法、Western blot分析Hbe/HBcAg的表达。结果感染的2.2.15细胞2周后,打点杂交证实可表达针对HBV C区双位点核酶。ELISA方法检测发现核酶抑制HBeAg表达68.6％,用免疫荧光、免疫组化、图象分析法、Western blot分析证实核酶可抑制HBV细胞内表达。结论该双位点核酶通过针对HBV C区基因的剪切作用,阻断C区基因表达,抑制了HBe/HBcAg的表达。     

U1 snRNA嵌合体核酶的抗丙型肝炎病毒作用

目的鉴定特异性抗丙型肝炎病毒(HCV)核酶与具有细胞核靶向性的U1 snRNA组成的嵌合体在体外切割HCV RNA的活性。方法通过聚合酶链反应及克降的力。法用HCV特异性核酶(Rz)序列戢代质粒pBSIISK U1(含有人类野生型U1 snRNA基因全序)中U1 snRNA第三个茎环结构，重组质粒命名为pBSIISK (U1-Rz)。再将pBSIlSK (U1-Rz)中核酶与U1 snRNA组成的嵌合体基因止向充隆于pGEM-T载体T7启动子的下游，重组质丰立命名为pGFM(U1-Rz)。质粒pGEM-(U1-Rz)和pGEM Rz[含有与pGEM(U1-Rz)相同的核酶序列]体外转录后，转录产物与靶RNAs在一定条件下进行切割反应，电泳后放射自显影。结果U1 snRNA嵌合体核酶构建成功。嵌合体核酶与核酶对靶RNAs均有切割活性，且二者的切割活性相似。随着时间的延长和工具酶体积比的增加切割效率增加。结论特异性抗HCV核酶与U1嵌合体在体外具有良好的持异性催化切割活性。     

抗内皮细胞表面主要组织相容性复合物Ⅱ类分子转录激活因子锤头状核酶的克隆及活性表达

目的 :拟克隆抗内皮细胞表面主要组织相容性复合物Ⅱ类分子转录激活因子 (CⅡTA)锤头状核酶 ,抑制血管内皮细胞表面主要组织相容性复合物 (MHC Ⅱ )分子的表达。方法 :设计并合成针对人类CⅡTA第 134、2 18、4 6 4位点的一组锤头状核酶 (Rz134、Rz2 18、Rz4 6 4 ) ,及其对应的CⅡTA靶基因m5 2 3。核酶体外切割实验鉴定Rz4 6 4活性最高 ,将Rz4 6 4克隆到真核表达载体pIRES2 EGFP(pIRES2 Rz4 6 4 ) ,并通过纳米载体介导其稳定转染人类脐静脉内皮细胞系 ,流式细胞术检测HLA DR、DP、DQ类抗原表达 ,RT PCR检测CⅡTAmRNA水平。结果 :pIRES2 Rz4 6 4阳性内皮细胞与对照组比较 ,HLA DR、DP、DQ抗原表达分别降低了 76 .2 6 %、87.75 %及 78.99% ,同时CⅡTAmRNA含量亦下降了 6 9%。结论 :Rz4 6 4抑制了CⅡTAmRNA含量 ,并阻止其调控的相应基因MHCⅡ分子的表达     

抗Caspase-3核酶的体外制备与活性鉴定

目的 研究针对大鼠凋亡基因Caspase-3设计的核酶（Rz107和Rz544）的体外转录,切割及活性鉴定。方法 大鼠Caspase-3基因的PCR片段克隆于T载体T7启动子下游,32^P标记的体外转录物作为靶RNA。设计合成并克隆针对大鼠Caspase-3 mRNA的核酶（Rz107和Rz544）,32^P标记转录,核酶与靶RNA按一定比例进行体外切割实验。结果 Rz107在37℃有活性。在一定温度范围内,其切割效率随温度升高而升高,最适温度为50℃。其米氏常数（km）为14.13mol/L,酶催化反应速度常数（kcat）为2.31/min。而Rz544则无切割活力。结论 体外制备的Rz107具有良好的特异催化切割活性,它有望通过切割（Caspase-3)而抑制凋亡发生,可发展为新一代抗肝炎症的核酸药物。     

不同长度丁型肝炎病毒核酶的体外自剪切活性及其意义

目的分析不同长度丁型肝炎病毒核酶的体外自剪切活性.方法用Xba Ⅰ,EcoRⅠ或BamHI将含有HDV.Rz序列的pRz277正向质粒(pRz277A)和pRz277反向质粒(pRz277B)线性化,以α-32p-UTP标志和T7噬菌体RNA聚合酶转录出不同长度的基因组核酶(g.Rz)和抗基因组(即复制中间体)核酶(ag.Rz),在一定条件下进行自剪切反应,然后作聚丙烯酰胺凝胶电泳(PAGE)和放射自显影.结果g.Rz24/100(自剪切位点5和3端分别含24nt和100nt,下同)、ag.Rz38/119在适当条件下绝大部分发生自剪切.g.Rz24/253和ag.Rz38/239也具有一定自剪切活性,但显著低于前两者,即使温度升至55℃或加入去离子甲酰胺也是如此.结论适宜长度的HDV.Rz在体外具有较高的自剪切活性.这些发现有助于我们下一步研究HDV.Rz的反式剪切作用.     

U1小核核糖核酸嵌合体核酶细胞内抑制丙型肝炎病毒表达的研究

目的观察以U1小核核糖核酸为靶向基因构建的嵌合体核酶在细胞内抑制丙型肝炎病毒（HCV）表达的作用。方法通过聚合酶链反应及克隆连接反应，将针对HCV核心区的特异性核酶序列代替了U1小核RNA的部分区域，构建出嵌合体核酶的真核表达载体；将其与带有荧光素酶报告基因的靶基因共转染Huh7细胞系，通过Western blot及发光检测仪来判定抑制效率并与相同序列核酶的效率进行比较。结果U1-嵌合体核酶构建成功；共转染结果显示核酶及嵌合体核酶对HCV表达均有抑制作用，但以嵌合体核酶的效率为高。结论以U1小核核糖核酸作为核酶载体可以增加核酶在细胞内的切割活性。     

特异性脱氧核酶对丙型肝炎病毒的体外消化作用

目的研究特异性脱氧核酶对丙型肝炎病毒（HCV）的体外消化作用。方法设计3个分别作用于HCV RNA 5＇非编码区（5＇ NCR）上157、168、173位点的脱氧核酶（DRz），分别命名为DRz1，DRz2,DRz3，均以5＇GGCTAGCTACAACGA-3’为脱氧核酶的催化活性中心，用内切酶XbaⅠ将含有HCV病毒序列的质粒pCMV/T7 NCRC,△Luc消化成线性，以此为模板体外转录出用α-^32PIUTP进行标记的靶HCV RNA。在37℃,PH7.5，Mg^2 10mmol/L等条件下，将HCV RNA（10nmol/L）与3个脱氧核酶（1μmol/L）分别混合进行切割反应，并进一步比较不同Mg^2浓度下DRz3的切割反应效率，反应产物在8%变性聚丙烯酰胺凝胶上电泳分离并行放射自显影，应用凝胶成像分析仪评价脱氧核酶的切割效率。结果在设定反应条件下，3个脱氧核酶对HCV病毒均有切割活性，随着Mg^2浓度的增加，DRz3的切割活性增强，结论特异性脱氧核酶可有效切割HCV RNA 5＇NCR的RNA，且切割效率与Mg^2浓度呈正相关。     

原癌基因c-myc核酶表达载体抑制血管平滑肌细胞的c-myc表达

目的构建原癌基因c-myc核酶表达载体以抑制心血管系统细胞异常增殖。方法采用DNASIS软件对大鼠c-mycmRNA进行二级结构和最低自由能分析,确定第4600位核苷酸作为核酶的切割位点,设计并合成c-myc核酶。通过体外切割实验证明该核酶的有效性。进一步构建成c-myc核酶表达载体,转染同步化血管平滑肌细胞(VSMC)以检测其对原癌基因c-myc表达的影响。结果体外切割实验显示该核酶能有效地将85bp的模板切割成60和25bp的二个片段。所构建载体经酶切法和基因测序法证明其序列的正确性。转染VSMC后能有效抑制血管紧张素Ⅱ诱导的c-myc的表达。结论c-myc核酶表达载体能有效抑制VSMC的c-myc过度表达,适合用于动脉增殖性疾病的基因治疗。     

Inhibition of hepatitis C virus (HCV)-RNA-dependent translation and replication of a chimeric HCV poliovirus using synthetic stabilized ribozymes

Ribozymes are catalytic RNA molecules that can be designed to cleave specific RNA sequences. To investigate the potential use of synthetic stabilized ribozymes for the treatment of chronic hepatitis C virus (HCV) infection, we designed and synthesized hammerhead ribozymes targeting 15 conserved sites in the 5' untranslated region (UTR) of HCV RNA. This region forms an internal ribosome entry site that allows for efficient translation of the HCV polyprotein. The 15 synthetic ribozymes contained modified nucleotides and linkages that stabilize the molecules against nuclease degradation. All 15 ribozymes were tested for their ability to reduce expression in an HCV 5' UTR/luciferase reporter system and for their ability to inhibit replication of an HCV-poliovirus (HCV-PV) chimera. Treatment with several ribozymes resulted in significant down-regulation of HCV 5' UTR/luciferase reporter expression (range 40% to 80% inhibition, P <.05). Moreover, several ribozymes showed significant inhibition (>90%, P <.001) of chimeric HCV-PV replication. We further show that the inhibitory activity of ribozymes targeting site 195 of HCV RNA exhibits a sequence-specific dose response, requires an active catalytic ribozyme core, and is dependent on the presence of the HCV 5' UTR. Treatment with synthetic stabilized anti-HCV ribozymes has the potential to aid patients who are infected with HCV by reducing the viral burden through specific targeting and cleavage of the viral genome.     

Identification of eIF2Bgamma and eIF2gamma as cofactors of hepatitis C virus internal ribosome entry site-mediated translation using a functional genomics approach

The 5'-untranslated region of hepatitis C virus (HCV) is highly conserved, folds into a complex secondary structure, and functions as an internal ribosome entry site (IRES) to initiate translation of HCV proteins. We have developed a selection system based on a randomized hairpin ribozyme gene library to identify cellular factors involved in HCV IRES function. A retroviral vector ribozyme library with randomized target recognition sequences was introduced into HeLa cells, stably expressing a bicistronic construct encoding the hygromycin B phosphotransferase gene and the herpes simplex virus thymidine kinase gene (HSV-tk). Translation of the HSV-tk gene was mediated by the HCV IRES. Cells expressing ribozymes that inhibit HCV IRES-mediated translation of HSV-tk were selected via their resistance to both ganciclovir and hygromycin B. Two ribozymes reproducibly conferred the ganciclovir-resistant phenotype and were shown to inhibit IRES-mediated translation of HCV core protein but did not inhibit cap-dependent protein translation or cell growth. The functional targets of these ribozymes were identified as the gamma subunits of human eukaryotic initiation factors 2B (eIF2Bgamma) and 2 (eIF2gamma), respectively. The involvement of eIF2Bgamma and eIF2gamma in HCV IRES-mediated translation was further validated by ribozymes directed against additional sites within the mRNAs of these genes. In addition to leading to the identification of cellular IRES cofactors, ribozymes obtained from this cellular selection system could be directly used to specifically inhibit HCV viral translation, thereby facilitating the development of new antiviral strategies for HCV infection.     

Ribozyme-mediated suppression of Moloney murine leukemia virus and human immunodeficiency virus type I replication in permissive cell lines.

Several hammerhead ribozymes targeted to different sites within the retroviral packaging (psi) sequences of the Moloney murine leukemia virus (Mo-MLV) and the human immunodeficiency virus type 1 (HIV-1) were designed and shown to cleave target RNA in vitro at the chosen sites. The engineered ribozymes, as well as antisense sequence complementary to the Mo-MLV psi packaging region, were cloned into the 3' untranslated region of the neomycin-resistance gene (neo). This was coupled to the simian virus 40 early promoter within the pSV2neo vector. For the ribozymes against the Mo-MLV psi site, the constructs were transfected into Mo-MLV-infected and virus-producing mouse NIH 3T3 cells. With the exception of one of the single ribozymes (the one least effective in cutting target RNA in vitro), all of the constructs effectively (70-80%) suppressed retrovirus production. These results demonstrate a direct correlation between in vitro cleavage and in vivo ribozyme-mediated virus suppression. In addition, a ribozyme targeted to the HIV-1 psi packaging site was engineered into the same vector and transfected into the human T-cell line SupT1. The transfectants were cloned and then challenged with HIV-1. When compared to vector-transfected control cells, a significant reduction in HIV-1 production was observed as measured by p24 and syncytia formation assays. This study demonstrates a feasible approach to the suppression of retrovirus replication by targeting the psi packaging site with hammerhead ribozymes.     

胃癌相关基因GCRG213正反义真核表达载体的构建及鉴定

目的:构建胃癌相关基因GCRG213正、反义真核表达载体．方法:从pGEM-T质粒上扩增出的胃癌相关基因GCRG213的DNA片段,两端分别引入限制性内切酶KpnI,BamHI和EcoRI, BamHI识别位点．按正向、反向克隆入真核表达载体pcDNA3．1( )．测序正确的重组子pcDNA3．1-a(含GCRG213正向克隆), pcDNA3．1-b(含GCRG213反向克隆)和空载体经脂质体转染人胃癌细胞系MKN45细胞, G418筛选获得稳定转染的细胞株,采用半定量RT-PCR及Wlestern blot比较转染不同质粒的 MKN45细胞中GCRG213在mRNA和蛋白质水平上的表达差异．结果:经测序证实,GCRG213正向克隆和反向克隆正确插入真核表达载体 pcDNA3．1( ),组成重组子pcDNA3．1-a(含正向克隆),pcDNA3．1-b(含反向克隆)．重组子 pcDNA3．1-a,pcDNA3．1-b和空载体经脂质体转染人胃癌细胞系MKN45细胞,G418筛选获得稳定转染的细胞株．与对应的空载体比较,RT-PCR结果显示转染pcDNA3．1-a的 MKN45细胞中其mRNA的表达上调35．4％,而转染pcDNA3．1-b的MKN45细胞中其mRNA 的表达下调32．1％;Western blot结果显示转染 pcDNA3．1-a的MKN45细胞中其蛋白的表达上调49．4％,而转染pcDNA3．1-b的MKN45细胞中其蛋白的表达下调50．3％．结论:成功构建胃癌相关基因GCRG213正、反义真核表达载体．     

Hammerhead ribozymes with cleavage site specificity for NUH and NCH display significant anti-hepatitis C viral effect in vitro and in recombinant HepG2 and CCL13 cells

BACKGROUND/AIMS: Four different ribozymes (Rz) targeting the hepatitis C virus (HCV) 5'-non-coding region (NCR) at nucleotide (nt) positions GUA 165 (Rz1), GUC 270 (Rz2), GUA 330 (Rz3) and GCA 348 (Rz1293) were compared for in vitro cleavage using a 455 nt HCV RNA substrate. The GUA 330 (Rz3) and GCA 348 (Rz1293) ribozymes, both targeting the HCV loop IV region, were found to be the most efficient, and were further analyzed in an in vitro translation system. METHODS: For this purpose RNA transcribed from a construct encoding a HCV-5'-NCR-luciferase fusion protein was used. Cleavage-inactive (Rz1426), mismatch (Rz1293m) or unrelated ribozymes (Rz1437) were synthesized as controls for Rz-1293. HCV specificity was analysed by competition experiments using sense and mismatch oligodeoxynucleotides HCVrzCI and HCVrzMM, respectively. RESULTS: A chemically modified nuclease-resistant variant of the GCA 348 cleaving ribozyme was selected for cell culture experiments using recombinant HepG2 or CCL13 cell lines stably transfected with a HCV-5'-NCR-luciferase target construct. CONCLUSIONS: This ribozyme (Rz1293) showed an inhibitory activity of translation of more than 70% thus verifying that the GCA 348 cleavage site in the HCV loop IV is an accessible target site in vivo and may be suitable for the development of novel optimized hammerhead structures.     

丙型肝炎病毒5＇端非编码区和NS3蛋白酶共调控外分泌性碱性磷酸酶表达细胞模型的建立及意义

目的 构建丙型肝炎病毒5'端非编码区(HCV 5'NCR)和NS3丝氨酸蛋白酶共调控外分泌性碱性磷酸酶(SEAP)表达细胞模型,并分析其用于抗HCV药物筛选和评价的可行性。 方法 用聚合酶链反应技术扩增HCV 5'NCR和NS3/4A片段,定向克隆至表达质粒pSEAP2-Control的SEAP基因上游,构建含HCV 5'NCR-NS3/4A-SEAP嵌合基因的重组表达质粒pNCR-NS3/4A-SEAP。将重组质粒转染至肝细胞株QSG7701,用化学发光法检测SEAP的表达,并观察HCV 5'NCR区对应的反义寡聚核苷酸(ASODN)和丝氨酸蛋白酶抑制剂TPCK对SEAP表达的影响。 结果 重组质粒pNCR-NS3/4A-SEAP有高强度SEAP表达,5μmol/L、10μmol/L ASODN和100μmol/L TPCK对SEAP表达有显著抑制作用(t值分别为4.315、6.985、6.949,P值均<0.01)。 结论 重组质粒pNCR-NS3/4A-SEAP的SEAP表达受HCV 5'NCR和NS 3蛋白酶共调控,建立的细胞模型可用于以HCV 5'NCR和NS3蛋白酶为靶位点的药物筛选和评价。