丙肝病毒核心基因靶向性M1GS 核酶的体外抗病毒活性

[目的]丙型肝炎病毒(Hepatitis C Virus,HCV)是引起病毒性肝炎的重要病原.目前临床HCV感染多采用干扰素联合病毒唑进行治疗,但其应答率低且感染易反复,探索新型抗HCV治疗策略与药物具有紧迫的现实意义.[方法]基于大肠埃希菌来源的核糖核酸酶P(RNase P),针对HCV核心基因的序列,设计一小段与之互补的外部引导序列(Guide Sequence,GS),通过PCR将其共价连接至大肠埃希菌RNase P催化性亚基(M1 RNA)的3'末端,从而构建一种靶向性的核酶——M1GS.[结果]构建的M1 GS-HCV/C52核酶在体外可对靶RNA片段产生特异性切割;在HCV感染的Huh7.5.1细胞,该人工核酶也能够显著抑制HCV核心基因的表达,并使培养上清中HCV RNA的拷贝数减少约1500倍.[结论]本研究构建的M1GS-HCV/C52核酶具有显著的体外抗病毒活性,从而为HCV的治疗研究提供了一条潜在途径.     

HCV特异性M1GS核酶的构建及体外切割活性研究

针对HCV基因组中较为保守的区域-5'UTR,设计一段GS引导序列,并与大肠杆菌RNase P的催化亚基-M1RNA的3'末端共价结合,构建序列特异性M1GS核酶-M1GS-HCV/C20。体外实验证实,所构建的人工核酶对HCV 5'UTR具有明显的靶向切割活性,且这种切割发生于靶序列的特定位点。本研究将为进一步阐明该核酶在胞内的活性、乃至动物模型内评价其抗病毒效果提供实验材料,从而为新型抗HCV药物及反义基因治疗的研究奠定基础。     

HCMV UL97 mRNA序列特异性M1GS的构建及其体外切割活性研究

HCMV UL97基因编码一种蛋白激酶,该酶参与调控病毒DNA的复制和衣壳的形成,且序列异常保守,可作为抗HCMV治疗的重要靶位。基于HCMV UL97 mRNA T3位点附近的序列,设计一段与该位点互补的引导序列（Guide Sequence,GS）,并将其与大肠杆菌核酶P催化亚基（M1 RNA）的3’末端共价连接,构建了一种序列特异性的M1GS（M1-T3）。体外实验证实,所构建的M1-T3可与UL97 mRNA的T3位点特异性结合并产生有效的切割作用。进一步研究M1-T3的结构与其对底物片段靶向切割活性的关系,结果发现在M1 RNA与GS之间增加一段88核苷酸桥连序列的M1-T3（即M1-T3’）,其靶向切割活性大大增强。此外,去除M1-T3 3’末端的CCA序列,其靶向切割活性将基本丧失。上述结果表明,这段桥连序列和3’末端的CCA序列是M1-T3重要的结构元件。这不仅有助于阐明M1GS与其底物的相互作用机制,同时也为进一步评价M1-T3在体内对UL97基因表达及病毒复制的抑制活性奠定了基础。     

M1RNA及其在基因治疗上的研究进展

M1RNA是E.coli体内一种具有催化活性的RNA分子,能特异性的切割靶RNA分子,可用来抑制基因的表达,由M1RNA发展而来的M1GS是一种潜在的基因治疗药物,在病毒性疾病和肿瘤治疗上有着广阔的应用前景。     

丙型肝炎病毒RNA特异性核酶的切割活性研究

针对丙型肝炎病毒RNA(HCV-RNA)的5′非编码区和部分C区的二级结构,设计并合成了四个不同的锤头型核酶(ribozyme A, ribozyme B, ribozyme C₁, ribozyme C₂).首先应用体外切割实验筛选出作用于HCV-RNA起始密码子上游GTA↓位点的核酶RzA有较好的活性.为初步验证核酶RzA在细胞内的切割活性,经脂质体介导,将RzA-RNA与另一携带该核酶靶基因的质粒表达载体pCl-neo-luciferase(载体中荧光素酶基因受核酶靶基因的调控)共转染HepG₂细胞.通过测定荧光素酶基因的表达证实了核酶在细胞内有较好的切割活性.在此实验基础上,把RzA基因克隆至pCl-neo质粒表达载体中,再次经脂质体介导,将重组的表达载体pCl-neo-RzA与携带该核酶靶基因的质粒表达载体pCl-neo-luciferase共转染HepG₂细胞,获得了更好的切割效果.     

细胞周期蛋白D1特异性核酶表达载体的构建及其活性

应用mfold程序对锤头状核酶(ribozyme,Rz)和大鼠细胞周期蛋白(cyclin)D1基因的二级结构进行分析,设计合成锤头状Rz基因,通过RT-PCR扩增获得大鼠细胞周期蛋白D1目的基因,将Rz基因和细胞周期蛋白D1基因分别克隆入载体pGEM-3Zf( )中,体外转录Rz基因和靶基因并进行切割实验;将Rz基因与逆转录病毒载体pLXSN重组得到Rz真核表达载体pLXSN-Rz,将其转染入HSC-T6细胞,G418筛选出阳性细胞克隆,用RT-PCR检测细胞周期蛋白D1基因的表达。结果显示:针对目的基因的832位点设计合成了Rz832,成功获得Rz832基因、细胞周期蛋白D1mRNA的体外转录载体pGEM3Zf-Rz832和pGEM3Zf-cD1,经体外转录出Rz832(105nt)及细胞周期蛋白D1mRNA(1079nt)。体外切割实验证实Rz832能够特异性切割细胞周期蛋白D1mRNA,产生1014nt和65nt的切割产物,切割效率为80%。所构建的pLXSN-Rz832经酶切电泳、PCR鉴定显示,插入的Rz832序列大小约为57bp,与预期结果相同,经测序证实Rz832序列正确。转染pLXSN-Rz832的肝星状细胞(hepaticstellatecells,HSCs)细胞周期蛋白D1mRNA的表达受到明显抑制,仅为对照组的42.22%(t=-193.443,P<0.01),结果表明:Rz832能够在体外特异性切割细胞周期蛋白D1mRNA、并在HSC-T6细胞内有效抑制细胞周期蛋白D1基因的表达。     

抗人组织金属蛋白酶抑制剂—1核酶的体外制备和活性鉴定

研究了特异切割人瘢痕组织中组织金属蛋白酶抑制剂 1(tissueinhibitorofmetalloproteinases 1,TIMP 1)的锤头状核酶 ,测定了其体外切割活性。制备特异切割TIMP 1的U6snRNA嵌合型核酶基因克隆。TIMP 1mRNA基因片段克隆至T载体。用体外转录法大量制备以 [α 3 2 P]UTP标记的核酶及靶RNA ,进行体外切割实验。结果表明 :核酶∶底物 =1∶1时 ,活性的U6snRNA嵌合型核酶 (U6Rz35 8)在 5 0℃具有最佳切割活性 ,切割效率为 76 .34% ;37℃时 ,切割效率为 5 5 .2 1%。5 0℃时 ,Km=39.6nmol/L ,kcat=0 .2 1min-1;而点突变型核酶U6Rz35 8m 没有切割活性。制备的U6 Rz35 8有良好的特异切割活性 ,有望在瘢痕成纤维细胞内抑制人TIMP 1的表达。     

核酶的体外合成及其对鸭乙型肝炎病毒S基因体外转录物的定点切割

选择鸭乙型肝炎病毒作为核酶的靶病毒,设计针对病毒前基因组Ｓ基因区第１２８８位和１４６９位“锤头状”核酶序列（ＲＺ１和ＲＺ２）。经体外转录及做核酶切割,ＲＺ１和ＲＺ２均有切割作用,以ＲＺ１作用更强。鉴于核酶在相当于鸭体温（４２℃）有切割作用,因此有可能在鸭体内研究其切割作用。     

双位点核酶对乙型肝炎病毒C基因体外转录物的剪切作用

为探讨双位点核酶对乙型肝炎病毒C基因体外转录物的剪切作用,观察双位点核酶对单一核酶体外剪切的增强作用,同时比较串联核酶和混合核酶的体外切割作用,构建了核酶Rz1,Rz3, Rz1和Rz3的串联核酶(Rz13)体外转录载体, 经体外转录后切割靶RNA. 结果表明:双位点核酶,无论是串联或混合核酶均可增强单一核酶的体外切割作用, 串联和混合核酶中的单一核酶可独立发挥作用;当串联和混合数目为2个时,两者的切割效率差别不大(P＞0.05).     

Genetic polymorphisms and antiviral activity in the bovine MX1 gene

Bovine MX1 cDNAs consisting of 2280 bp from 11 animals of five breeds and from a cultured cell line were sequenced and compared with previously reported data. Ten nucleotide substitutions were synonymous mutations, and a single nucleotide substitution at 458 resulted in an amino acid exchange of Ile (ATT) and Met (ATG). A 13-bp deletion-insertion mutation was also found in the 3'-UTR. Based on the nucleotide substitutions found in this study, bovine MX1 cDNA was classified into 11 genotypes. A phylogenetic tree of the 11 genotypes suggested that the genotypes observed in Brahman were a great genetic distance from other genotypes. An 18-bp deletion-insertion variation at position 171 was found to be the result of alternative splicing. The 18-bp deletion-insertion is located at the boundary between exon 3 and intron 3. Permanently transfected 3T3 cell lines expressing bovine MX1 mRNA were established to analyse the antiviral potential against VSVDeltaG*-G infection. Transfected cell clones expressing bovine MX1 mRNA showed a significantly smaller number of cells infected with VSVDeltaG*-G compared with the control cells. These results indicate that the bovine MX1 protein has potent antiviral activity.     

Expression of an RNase P ribozyme against the mRNA encoding human cytomegalovirus protease inhibits viral capsid protein processing and growth

A sequence-specific ribozyme (M1GS RNA) derived from the catalytic RNA subunit of RNase P from Escherichia coli was used to target the mRNA encoding human cytomegalovirus (HCMV) protease (PR), a viral protein that is responsible for the processing of the viral capsid assembly protein. We showed that the constructed ribozyme cleaved the PR mRNA sequence efficiently in vitro. Moreover, a reduction of about 80% in the expression level of the protease and a reduction of about 100-fold in HCMV growth were observed in cells that expressed the ribozyme stably. In contrast, a reduction of less than 10% in the expression of viral protease and viral growth was observed in cells that either did not express the ribozyme or produced a catalytically inactive ribozyme mutant. Further examination of the antiviral effects of the ribozyme-mediated cleavage of PR mRNA indicates that (1) the proteolytic cleavage of the capsid assembly protein is inhibited significantly, and (2) the packaging of the viral genomic DNA into the CMV capsids is blocked. These observations are consistent with the notion that the protease functions to process the capsid assembly protein and is essential for viral capsid assembly. Moreover, our results indicate that the RNase P ribozyme-mediated cleavage specifically reduces the expression of the protease, but not other viral genes examined. Thus, M1GS ribozyme is highly effective in inhibiting HCMV growth by targeting the PR mRNA and may represent a novel class of general gene-targeting agents for the studies and treatment of infections caused by human viruses, including HCMV.     

牛γ-干扰素在重组杆状病毒中的表达及其抗病毒活性的测定

研究利用Bac-To-Bac杆状病毒表达系统构建含有牛γ-干扰素(Bovine interferon-γ,BoIFN-γ)完整开放阅读框的供体质粒pFastBacTM1-BoIFN-γ,转化DH10Bac感受态细胞获得重组穿梭质粒rBacmid-BoIFN-γ,转染sf9昆虫细胞救获表达BoIFN-γ的重组杆状病毒rBac-BoIFN-γ。采用抗BoIFN-γ单克隆抗体作为一抗进行间接免疫荧光(IFA)及间接ELISA检测,表明BoIFN-γ在重组杆状病毒rBac-BoIFN-γ感染的sf9昆虫细胞中获得正确表达。利用VSV*GFP-MDBK细胞系统测定rBoIFN-γ抗病毒活性,重组杆状病毒表达重组BoIFN-γ(rBoIFN-γ)能有效抑制水疱性口炎病毒(VSV)在牛肾细胞(MDBK)上的复制,rBac-BoIFN-γ感染sf9昆虫细胞上清抗病毒活性为2×105IU/mL,而且其抗病毒活性可以被鼠抗原核表达重组BoIFN-γ免疫血清阻断。结果表明:rBoIFN-γ在重组杆状病毒rBac-BoIFN-γ感染的sf9昆虫细胞中获得良好表达,并具有高效抗病毒活性。     

丙型肝炎病毒C+E1区基因在原核细胞中的克隆与表达

将丙型肝炎病毒Ｃ＋Ｅ１区基因插入到原核高效表达载体ｐＢＶ２２１质粒中,构建了质粒ｐＢＶ２２１ＨＣＶ／Ｃ＋Ｅ１作为表达载体,然后,将含有该质粒的宿主大肠杆菌进行升温诱导表达ＨＣＶ／Ｃ＋Ｅ１区基因,并对表达产物进行了生物活性的检测。结果表明,插入到表达载体ｐＢＶ２２１中的ＨＣＶ／Ｃ＋Ｅ１基因片段能够得到有效的表达,表达产物主要为非融合蛋白形式存在于细胞中,同时这种Ｃ区和Ｅ１区连接共表达的产物保持了良好的抗原活性     

Synthesis,in vitro and in vivo antitumor and antiviral activity of novel 1-substituted benzimidazole derivatives

Abstract

A novel series of 5-nitro-1H-benzimidazole derivatives substituted at position 1 by heterocyclic rings was synthesized. Cytotoxicity and antiviral activity of the new compounds were tested. Compound 3 was more active than doxorubicin against A-549, HCT-116 and MCF-7. However, compound 3 showed no activity against human liver carcinoma Hep G-2 cell line. Compounds 9 and 17b (E) showed potency near to doxorubicin against the four cell lines. The acute toxicity of compound 9 on liver cancer induced in rats was determined in vivo. Interestingly, it showed restoration activity of liver function and pathology towards normal as compared to the cancer-bearing rats induced by DENA. Compounds 17a (Z), 17b (E) and 18a (Z) were the most promising compounds for their antiviral activity against rotavirus Wa strain.     

表面活性素体外抗伪狂犬病毒活性研究

研究了表面活性素（surfactin）体外抗伪狂犬病毒（Pseudorabies Virus,PRV）效果。观察表面活性素的细胞毒性、对PRV直接灭活作用、抗PRV吸附作用及对PRV生物合成抑制作用。结果表明表面活性素对猪肾（porcinekidney,PK-15）细胞的TD50和TD0分别为31．25、4．03μg／mL;具有直接灭活PRV效果,不具有抗PRV吸附作用,对PRV生物合成无显著影响.