一个双价锤头型核酶在体外对两种不同植物病毒RNA的专一切割作用

根据锤头核酶模型,设计合成了一个以黄瓜花叶病毒（ＣＭＶ）外壳蛋白（ＣＰ）亚基因组ＲＮＡ为底物的锤头型核酶（ＲＺＣ）,在证明它能有效切割该底物后,再将这个核酶与一个能专一性切割烟草花叶病毒（ＴＭＶ）移动蛋白（ＭＰ）亚基因组ＲＮＡ的锤头型核酶（ＲＺ１）相互串联构成了一个双价核酶（ＲＺ１Ｃ）,体外结果表明,这个双价核酶能与相应的单价核酶ＲＺ１和ＲＺＣ一样专一而有效地切割ＣＭＶ ＣＰ和ＴＭＶ ＭＰ ＲＮＡ     

特异切割马铃薯纺锤形块茎类病毒负链的多价核酶的构建和体外活 …

根据锤头型核酶的作用模式,设计、合成并克隆了特异切割马铃薯纺锤形块茎类病毒（ＰＳＴＶｄ）负链ＲＮＡ不同区域位点的双价和三价锤头型核酶基因。通过体外转录,将ＰＳＴＶｄ负链ＲＮＡ分别与双价和三价核酶混合,３７℃温育２ｈ。结果表明,双价核酶和三价核酶均表现出较高的切割活性,其中双价核酶处理的切割产物的大小与理论值相符合。三价核酶虽表现出较高的切割活性,但只是其中一价核酶在起作用,讨论了二价和三价核酶的应     

特异切割马铃薯卷叶病毒复制酶基因负链的核酶研究

根据锤头状核酶的作用模式,设计、合成并克隆了特异性切割马铃薯地病毒中国分离株（ＰＬＲＶ－Ｃｈ）复制酶基因负链ＲＮＡ的核酶序列,以体外转录的ＰＬＲＶ－Ｃｈ复制酶基因负链ＲＮＡ作为底物,与转录的核酶ＲＮＡ共同保温,以检测核酶对底和的体外切割作用。实验结果表明,核酸疼 ＲＮＡ对ＰＬＲＶ－Ｃｈ复制酶基因负锭ＲＮＡ具有特异切割作用。     

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。     

对烟草花叶病毒外壳蛋白亚基因组RNA生成机制的进一步研究

用原生质体瞬间表达的方法证明,在烟草细胞中ＴＭＶ外壳蛋白亚基因组ＲＮＡ的生成,不是通过核酸酶在基因组ＲＮＡ上直接切割,也不是通过在生成负链ＲＮＡ时预成性终止,再从这种负链ＲＮＡ终止部位起始产生的,ＴＭＶ外壳蛋白亚基因组ＲＮＡ,是ＴＭＶ识别利用负链ＲＮＡ中间的亚基因组启动子后产生的,这个启动子位于外壳蛋白读码框５′端２５６碱基范围之内,但大于４８个碱基,虽然ＴＭＶ能利用单链的负链ＲＮＡ上的亚基因组启     

发夹核酶的研究与应用

核酶 (ｒｉｂｏｚｙｍｅ)是既能特异识别又能特异切割小分子ＲＮＡ的核酸内切酶 ,其本身也是ＲＮＡ ,主要包括发夹核酶、锤头核酶、丁肝病毒、链孢霉属ＶＳ和铅依赖性ＲＮＡ ,共同特点是可逆地切割底物ＲＮＡ的磷酸二酯键 ,生成 5′ ＯＨ和 2′ ,3′ 环磷酸末端。虽然催化产物相似 ,但它们的结构和催化机制却是很不相同的。发夹核酶 (ｈａｉｒｐｉｎｒｉｂｏｚｙｍｅ)发现于三种不同植物ＲＮＡ病毒 ,即烟草环点病毒 (ｔｏｂａｃ ｃｏｒｉｎｇｓｐｏｔｖｉｒｕｓ) ,菊苣黄色斑点病毒型 (ｃｈｉｃｏ ｒｙｙｅｌｌｏｗｍｏｔｔｌｅｖｉｒｕｓｔ…     

抗水稻条纹叶枯病毒核酶的设计,克隆及体外活性测定

为探索控制水稻条纹叶枯病毒（Ｒｉｃｅｓｔｒｉｐｅｖｉｒｕｓ,ＲＳＶ）设计合成了特异切割该病毒ＲＮＡ保守区及编码病害特异性蛋白（ＤｉｓｅａｓｅＳｐｅｃｉｆｉｃＰｒｏｔｅｉｎ,ＤＳＰ）基因的核酶,核酶基因的长度均为４０个碱基,用化学合成方法合成其正链及与其３＇－末端互补的１５个碱基引物,用ＴａｇＤＮＡ多聚酶合成其互补链。双链ＤＮＡ直接插入克隆载体ＰＧＥＭ３ｚｆ（＋）的Ｓｍａｌ位点。序列测定表明,克隆得到的核酶序列与设计的核酶序列完全一致。经ＳＰ６ＲＮＡ多聚酶体外转录得到核酶ＲＮＡ。当核酶ＲＮＡ与以同样方法转录得到的靶基因ＲＮＡ混合反应,可得到预期结果相同的切割片段,表明两种核酶在体外均具有特异性切割活性。     

利用TMV—U1作为载体表达鼠肝炎病毒S糖蛋白片段

为了验证转基因烟草中表达的外壳蛋白（ＣＰ）能够重新包被侵入的烟草花叶病毒（ＴＭＶ）的假设,利用抗原表位标记的方法观察ＣＰ亚单位在病毒５′端的交换。通过ＰＣＲ 方法将来源于鼠肝炎病毒（ＭＨＶ） Ｓ蛋白的两个小肽段（１１ ａ．ａ．和１５ ａ．ａ．）的ＤＮＡ序列分别插入ＴＭＶ－Ｕ１ ＣＰ基因邻近３′端的两个位点,并构建了带有外源序列的ＴＭＶ 侵染克隆Ｖ９ （１１ ａ．ａ．）和Ｅ１５ （１５ ａ．ａ．）。通过体外转录反应,得到Ｖ９ ＲＮＡ 及Ｅ１５ ＲＮＡ。突变病毒ＲＮＡ 侵染烟草（Ｎｉｃｏｔｉａｎａ ｔａｂａｃｕｍ ）后表现不同特性。Ｖ９ 和Ｅ１５ 侵染ＸａｎｔｈｉＮＮ烟草后同野生型ＴＭＶ一样产生枯斑。但是,当它们侵染Ｘａｎｔｈｉｎｎ 烟草时,Ｖ９ 产生同侵染ＸａｎｔｈｉＮＮ 烟草相同的枯斑,而Ｅ１５的特性同ＴＭＶ－Ｕ１几乎完全相同,能对Ｘａｎｔｈｉｎｎ 烟草进行系统侵染并在叶片中聚集大量的带有外源片段的外壳蛋白,而且病毒的结构极其稳定。Ｖ９ 和Ｅ１５ 特性的差异可能是由于外源片段在外壳蛋白中存在位置的不同影响了外壳蛋白的结构所致     

核酶对对虾白斑病毒的一个可能的早晚期基因的体 …

报道了对虾白斑杆状病毒的一个可能的早晚期基因（１１９７ｂｐ基因）的克隆和序列分析,并针对此基因的转录产物,用计算机软件设计了二个锤头状核酶（ＲＺ１和ＲＺ２）。体外切不两个核酶均能在合适条件下,对靶基因进行完全的切割。这些研究表明,有可能利用核酶对对虾杆状病毒的复制和增殖进行抑制。     

特异切割12-脂加氧酶mRNA的核酶体外活性及动力学研究

根据锤头状核酶（Ｒｉｂｏｚｙｍｅ）的作用模式,设计、合成并克隆了特异性切割１２－脂加氧酶（１２－ＬＯ）ｍＮＲＡ的核酶基因。以合成的２５个核苷酸长的１２－脂加氧酶ＲＮＡ片段为底物与转录的核酶ＲＮＡ一起保温检测其体 割活性。实验结果表明,在３７℃保温时,核酶在体外对１２－脂加氧酶具有较高的特异切割活性,其Ｋｍ值为１３００ｎｍｏｌ／Ｌ,其ｋｃａｔ值为０．０８３／ｍｉｎ,在５０℃保温时,核酶具有很高的切割     

A ribozyme gene and an antisense gene are equally effective in conferring resistance to tobacco mosaic virus on transgenic tobacco

Ribozymes of the hammerhead class can be designed to cleave a target RNA in a sequence-specific manner and can potentially be used to specifically modulate gene activity. We have targeted the tobacco mosaic virus (TMV) genome with a ribozyme containing three catalytic hammerhead domains embedded within a 1 kb antisense RNA. The ribozyme was able to cleave TMV RNA at all three target sites in vitro at 25°C. Transgenic tobacco plants were generated which expressed the ribozyme or the corresponding antisense constructs directed at the TMV genome. Six of 38 independent transgenic plant lines expressing the ribozyme and 6 of 39 plant lines expressing the antisense gene showed some level of protection against TMV infection. Homozygous progeny of some lines were highly resistant to TMV; at least 50% of the plants remained asymptomatic even when challenged with high levels of TMV. These plants also displayed resistance to infection with TMV RNA or the related tomato mosaic virus (ToMV). In contrast, hemizygous plants of the same lines displayed only very weak resistance when inoculated with low amounts of TMV and no resistance against high inoculation levels. Resistance in homozygous plants was not overcome by a TMV strain which was altered at the three target sites to abolish ribozyme-mediated cleavage, suggesting that the ribozyme conferred resistance primarily by an antisense mechanism.     

丙型肝炎病毒特异性核酶对HepG2.9706细胞HCV5′NCR基因表达的抑制作用

 为探讨锤头型核酶抗丙型肝炎病毒的作用 ,根据HCV 5′NCR及翻译起始区的二级结构 ,设计及合成了 1个锤头型核酶RzA .将其插入pCI neo表达载体的多克隆位点 ,构建了表达RzA的质粒pHCV RzA .采用转基因细胞模型HepG2 970 6细胞 ,评价了pHCV RzA质粒对HCV 5′NCR调控荧光素酶表达的抑制活性 .结果表明 ,在HepG2 970 6细胞中 ,pHCV RzA以序列特异性、剂量依赖性方式抑制荧光素酶基因的表达 ,抑制率达 80 % ,提示核酶有可能作为HCV感染基因治疗的一种有效途径 .     

特异切割苹果锈果类病毒的核酶基因的克隆和转录物的体外活性测定

根据锤头型核酶的作用模式 ,设计、合成和克隆了特异切割苹果锈果类病毒ASSVd正链 (194-196位点 )或负链 (89- 91位点 )RNA的 2个短臂锤头型核酶基因 :42nt的RzASSVd(+)和 40nt的RzASSVd(- )。经转录获得核酶转录物和³²P标记的ASSVd正、负链转录物。将核酶与ASSVd混合 ,50℃或 37℃保温 3～ 4h ,进行 8%PAGE(含8mol L尿素 )和放射自显影分析。体外切割检测表明 :2个核酶均具有特异切割活性 ,其中RzASSVd(- )对ASSVd负链的切割活性较高 ,对ASSVd正链不起作用。RzASSVd(+)对ASSVd正链的切割活性较弱 ,对ASSVd负链亦不起作用。在此基础上 ,构建得到双价核酶基因pGEMRzASSVd(± )。     

Activity identification of chimeric anti-caspase-3 mRNA hammerhead ribozyme in vitro and in vivo

To study the expression activity of various vectors containing anti-caspase-3 ribozyme cassettes in vivo, and to further study the role of caspas-3 in the apoptotic pathway, we constructed anti-caspase-3 hammerhead ribozyme embedded into the human snRNA U6, and detected the activity of the ribozyme in vitro and in vivo. Meanwhile we compared it with the self-cleaving hammerhead ribozymes that we previously studied, and with the general ribozyme, cloned into RNA polymerase II expression systems. The results showed that the three ribozymes, p1.5RZ107, pRZ107 and pU6RZ107 had the correct structure, and that they could cleave cas-pase-3 mRNA exactly to produce two fragments: 143nt/553nt. p1.5RZ107 has the highest cleavage efficiency in vitro, almost 80%. However, the U6 chimeric ribozyme, pU6RZ107, has the highest cleavage activity in vivo, almost to 65%, though it has lower cleavage activity in vitro. The cleavage results demonstrated that the pU6RZ107, the U6 chimeric ribozyme, could more efficiently expre     

BCR3/ABL2核酶的设计、构建及对K562细胞增殖与集落形成的抑制作用和凋亡诱导作用

利用计算机模拟设计合成了针对 K5 62细胞致癌融合 bcr3/abl2 m RNA的锤头状核酶 .该核酶以融合点附近 UUC为识别切割三联体 ,在核酶的 3′端增加一段 T7噬菌体终止子序列 .用基因克隆结合体外转录的方法 ,肯定了核酶的体外切割活性 .进而将核酶基因克隆到 p CEP4真核细胞高效表达载体上 ,利用脂质体 Lipofectin AMINE介导的转染技术将核酶与核酶基因导入靶细胞 ,从抑制靶细胞 K5 62的增殖与集落形成及引起靶细胞凋亡等方面验证了核酶在细胞水平上对融合基因 bcr3/abl2 m RNA的特异切割作用 ,并观察到了 T7噬菌体终止子序列对核酶切割效率的增强影响 .     

Activity identification of chimeric anti-caspase-3 mRNA hammerhead ribozyme in vitro and in vivo

To study the expression activity of various vectors containing anti-caspase-3 ribozyme cassettesin vivo, and to further study the role of caspas-3 in the apoptotic pathway, we constructed anti-caspase-3 hammerhead ribozyme embedded into the human snRNA U6, and detected the activity of the ribozymein vitro andin vivo. Meanwhile we compared it with the self-cleaving hammerhead ribozymes that we previously studied, and with the general ribozyme, cloned into RNA polymerase II expression systems. The results showed that the three ribozymes, p1.5RZ107, pRZ107 and pU6RZ107 had the correct structure, and that they could cleave caspase-3 mRNA exactly to produce two fragments: 143nt/553nt. p1.5RZ107 has the highest cleavage efficiencyin vitro, almost 80%. However, the U6 chimeric ribozyme, pU6RZ107, has the highest cleavage activityin vivo, almost to 65%, though it has lower cleavage activityin vitro. The cleavage results demonstrated that the pU6RZ107, the U6 chimeric ribozyme, could more efficiently express and downregulate the level of caspase-3in vivo, and the ribozyme could provide an alternative approach to the research into the mechanism of apoptosis and human gene therapy also.     

Activity identification of chimeric anti-caspase-3 mRNA hammerhead ribozymein vitro andin vivo

To study the expression activity of various vectors containing anti-caspase-3 ribozyme cassettesin vivo, and to further study the role of caspas-3 in the apoptotic pathway, we constructed anti-caspase-3 hammerhead ribozyme embedded into the human snRNA U6, and detected the activity of the ribozymein vitro andin vivo. Meanwhile we compared it with the self-cleaving hammerhead ribozymes that we previously studied, and with the general ribozyme, cloned into RNA polymerase II expression systems. The results showed that the three ribozymes, p1.5RZ107, pRZ107 and pU6RZ107 had the correct structure, and that they could cleave caspase-3 mRNA exactly to produce two fragments: 143nt/553nt. p1.5RZ107 has the highest cleavage efficiencyin vitro, almost 80%. However, the U6 chimeric ribozyme, pU6RZ107, has the highest cleavage activityin vivo, almost to 65%, though it has lower cleavage activityin vitro. The cleavage results demonstrated that the pU6RZ107, the U6 chimeric ribozyme, could more efficiently express and downregulate the level of caspase-3in vivo, and the ribozyme could provide an alternative approach to the research into the mechanism of apoptosis and human gene therapy also.     

抗HPV11 E2核酶的计算机设计

 借助计算机软件分析 ,设计出能特异性切割HPV11型 6 4 4ntE2mRNA的核酶 (ribozyme) .遵循Symon′s锤头状核酶结构和GUX剪切位点原则 ,靶序列存在 32个这样的剪切位点 .通过计算机软件分析出核酶的最佳剪切位点 ,并对底物及核酶的二级结构进行预测及进行相应基因生物学功能和基因同源性分析 ,筛选出 2个锤头结构核酶 .针对这两位点设计的核酶分别命名为RZ2 777和RZ32 81.计算机分析显示 ,两核酶与底物切点两翼碱基形成锤头状结构 ,切点所在基因序列具有相对松弛的二级结构 ,位于该基因重要生物功能区内 ,是核酶的理想攻击区域 .通过基因库检索 ,在已知人类基因排除了与上述两核酶切点两翼碱基有基因同源性序列的可能性 .将两核酶用于体外剪切实验取得了良好的实验结果 ,认为借助计算机分析可帮助尽快从多个剪切位点选择出最适核酶     

Inhibition of hepatitis B virus by hammerhead ribozyme targeted to the poly(A) signal sequence in cultured cells

The deviant poly(A) signal of hepatitis B virus (HBV) not only controls the formation of the 3' end of all the viral RNA, but is also crucial for HBV replication. Hence, a cis-releasing hammerhead ribozyme (RzA) targeted to the poly(A) signal region of HBV subtype adr was investigated for its antiviral effects. In vitro, RzA cleaved HBV RNA at its target site up to 70%, while the disabled ribozyme (dRzA), which had a one-base mutation in the catalytic site, did not cleave the target RNA at all. When the ribozymes were cotransfected into HepG2 cells with the HBV genome-containing plasmid p3.6II, the wild-type ribozyme RzA could effectively decrease HBV RNA levels and inhibit HBV replication, whereas its disabled form, dRzA, had much weaker effects, indicating that the active catalytic domain of the hammerhead ribozyme could markedly increase the extent of antisense-mediated inhibition. In addition, there was a gradient of effectiveness: the higher the amount of released ribozyme, the more the reduction in target HBV RNA in cells as well as progeny DNA reduction. These results suggest the possibility of the hammerhead ribozyme RzA to be used for the gene therapy of HBV infection.