作用于HBV（adr亚型）RNA的tRNA—包埋锤头状核酶的研究

设计了针对ＨＢＶ（ａｄｒ亚型）ＲＮＡ的二个锤头状核酶（ＲＳ３和ＲＣ２）,并将其插入ｔＲＮＡ反密码环中（ＲｔＳ３和ＲｔＣ２）,以增加其稳定性。实验表明,虽然插入ｔＲＮＡ中的核酶与裸露核酶相比,催化活性有所下降,但在胎牛血清和ＨｅｐＧ２细胞抽提液中的稳定性却明显提高。因此,ｔＲＮＡ－包埋核酶有可能提高在体内的抗病毒能力。     

反义寡聚脱氧核苷酸（ODN）衍生物抑制乙肝病毒抗原表达的研究

为比较针对中不同基因的ＯＤＮ硫代衍生物阻断乙型肝炎病毒（ＨＢＶ）的抗原表达,合成了与核心蛋白编码基因起始码上游序列,多聚酶蛋白编码基因起码上游及内部序列３．５ｋｂＲＮＡ３＇端起始负链ＤＮＡ合成序列互补的寡聚脱氧核苷酸硫代衍生物,分别在ＨＢＶ短暂表达和稳定表达细胞培养系统中观察其抑制ＨＢＶ抗原表达的作用。结果发现,当培养液中ＯＤＮ浓度为２０μｍｏｌ／ＬＪＦ ,四种ＯＤＮ均能抑ＨｅｐＧ２．２．１５细胞     

反义寡聚硫代磷酸脱氧核苷抑制体外细胞培养系统中乙肝抗原表达的研究

设计合成了两个分别互补于乙肝病毒２．１ｋｂ ｍＲＮＡ起始区（片段Ａ）和增强子区（片段Ｂ）的硫代磷酸的ＤＮＡ片段,在经克隆ＨＢＶ ＤＮＡ转染ＨｅｐＧ２细胞建立的ＨＢＶ短暂表达系统及稳定产生ＨＢＶ的２２１５细胞中研究二者对ＨＢｓＡｇ及ＨＢｅＡｇ表达的抑制作用。结果表明反义寡聚物能不同程序抑制乙肝抗原表达,并与剂量呈一定正相关。在ＨｅｐＧ２细胞ＨＢＶ短暂表达系统中,６μｍｏｌ／Ｌ浓度时,片段Ａ、Ｂ对ＨＢ     

反义寡核苷酸对丙型肝炎病毒调控基因表达的体外抑制活性研究

为探讨反义寡核苷酸对丙型肝炎病毒的抑制活性,研究和开发新型抗ＨＣＶ药物。采用ＨＣＶ５’ＮＣＲ调控荧光素酶基因的稳定表达细胞株ＨｅｐＧ２．９７０６,评价了３条针对ＨＣＶ调控基因的ＡＳＯＤＮ,即ＨＣＶ３６３,ＨＣＶ３４９及ＨＣＶ２７９。将Ｌｉｐｏｆｅｃｔｉｎ包封的ＡＳＯＤＮ与ＨｅｐＧ２．９７０６细胞株每天作用５小时,连续３天后检测荧光素酶活性。     

HBV整合片段对人PCNA基因启动子的调控

Ｈ７Ｃ是从一例肝癌组织中克隆得到的ＨＢＶ整合片段,其中保留有ｐｒｅＳ２基因的启动子及Ｃ端缺失的ｐｒｅＳ／Ｓ蛋白的阅读框架,我们用共转染方法研究了该整合片段地增殖细胞核抗原启动子的影响。结合表明在ＨｅｐＧ２细胞中,含有上述ＨＢＶ整合片段ＤＮＡ的质粒ＰＫＳＨ７Ｃ－ＨｐａＩ能以剂量依赖方式激活ＰＣＮＡ启动子。对ＳＶ４０启动子而言,ＰＣＮＡ启动子有１－２倍的更高激活。而破坏ｐｒｅＳ／Ｓ表达的两个亚克隆ＰＫ     

硫代磷酸反义寡脱氧核苷酸抗HBV细胞的实验研究

针对ＨＢＶ的５个基因位点作为靶序列,设计合成硫代反义寡聚核苷酸（Ｓ－ａｓＯＤＮ）．应用ＥＬＩＳＡ方法、ＭＴＴ比色法、电子显微镜等手段,观察Ｓ－ａｓＯＤＮ对ＨｅｐＧ２２２１５细胞ＨＢｓＡｇ、ＨＢｅＡｇ抗原的表达,及细胞的毒性、细胞形态和超微结构的影响．结果显示不同靶位点的Ｓ－ａｓＯＤＮ对ＨＢｓＡｇ、ＨＢｅＡｇ表达都有显著的抑制作用,且表现为序列特异性、剂量相关性、联合协同性和一定的抗核酸酶性,在浓度为２０μｍｏｌ／Ｌ时,对细胞无明显杀伤作用,对细胞的超微结构无显著的改变．结果提示Ｓ－ａｓＯＤＮ有望发展为抗ＨＢＶ的有效药物,但靶序列的选择、透细胞膜性及联合用药配伍等仍值得进一步研究和解决．     

利用PCR技术行反义寡核苷酸体外抗丁型肝炎病毒基因组RNA中核酶的研究

设计一对ＰＣＲ引物,其中上游引物的５’端除目的基因外,还加Ｔ７ＲＮＡ聚合酶启动子序列,以质粒（ｐＳＶＬＤ３）为模板,通过ＰＣＲ扩增出带有Ｔ７ＲＮＡ聚合酶启动子序列的１３９ｂｐ的ｃＤＮＡ片段,它含有丁型肝炎病毒（ＨＤＶ）基因组ＲＮＡ中核酶（Ｒｉｂｏｚｙｍｅ）区的ｃＤＮＡ该核酶具有自身裂解功能,经测序发现该ｃＤＮＡ有２个碱基变异,以此ＰＣＲ产物为模板,通过Ｔ７ＲＮＡ聚合酶,转录出核酶的前体,并观察到其     

CO2浓度倍增对小麦生育性状和产量构成的影响

ＣＯ_２浓度倍增对小麦生育性状和产量构成的影响王修兰,徐师华,李佑祥（中国农业科学院农业气象研究所,北京,１０００８１）ＴＨＥＥＦＦＥＣＴＳＯＦＣＯ_２ＤＯＵＢＬＩＮＧＯＮＧＲＯＷＩＮＧＡＮＤＤＥＶＥＬＯＰＩＮＧＣＨＡＲＡＣＴＥＲＳＡＮＤＹＩＥＬＤＦＯ...     

r－干扰素对喉癌细胞系HEP－2HLA－DR抗原和增殖细胞核抗原表达的影响

本实验用人重组ｒ－干扰素（ｒｈｕ－ＩＦＮ）作用ＨＥＰ－２细胞后ＨＬＡ－ＤＲ抗原和增殖细胞核抗原（ＰＣＮＡ）表达的检测来探讨ｒ－干扰素对ＨＥＰ－２细胞ＨＬＡ－ＤＲ抗原表达诱导作用及体外抗增殖活性。用单克隆抗体ＣＲ３／４３（抗ＨＬＡ－ＤＲ）和Ｋｉ－６７（抗ＰＣＮＡ）。以链霉素一生物素技术（ＬＳＡＢ）检测ＨＥＰ－２细胞ＨＬＡ－ＤＲ抗原和ＰＣＮＡ表达,结果显示：ｒ－ＩＥＮ诱导ＨＬＡ－ＤＲ抗原和抑制ＰＣＮＡ表达其强弱与ｒ－ＩＦＮ剂量有关。资料提示：ｒ－ＩＦＮ不仅对ＨＥＰ－２细胞有细胞毒作用,同时能调节其细胞膜特性,因而在喉癌的治疗中是有效的。     

庚型肝炎病毒包膜糖蛋白E2基因在昆虫细胞中的表达

用ＰＣＲ扩增出ＨＧＶＥ２全基因,克隆进杆状病毒表达载体ｐＦＡＳＴＢＡＣＨＴａ中,构建成重组转座载体ｐＦＡＳＴＢＡＣＥ２,转化ＤＨ１０ＢＡＣ大肠杆菌感受态细胞,筛选阳性菌落,抽提大分子质粒ＤＮＡ,获得含ＨＧＶＥ２基因的重组杆状病毒穿梭载体,转染昆虫草地夜蛾Ｓｆ９细胞,出现细胞病变后,收集含有重组病毒颗粒的培养上清,重新感染草地夜蛾Ｓｆ９单层细胞及甜菜夜蛾幼虫,分别收集Ｓｆ９细胞和甜菜夜蛾幼虫体内的血淋巴细胞,进行１２％ＳＤＳ聚丙烯酰胺凝胶电泳,可见表达的融合蛋白带,经亲和层析进行蛋白纯化,用ＥＬＩＳＡ方法检测各类血清标本,初步研究ＨＧＶＥ２糖蛋白的抗原性     

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).     

Polymerase ribozyme efficiency increased by G/T-rich DNA oligonucleotides

The RNA world hypothesis states that the early evolution of life went through a stage where RNA served as genome and as catalyst. The replication of RNA world organisms would have been facilitated by ribozymes that catalyze RNA polymerization. To recapitulate an RNA world in the laboratory, a series of RNA polymerase ribozymes was developed previously. However, these ribozymes have a polymerization efficiency that is too low for self-replication, and the most efficient ribozymes prefer one specific template sequence. The limiting factor for polymerization efficiency is the weak sequence-independent binding to its primer/template substrate. Most of the known polymerase ribozymes bind an RNA heptanucleotide to form the P2 duplex on the ribozyme. By modifying this heptanucleotide, we were able to significantly increase polymerization efficiency. Truncations at the 3'-terminus of this heptanucleotide increased full-length primer extension by 10-fold, on a specific template sequence. In contrast, polymerization on several different template sequences was improved dramatically by replacing the RNA heptanucleotide with DNA oligomers containing randomized sequences of 15 nt. The presence of G and T in the random sequences was sufficient for this effect, with an optimal composition of 60% G and 40% T. Our results indicate that these DNA sequences function by establishing many weak and nonspecific base-pairing interactions to the single-stranded portion of the template. Such low-specificity interactions could have had important functions in an RNA world.     

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.     

Combinatorial Screening and Intracellular Antiviral Activity of Hairpin Ribozymes Directed against Hepatitis B Virus

A combinatorial screening method has been used to identify hairpin ribozymes that inhibit hepatitis B virus (HBV) replication in transfected human hepatocellular carcinoma (HCC) cells. A hairpin ribozyme library (5 x 10(5) variants) containing a randomized substrate-binding domain was used to identify accessible target sites within 3.3 kb of full-length in vitro-transcribed HBV pregenomic RNA. Forty potential target sites were found within the HBV pregenomic RNA, and 17 sites conserved in all four subtypes of HBV were chosen for intracellular inhibition experiments. Polymerase II and III promoter expression constructs for corresponding hairpin ribozymes were generated and cotransfected into HCC cells together with a replication-competent dimer of HBV DNA. Four ribozymes inhibited HBV replication by 80, 69, 66, and 49%, respectively, while catalytically inactive mutant forms of these ribozymes affected HBV replication by 36, 28, 0, and 0%. These findings indicate that the inhibitory effects on HBV replication were largely mediated by the catalytic activity of the ribozymes. In conclusion, we have identified catalytically active RNAs by combinatorial screening that mediate intracellular antiviral effects on HBV.     

An in vitro evolved precursor tRNA with aminoacylation activity

A set of catalysts for aminoacyl-tRNA synthesis is an essential component for translation. The RNA world hypothesis postulates that RNA catalysts could have played this role. Here we show an in vitro evolved precursor tRNA consisting of two domains, a catalytic 5'-leader sequence and an aminoacyl-acceptor tRNA. The 5'-leader sequence domain selectively self-charges phenylalanine on the 3'-terminus of the tRNA domain. This cis-acting ribozyme is susceptible to RNase P RNA, generating the corresponding 5'-leader segment and the mature tRNA. Moreover, the 5'-leader segment is able to aminoacylate the mature tRNA in trans. Mutational studies have revealed that C(74) and C(75) at the tRNA aminoacyl-acceptor end form base pairs with G71 and G70 of the trans-acting ribozyme. Such Watson-Crick base pairing with tRNA has been observed in RNase P RNA and 23S rRNA, suggesting that all three ribozymes use a similar mechanism for the recognition of the aminoacyl-acceptor end. Our demonstrations indicate that catalytic precursor tRNAs could have provided the foundations for the genetic coding system in the proto-translation system.     

Construction of a ribozyme-expression system that effectively transports ribozymes to the cytoplasm.

In our previous studies, it was demonstrated that the activity of a ribozyme in vivo was governed by several parameters, which include a high level-expression of ribozyme, the intracellular stability of the ribozyme and colocalization of the ribozyme with its target RNA in the same cellular compartment. To generate ribozymes with significant activity in vivo, we have developed a ribozyme-expression system based on a human tRNA(Val) promoter. Our tRNA-embedded ribozymes produced by our ribozyme-expression system remain relatively stable in cultured cells with half-lives longer than 30 min. Moreover, tRNA-ribozymes with a cloverleaf structure were efficiently exported from the nucleus to the cytoplasm, where they would effectively cleave target RNAs. In the present study, we investigated the relationship between the secondary structure of the tRNA-ribozymes and the transport efficacy of them in mammalian cells by using a screening system in vivo. Furthermore, we also investigated the mechanism of the export of tRNA-embedded ribozymes both in mammalian cells and in Xenopus oocytes.     

抗HPV16 E7-ribozyme在CV-1细胞中的稳定表达

构建了由RSV—LTR启动子带动并能在细胞内稳定复制的Ribozyme的自身修剪表达质粒pRSV—Rz523、Ribozyme反义对照质粒pRSV—AE7及人增殖细胞核抗原基因(PCNA)启动子带动的HPVl6 E7片段(+554～+686)的真核表达质粒pPCNA—E7。经G418抗性筛选获得了稳定表达Ribozyme的CV-1细胞克隆,其表达水平约为9．Opmol／lO6个细胞,其中活性Ribozyme的量大于50fmol／lO6个细胞,分离得到的Ribozyme可在体外特异切割E7靶RNA片段。通过共转染Ribozyme(或反义对照)和底物表达质粒并筛选出细胞克隆．研究了Ribozyme在细胞中对底物表达水平的影响。初步结果显示．Rihozyme的导人可使细胞内底物E7的RNA表达水平降低了90％(反义对照使E7 RNA表达降低20％)。上述结果提示：在CV-1细胞中表达的Ribozyme不仅在体外,同时在细胞内具有一定的生物学活性,有可能应用于逆转官颈癌细胞的恶性表型。