猕猴肉瘤病毒SV40 T-ag-C 基因克隆及序列分析

摘要: 目的对来源于一株自然感染猕猴肉瘤病毒SV40 的猴肾细胞培养物进行大T 抗原C-羧基端( T-ag-C) 基因 克隆及核苷酸序列分析。方法采用PCR 法分别从一株自然感染SV40 病毒的猴肾细胞培养物和SV40776 标准 株接种的vero 细胞培养物提取的总DNA 中扩增出441bp 的SV40 大T 抗原C-羧基端( T-ag-C) 基因片段,分别将其 克隆到PMD18-T 载体中,转化至JM109 感受态大肠杆菌细胞后,挑取阳性克隆进行测序鉴定,并对获得的目的基 因核苷酸序列进行序列分析及同源性分析。结果来源于猴肾细胞培养物的SV40 大T 抗原片段与本实验室来源 于云南野生猴群的猕猴外周血所得到的SV40 大T 抗原片段同源性为97. 31% ,与GenBank 中登录号为NC _ 001669. 1 序列进行比对,同源性为96. 33% ; 与SV40-776 标准株接种的vero 细胞培养物所扩增的大T 抗原片段同 源性为97. 55% 。结论对大T 抗原基因克隆和序列分析是了解和掌握SV40 病毒分子流行病学及其变异趋势的 重要手段。     

猿猴空泡病毒40病毒样颗粒的制备及纯化

本研究将猿猴空泡病毒40(Simian virus 40,SV40)的主要衣壳蛋白VP1通过Bac-toBac杆状病毒表达系统在昆虫细胞中大量表达,并自我装配成形态结构及免疫原性均与天然病毒粒子相同或相似的SV40病毒样颗粒(SV40virus-like particles,SV40VLPs),经表达条件优化及分子筛纯化,成功制备出高纯度的VLPs.聚丙烯酰胺凝胶电泳结果可见大小约为46kDa的VP1特异性条带.间接免疫荧光试验(IFA)证实VP1蛋白能够与异硫氰酸荧光素标记的羊抗鼠抗体发生反应,出现明显的特异性绿色荧光,具有良好的抗原性.纯化产物在透射电镜下可见直径约45nm的病毒样颗粒,显示出成功组装了SV40VLPs,免疫印迹试验证明VLPs能够与人抗SV40阳性血清发生反应,具有良好的抗原性.     

Activation of SV40 genome by 72-base pair tandem repeats of Moloney sarcoma virus

The simian virus 40 (SV40) 72-base pair (bp) tandem-repeated sequences have a crucial role as an activator element in viral gene expression. We replaced the SV40 72-bp repeat with a 72-bp repeat derived from the long terminal repeat (LTR) of cloned Moloney murine sarcoma virus (MSV) DNA. Although there is no detectable sequence homology to SV40, the MSV repeats can substitute functionally for the SV40 repeats and generate a viable virus in monkey kidney cells.     

SV40LT 基因过表达慢病毒载体的构建与包装

摘要: 目的构建SV40LT 基因过表达慢病毒载体,并对其进行慢病毒包装,为建立永生化的uncv 小鼠胚胎成纤维细胞奠定基础。方法从293T 细胞中获得SV40LT 基因,将其克隆到pLenti-GFP 质粒中,构建重组穿梭质粒pLenti-GFP-SV40LT,测序鉴定后分别将鉴定的阳性pLenti-GFP-SV40LT 和包装质粒pMD2. 0G 和psPAX2 共转染293T 细胞,包装产生慢病毒。结果SV40LT 基因过表达慢病毒载体的构建与包装成功。结论SV40LT 基因过表达慢病毒载体构建与包装的成功为uncv 小鼠胚胎成纤维细胞的永生化提供了工具。     

Simian virus 40 replication in adenovirus-transformed human cells antagonizes gene expression

Simian virus 40 (SV40) replicates efficiently in monkey kidney cells. However, we have now found that SV40-based vectors transfected into most human cells replicate poorly, if at all. In contrast, strong SV40 replication is observed in human embryonic kidney (HEK) cells transformed with the adenovirus early region, but not in untransformed HEK cells. Vector replication in adenovirus-transformed cells is dependent on the presence of the SV40 origin of replication and large-T antigen. However, vigorous replication occurs at levels of large-T antigen that are undetectable by immunofluorescence. These data suggest that the adenovirus oncogenes create a replication-permissive environment to which the SV40 replicon responds. Furthermore, replication and gene expression seem to be antagonistic on our vectors. High levels of large-T antigen are observed only when vector replication is blocked by mutations in the gene for large-T antigen or the origin of replication, or by direct inhibition of DNA polymerase with aphidicolin.     

N-Acetoxy-acetylaminofluorene reacts preferentially with a control region of intracellular SV40 chromosome

Many chemical carcinogens or their metabolites react with DNA; thus it is of interest to determine what effect chromosomal structure has on these reactions. The chromosome of simian virus 40 (SV40) is well suited for such studies; like chromatin of eukaryotic cells, it is organized into nucleosomes. The nucleotide sequence of SV40 is known, together with much about the pattern of viral gene expression and DNA replication, and the structure of the viral chromosome. We have investigated the binding of the ultimate carcinogen, N-acetoxy-acetylaminofluorene (AAAF), to specific regions of the SV40 chromosome in situ in the intact infected cell. The results, reported here, indicate that a region containing regulatory functions on the intracellular SV40 chromosome has unique structural properties which render it more susceptible to attack by AAAF than the rest of the SV40 genome. The preferential binding of AAAF to regulatory regions of chromatin may have implications for the mechanism of action of this and similar carcinogens.     

An embryo protein induced by SV40 virus transformation of mouse cells

A specific protein of molecular weight (MW) approximately 55,000 (55K) was found recently by immunoprecipitation in all SV40 virus-transformed mammalian cells, in addition to the SV40 large T antigen (appoximately 94K) and small antigen (approximately 17K), which are the only proteins coded by the 'early half' of the SV40 genome. The 55K protein is encoded by cellular DNA; its peptide pattern is different from that of the SV40 antigens and it is species specific in mouse, rat, hamster, monkey and human SV40-transformed (or infected) cells. A 55K protein with a similar peptide pattern was found in mouse embryonal carcinoma cells not exposed to SV40. Similar proteins were reported in mouse sarcomas and leukaemias induced by a great variety of aetiological agents and also in a spontaneously transformed mouse fibroblast cell line, and it has been suggested that the protein may be a general correlated of cellular tumorigenicity. We now report that the approximately 55K protein is present in primary cell cultures from 12-14 day old mouse embryos, but not in 16-day old mouse embryos. The embryo protein has a peptide pattern virtually indistinguishable from that of the SV40-induced protein. We also show by comparing closely related cell families that spontaneously transformed highly tumorigenic mouse cells do not possess the 55K protein.     

Deregulation of c-myc and SV40Tag causing brain tumor in mice

Deregulated expressions of both c-myc and simian virus 40 large T antigen （SV40Tag） are consistent features of lots of tumors. To investigate whether the expression of c-myc and SV40Tag in mouse might help develop a model of human tumor, we generated c-myctransgenics by inserting human c-mycgene into pTRE2 of Tet-On system. We obtained conditional expression of SV4OTag transgenics by the Tet-On system from Yangzhou University. Crossing the c-myc transgenic mouse with the SV40Tag transgenic mice to generate bitransgenics we got double-transgenic mice expressing c-myc and SV40Tag by the Tet-On system. After being treated with doxycycline continuously, single-transgenic SV40Tag mice developed brain tumor infrequently （3 of 84, 3.6%） with a long onset （185 d on average）. In contrast, double-transgenic c-mydSV4OTag mice developed brain tumor with a short onset （96 days on average） and a 41% brain tumor incidence rate （7 of 17, 41%）. This tumor was assumed to be medulloblastoma. Our experiments suggest that deregulated expression of c-myc and SV4OTag in brain might generate a mouse model of human brain tumor that recapitulates some features of human medulloblastoma.     

The cell-cycle regulated proliferating cell nuclear antigen is required for SV40 DNA replication in vitro

Cell-free extracts prepared from human 293 cells, supplemented with purified SV40 large-T antigen, support replication of plasmids containing the SV40 origin of DNA replication. A cellular protein (Mr approximately 36,000) that is required for efficient SV40 DNA synthesis in vitro has been purified from these extracts. This protein is recognized by human autoantibodies and is identified as the cell-cycle regulated protein known as proliferating cell nuclear antigen (PCNA) or cyclin.     

Functional identity of proliferating cell nuclear antigen and a DNA polymerase-delta auxiliary protein

The mechanism of replication of the simian virus 40 (SV40) genome closely resembles that of cellular chromosomes, thereby providing an excellent model system for examining the enzymatic requirements for DNA replication. Only one viral gene product, the large tumour antigen (large-T antigen), is required for viral replication, so the majority of replication enzymes must be cellular. Indeed, a number of enzymatic activities associated with replication and the S phase of the cell cycle are induced upon SV40 infection. Cell-free extracts derived from human cells, when supplemented with immunopurified SV40 large-T antigen support efficient replication of plasmids that contain the SV40 origin of DNA replication. Using this system, a cellular protein of relative molecular mass 36,000 (Mr = 36K) that is required for the elongation stage of SV40 DNA replication in vitro has been purified and identified as a known cell-cycle regulated protein, alternatively called the proliferating cell nuclear antigen (PCNA) or cyclin. It was noticed that, in its physical characteristics, PCNA closely resembles a protein that regulates the activity of calf thymus DNA polymerase-delta. Here we show that PCNA and the polymerase-delta auxiliary protein have similar electrophoretic behaviour and are both recognized by anti-PCNA human autoantibodies. More importantly, both proteins are functionally equivalent; they stimulate SV40 DNA replication in vitro and increase the processivity of calf thymus DNA polymerase-delta. These results implicate a novel animal cell DNA polymerase, DNA polymerase-delta, in the elongation stage of replicative DNA synthesis in vitro.     

SV40 enhancer and large-T antigen are instrumental in development of choroid plexus tumours in transgenic mice

We have shown recently that choroid plexus tumours frequently develop in transgenic mice which have developed from fertilized eggs injected with DNA molecules containing both simian virus 40 (SV40) early-region genes and metallothionein (MT) fusion genes, and several lines of mice have now been established in which all of the offspring that inherit the foreign DNA succumb to these tumours at 3-5 months of age (ref. 1 and our unpublished data). Several other tissues, notably thymus and kidney, occasionally also show pathological changes. SV40 large-T antigen protein and messenger RNA are always present in affected tissues at much greater concentrations than in unaffected tissues, suggesting that SV40 early-region genes are preferentially activated in choroid plexus, thymus and kidney and that this activation frequently leads to tumorigenesis in the choroid plexus. To determine which regions of the original constructs are important for this tumorigenesis, we have now tested several derivatives and report here that the large-T antigen is sufficient, that the MT fusion gene is dispensable and that the SV40 enhancer (72-base-pair repeat region) has an important role in directing tumours to the choroid plexus. Deletion of the SV40 enhancer region alone commonly leads to peripheral neuropathy, as well as liver and pancreatic tumours, which are the subject of the accompanying paper. Evidence is presented that these pathologies may result from an enhancing effect of the MT sequences on large-T antigen genes, made possible by removal of the otherwise dominant SV40 enhancer.     

Tightly associated lipids may anchor SV40 large T antigen in plasma membrane

Simian virus 40 (SV40) large T antigen, a multifunctional protein necessary for lytic growth and cell transformation, is located mainly in the nucleus and in small amounts on the cell surface (surface T). Surface T may have a passive role in SV40 tumour rejection by cytotoxic T cells as a component of SV40-TSTA (tumour-specific transplantation antigen). The unusual induction of this immune response by immunizing mice with soluble T antigen led us to investigate the in vitro binding of T antigen to the surface of living cells in more detail. Our results show that native surface T and a minor subset of large T antigen having a high cell surface binding affinity in vitro, behave like integral membrane proteins. Several viral proteins including SV40 T antigen and cellular proteins seem to be linked to fatty acids (acylation). To analyse whether this mechanism is involved in the stable attachment of in vitro-bound T antigen to the plasma membrane of living target cells, we determined the degree of labelling of this molecule by using target cells prelabelled with 3H-fatty acid. Here we report that T antigen extracted from unlabelled SV40-transformed cells (SV80) becomes 3H-labelled after in vitro binding to the cell surface of 3H-palmitate-prelabelled HeLa cells. These results suggest that T antigen attached externally to living cells, may be anchored by tightly linked lipids.     

梅花鹿鹿茸软骨细胞和间充质干细胞永生化细胞株的构建及鉴定

分离梅花鹿鹿茸软骨细胞和间充质干细胞,使用SV40 LT抗原慢病毒载体建立永生化软骨细胞和间充质干细胞系并鉴定。将含有SV40 LT基因片段的慢病毒载体,转染人胚肾细胞293T获得包装后的病毒粒子,感染鹿茸软骨细胞及间充质干细胞,连续传代培养,通过形态观察、细胞增殖、real-time PCR、甲苯胺蓝染色法和流式细胞术等方法检测SV40 LT抗原表达以及细胞性质。实验所建立的永生化鹿茸软骨细胞系与间充质干细胞系能够稳定传代并具有较强的体外增值活性。RT-PCR检测到SV40T抗原的表达,通过甲苯胺蓝染色法和流式细胞术鉴定所得细胞系具有原代细胞的基本性质。成功获得永生化的软骨细胞与间充质干细胞系,为后续鹿茸生长及发育研究奠定了基础。     

Yeast replication factor-A functions in the unwinding of the SV40 origin of DNA replication

Cell-free replication systems for simian virus 40 (SV40) DNA are taken to be a model for the replication of eukaryotic chromosomes, because only one viral protein is required to supplement the replication proteins provided by a human cell extract. To prove that these cellular proteins function in chromosomal DNA replication we have begun to identify homologous proteins in an organism that can be genetically manipulated. Here we report the identification of yeast replication factor-A (yRF-A) from Saccharomyces cerevisiae and show that it is functionally and structurally related to a human protein that is required for the initiation and elongation of SV40 DNA replication. Yeast RF-A, a multi-subunit phosphoprotein, is similar to the human protein in its chromatographic behaviour, subunit structure and DNA-binding activity. The yeast protein will fully substitute for the human protein in an early stage of the initiation of SV40 DNA replication. Substitution of yRF-A in the complete SV40 replication system, however, results in reduced DNA replication, presumably due to a requirement for species-specific interactions between yeast RF-A and the DNA polymerase complex.