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陕西医学杂志 2011年第9期40卷 1127-1130页

作者：刘蕊李志奎西安市第四医院呼吸科西安710004

目的:构建以乙肝病毒核心蛋白(HBC)为载体的甲型流感病毒M2基因胞外区(M2e)通用疫苗杆粒,利用昆虫细胞杆状病毒表达系统,进行真核表达。方法:利用PCR技术将甲型流感病毒M2e基因序列与HBC相连。经酶切、酶联将融合基因插入pFastBacHTA载... 详细信息

目的:构建以乙肝病毒核心蛋白(HBC)为载体的甲型流感病毒M2基因胞外区(M2e)通用疫苗杆粒,利用昆虫细胞杆状病毒表达系统,进行真核表达。方法:利用PCR技术将甲型流感病毒M2e基因序列与HBC相连。经酶切、酶联将融合基因插入pFastBacHTA载体,在DH10Bac细胞中进行同源重组,经测序鉴定后构建M2e-HBC杆粒,脂质体转染sf9昆虫细胞,收获并扩增含有M2e-HBC的杆状病毒,经扩增后获得高效价的重组杆状病毒,用SDS-PAGE、免疫荧光法和电镜检测目的蛋白的表达。结果:构建了以HBC为载体的M2e通用疫苗杆粒,通过转染sf9昆虫细胞得到M2e-HBC融合蛋白真核表达。结论:成功构建了HBC与甲型流感病毒M2e融合蛋白的病毒样颗粒,为流感通用疫苗的研制奠定了基础。

关键词：正粘病毒科病毒融合蛋白质类/代谢昆虫病毒聚合酶链反应

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Expression of receptor for ecotropic murine leukemia virus on hematopoietic cells

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ARCHIVES OF VIROLOGY 2001年第3期146卷 507-519页

作者： Suzuki, T Aizawa, S Ikeda, H Natl Inst Anim Hlth Immunogenet Lab Tsukuba Ibaraki 3050856 Japan Natl Inst Radiol Sci Div Radiobiol & Biodosimetry Chiba 260 Japan Natl Inst Anim Hlth Lab Virol Prod Tsukuba Ibaraki 305 Japan

A fusion protein (F-SU/GFP) which is comprised of the surface (SU) subunit of the Friend MuLV envelope glycoprotein and the green fluorescence protein (GFP) was generated by a baculovirus expression system. The F-SU/GFP specifically bound to mammalian tissue cultured cells expressing the mCAT-1, the receptor for ecotropic murine leukemia virus (Eco-MuLV). The expression level of mCAT-1 on hematopoietic cells was measured based on the capacity of cells to absorb the F-SU/GFP. In BALB/c mice susceptible to Eco-MuLV infection, all hematopoietic cell subpopulations absorbed the F-SU/GFP with the most prominent absorption observed in the TER119(+) erythroblasts. Hematopoietic cells from C4W and AKR mice did not absorb the F-SU/GFP as readily as the BALB/c cells, probably due to expression of the envelope glycoproteins of endogenous Eco-MuLVs in these mice. Although AKR mice are susceptible to Eco-MuLV infection while C4W mice are resistant, these mice showed no apparent difference in the F-SU/GFP-absorbing capacity.

关键词：氨基酸转运系统碱性杆状病毒科/遗传学载体蛋白质类/代谢细胞系幼红细胞/代谢弗林德鼠白血病病毒/代谢遗传载体膜糖蛋白类/代谢膜蛋白质类/代谢小鼠近交AKR 小鼠近交BALBC 蛋白质结合受体病毒/代谢重组蛋白质类/代谢病毒包膜蛋白质类/遗传学病毒包膜蛋白质类/代谢病毒融合蛋白质类/代谢动物小鼠

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Identification and characterization of a structural protein of hepatitis B virus: A polymerase and surface fusion protein encoded by a spliced RNA

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VIROLOGY 2000年第2期275卷 398-410页

作者： Huang, HL Jeng, KS Hu, CP Tsai, CH Lo, SJ Chang, CM Natl Yang Ming Univ Sch Life Sci Inst Microbiol & Immunol Taipei 112 Taiwan Natl Hlth Res Inst Div Mol & Genom Med Res Taipei 115 Taiwan Vet Gen Hosp Dept Med Res Taipei 112 Taiwan

The hepatitis B virus (HBV) genome is known to contain four conserved and overlapped open reading frames (ORFs) encoding the viral core, polymerase (P), surface (S), and X proteins. Whether HBV encodes other proteins has long been a major interest in the field. Using P-32-labeling of an introduced protein kinase A site attached to the N- or C-terminus of the HBV polymerase gene, a 43-kDa P-S fusion protein was detected in cell lysate, secreted virions, and 22-nm subviral particles. Immunobiochemical studies showed that the 43-kDa protein contains the epitopes of the N-terminus of polymerase and most parts of the surface proteins This 43-kDa protein was shown to be a glycoprotein, similar to the surface protein. RT-PCR and sequence analyses identified a spliced mRNA which was derived from pregenomic RNA with a deletion of 454 nucleotides (nt) from nt 2447 to 2902. This splice event creates a P-S fusion ORF This finding is consistent with the result obtained from an immunobiochemical study. Mutations at the splice donor or acceptor site on the HBV genome abrogated the production of the 43-kDa protein. These mutants had no effect on viral replication in transfected HuH-7 cells. However, this P-S fusion protein is able to substitute for the LS protein in Virion maturation. On the basis of these results, we conclude that the 43-kDa protein is a polymerase-surface fusion protein encoded by a spliced RNA Similar to the LS protein, the 43-kDa P-S fusion protein is a structural protein of HBV and might play a role in the HBV life cycle. (C) 2000 Academic Press.

关键词：天冬氨酸内肽酶类/遗传学细胞系电泳聚丙烯酰氨凝胶表位基因产物 pol/遗传学糖蛋白类/遗传学糖蛋白类/代谢糖基化肝炎表面抗原乙型/遗传学肝炎病毒乙型/遗传学肝炎病毒乙型/代谢分子量开放读码框架沉淀素试验 RNA剪接逆转录聚合酶链反应病毒融合蛋白质类/遗传学病毒融合蛋白质类/代谢病毒结构蛋白质类/遗传学病毒结构蛋白质类/代谢人类

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Cleavage of hepatitis C virus nonstructural protein 5A by a caspase-like protease(s) in mammalian cells

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VIROLOGY 2000年第2期270卷 476-487页

作者： Satoh, S Hirota, M Noguchi, T Hijikata, M Handa, H Shimotohno, K Tokyo Inst Technol Fac Biosci & Biotechnol Midori Ku Yokohama Kanagawa 2268501 Japan Kyoto Univ Inst Virus Res Sakyo Ku Kyoto 6068507 Japan

Nonstructural 5A protein (NS5A) of hepatitis C virus (HCV) is localized in the cytoplasm although it has a functional nuclear localization signal. To clarify the determinant of NS5A cytoplasmic localization, various N- or C-terminal deleted NS5A mutants were generated and their subcellular localization was analyzed in cell lines after transient expression. N-terminal deleted forms of NS5A were localized in the nucleus, and the sequence of the N-terminal 27 amino acids of NS5A had sufficient function to cause retention of a normally nuclear protein in the cytoplasm. These observations indicated that cytoplasmic localization of NS5A is determined primarily by the N-terminal region of the molecule. In addition, we found proteolytic processing of NS5A in transiently expressing cells. In these cells, cleavage occurred at a few sites located in the N- and C-terminal regions of NS5A. This cleavage in cells was enhanced by apoptotic stimuli and was Inhibited by the caspase inhibitor Z-VAD-FMK, suggesting that a caspase-like protease(s) contributes to the cleavages of NS5A Based on the results of mutational analysis of NS5A, we predicted one cleaved form, which had lost both the N- and the C-terminal portions of NS5A, to be composed of amino acid residues 155 to 389. Peptide containing the same amino acid sequence as this cleaved product was localized in the nucleus. Furthermore, we found that a fusion protein consisting of Gal4 DNA-binding domain fused with this cleaved form showed transcriptional activity only when the cu-catalytic subunit of protein kinase A (PKA) was coproduced, suggesting that the transcriptional activity of this product was regulated by PKA. These results suggested that the cleavage product of NS5A by a caspase-like protease(s) plays a role in transcriptional regulation of the host cell gene(s) in HCV-infected cells. (C) 2000 Academic Press.

关键词：氨基酸序列半胱氨酸天冬氨酸蛋白酶/代谢肝炎病毒属/代谢肝炎丙型/代谢肝炎丙型/病毒学分子序列数据突变 RNA复制酶/代谢序列比对信号传导肿瘤细胞培养的病毒融合蛋白质类/遗传学病毒融合蛋白质类/代谢病毒非结构蛋白质类/遗传学病毒非结构蛋白质类/代谢人类

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Oligomerization, secretion, and biological function of an anchor-free parainfluenza virus type 2 (PI2) fusion protein

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VIROLOGY 2000年第2期270卷 368-376页

作者： Tong, SX Compans, RW Emory Univ Sch Med Dept Microbiol & Immunol Atlanta GA 30322 USA

A number of studies indicate that the transmembrane domain, the cytoplasmic domain, or both regions of viral surface glycoproteins are involved in quaternary structure formation. In this report, the transmembrane domain and cytoplasmic tail coding sequence of the fusion (F) glycoprotein gene from parainfluenza type 2 virus was truncated by PCR and the resulting gene (PI2F') was expressed in HeLa-T4 cells by using the vaccinia virus-T7 transient expression system. Pulse-chase experiments indicated that the anchor-free P12F' was expressed and processed into F-1 and F-2 subunits. Both the processed and the unprocessed anchor-free P12F' proteins were found to be efficiently secreted into the culture medium. Examination of the oligomeric form of the anchor-free P12F' by chemical cross-linking demonstrated that it assembles posttranslationally into dimers and trimers with a pattern similar to that of the wild-type PI2F protein. In an effort to better understand the biological properties of the truncated form of P12F', we anchored PI2F' by a glycosyl-phosphatidylinositol (GPI) linkage. The GPI-anchored P12F' protein, when coexpressed with PI2HN, did not induce cell fusion seen as syncytium formation, but was found to initiate lipid mixing (hemifusion) as observed by transfer of R-18 rhodamine from red blood cells to the GPI-PI2F'/PI2HN cotransfected cells. The results therefore indicate that the extracellular domain of the PI2 fusion protein contains not only the structural information sufficient to direct assembly into higher oligomers, but also is competent to initiate membrane fusion, suggesting that the anchor-free P12F' may be useful for further structural studies. (C) 2000 Academic Press.

关键词：突变副流感病毒2型人/代谢蛋白质构象蛋白质加工转译后病毒融合蛋白质类/化学病毒融合蛋白质类/遗传学病毒融合蛋白质类/代谢

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Presentation of antigenic sites from foot-and-mouth disease virus on the surface of baculovirus and in the membrane of infected cells

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ARCHIVES OF VIROLOGY 2000年第9期145卷 1815-1828页

作者： Tami, C Farber, M Palma, EL Taboga, O INTA CICVyA Inst Biotecnol Buenos Aires DF Argentina

We describe the construction of recombinant baculoviruses displaying on their surface and in the membrane of infected cells the small, immunodominant antigenic site (site A) or the large polyprotein (P1) coding for the four structural proteins of foot-and-mouth disease virus (FMDV). The coding sequences were inserted in the amino-terminus of gp64, the major glycoprotein of the baculovirus Autographa californica nuclear polyhedrosis virus (AcNPV). Following infection of insect cells with the recombinant baculoviruses, the cellular localization of the chimaeric proteins as well as their presence in the surface of extracellular viruses was assessed by immunofluorescence microscopy and Western blot. The antigenicity of the recombinant viruses was studied by competitive ELISAs, which showed that although both recombinant viruses were able to compete with FMDV-specific monoclonal antibodies (MAbs), their patterns of reactivity were different. The results suggest that this eukaryotic display system could be an alternative method of presentation of foreign antigens in a multimeric form as a new approach to biosynthetic vaccines.

关键词：抗原病毒/遗传学抗原病毒/免疫学抗原病毒/代谢结合部位印迹法蛋白质病毒壳体/遗传学病毒壳体/免疫学病毒壳体/代谢衣壳蛋白质类细胞系细胞膜/病毒学酶联免疫吸附测定荧光抗体技术核多角体病毒属/遗传学核多角体病毒属/代谢细小RNA病毒科/遗传学细小RNA病毒科/免疫学重组融合蛋白质类/代谢转化遗传病毒融合蛋白质类/遗传学病毒融合蛋白质类/代谢病毒结构蛋白质类/遗传学病毒结构蛋白质类/免疫学病毒结构蛋白质类/代谢动物

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Baculoviral display of functional scFv and synthetic IgG-binding domains

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BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 2000年第1期275卷 84-90页

作者： Mottershead, DG Alfthan, K Ojala, K Takkinen, K Oker-Blom, C Univ Jyvaskyla Dept Biol & Environm Sci FIN-40351 Jyvaskyla Finland Univ Helsinki Dept Biosci Div Biochem FIN-00014 Helsinki Finland

Viral vectors displaying specific ligand binding moities such as scFv fragments or intact antibodies hold promise for the development of targeted gene therapy vectors. In this report we describe baculoviral vectors displaying either functional scFv fragments or the synthetic Z/ZZ IgG;binding domain derived from protein A. Display on the baculovirus surface was achieved tia fusion of the scFv fragment or Z/ZZ domain to the N-terminus of gp64, the major envelope protein of the Autographa californica nuclear polyhedrosis virus, AcNPV. As examples of scFv fragments we have used a murine scFv specific for the hapten 2-phenyloxazolone and a human scFv specific for carcinoembryonic antigen. In principle, the Z/ZZ IgG binding domain displaying baculoviruses could be targeted to specific cell types via the binding of an appropriate antibody. We envisage applications for scFv and Z/ZZ domain displaying baculoviral vectors in the gene therapy field. (C) 2000 Academic Press.

关键词：抗体特异性杆状病毒科/遗传学杆状病毒科/代谢结合部位抗体/遗传学结合部位抗体/免疫学印迹法蛋白质癌胚抗原/遗传学癌胚抗原/免疫学细胞培养的酶联免疫吸附测定半抗原/遗传学半抗原/免疫学免疫球蛋白碎片/遗传学免疫球蛋白碎片/免疫学免疫球蛋白G/遗传学免疫球蛋白G/免疫学恶唑酮/类似物和衍生物恶唑酮/免疫学肽库重组融合蛋白质类/遗传学重组融合蛋白质类/免疫学重组融合蛋白质类/代谢斜纹夜蛾属/病毒学病毒融合蛋白质类/遗传学病毒融合蛋白质类/代谢动物人类小鼠

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Selection of peptides with semiconductor binding specificity for directed nanocrystal assembly

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NATURE 2000年第6787期405卷 665-668页

作者： Whaley, SR English, DS Hu, EL Barbara, PF Belcher, AM Univ Texas Dept Chem & Biochem Austin TX 78712 USA Univ Texas Texas Mat Inst Austin TX 78712 USA Univ Calif Santa Barbara Ctr Quantized Elect Struct Santa Barbara CA 93106 USA

In biological systems, organic molecules exert a remarkable level of control over the nucleation and mineral phase of inorganic materials such as calcium carbonate and silica, and over the assembly of crystallites and other nanoscale building blocks into complex structures required for biological function(1-4). This ability to direct the assembly of nanoscale components into controlled and sophisticated structures has motivated intense efforts to develop assembly methods that mimic or exploit the recognition capabilities and interactions found in biological systems(5-10). Of particular value would be methods that could be applied to materials with interesting electronic or optical properties, but natural evolution has not selected for interactions between biomolecules and such materials. However, peptides with limited selectivity for binding to metal surfaces and metal oxide surfaces have been successfully selected(10,11). Here we extend this approach and show that combinatorial phage-display libraries can be used to evolve peptides that bind to a range of semiconductor surfaces with high specificity, depending on the crystallographic orientation and composition of the structurally similar materials we have used. As electronic devices contain structurally related materials in close proximity, such peptides may rnd use for the controlled placement and assembly of a variety of practically important materials, thus broadening the scope for 'bottom-up' fabrication approaches.

关键词：氨基酸序列细菌噬菌体M13 DNA结合蛋白质类/分析 DNA结合蛋白质类/代谢显微镜检查原子力显微镜检查荧光肽库肽类/分析肽类/代谢蛋白质结合半导体病毒融合蛋白质类/分析病毒融合蛋白质类/代谢

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Amino acid substitutions at position 481 differently affect the ability of the measles virus hemagglutinin to induce cell fusion in monkey and marmoset cells co-expressing the fusion protein

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ARCHIVES OF VIROLOGY 1999年第9期144卷 1689-1699页

作者： Xie, MF Tanaka, K Ono, N Minagawa, H Yanagi, Y Kyushu Univ Fac Med Dept Virol Fukuoka 8128582 Japan

The hemagglutinin (H) protein of the measles virus (MV) Edmonston strain induced cell fusion in Cos (monkey) and B95a (marmoset) cells, when co-expressed with the fusion (F) protein, whereas the H protein of the wild-type KA strain induced fusion in B95a cells, but not in Cos cells. Asparagine residue at position 481 of the KA H protein was replaced by various amino acids through site-directed mutagenesis. Substitution with tyrosine, which was found at position 481 of the Edmonston H protein, enabled the mutant KA H protein (N481Y) to induce cell fusion in Cos cells co-expressing the F protein, which could be completely blocked by anti-CD46 antibody. This mutant, however, did not cause CD46 downregulation, unlike the Edmonston H protein. The other H protein mutants (N481S, N481T, N481D, N481H, N481F) did not produce syncytia in Cos cells. On the other hand, all of the mutants retained the ability to induce cell fusion in B95a cells. Thus, while tyrosine at position 481 was indispensable for the MV H protein's interaction with CD46, the residue at this position does not appear to be critically involved in the interaction with the receptor for wild-type strains present on B95a cells.

关键词：氨基酸取代抗原 CD/代谢细胞融合细胞系克隆分子流式细胞术基因病毒血凝素类病毒/遗传学血凝素类病毒/代谢麻疹病毒/遗传学麻疹病毒/代谢麻疹病毒/生理学分子序列数据诱变定点序列分析 DNA 转染病毒融合蛋白质类/遗传学病毒融合蛋白质类/代谢动物人类

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The fusion protein of peste des petits ruminants virus is a hemolysin

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ARCHIVES OF VIROLOGY 1999年第6期144卷 1241-1247页

作者： Devireddy, LR Raghavan, R Ramachandran, S Shaila, MS Vet Coll Dept Vet Microbiol Bangalore Karnataka India Univ Edinburgh Ctr Trop Vet Med Roslin Midlothian Scotland Indian Inst Sci Dept Microbiol & Cell Biol Bangalore 560012 Karnataka India

The fusion glycoprotein (F protein) of paramyxoviruses plays a vital role in virus-induced cytopathology. To explore the role of the F protein in peste des petits ruminants virus (PPRV)-induced cytopathology, the F protein of PPRV was purified by immunoaffinity chromatography. The purified F protein, when incubated with chicken erythrocytes, caused lysis suggesting that PPRV F protein is a hemolysin. Furthermore, the hemolysis can be inhibited by hyperimmune serum against F protein. The virus-induced cell fusion (syncytia) was also inhibited by the hyperimmune serum against the F protein. In summary, these results indicate that the purified PPRV F protein is biologically active and is involved in virus-induced hemolysis, cell-fusion and the initiation of infection.

关键词：色谱法亲和溶血素蛋白质类/分离和提纯溶血素蛋白质类/代谢溶血小反刍动物瘟疫病毒/代谢病毒融合蛋白质类/分离和提纯病毒融合蛋白质类/代谢动物

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