Efficient eukaryotic expression of fluorescent scFv fusion proteins directed against CD antigens for FACS applications

Two sets of expression vectors were constructed that permitted the efficient expression of single-chain Fv fragments (scFvs) fused N-terminally to an enhanced mutant of the green fluorescent protein GFP+ or the red fluorescent protein DsRed in insect and mammalian cells. The vectors allowed rapid cloning of scFv fragments and secretion of the fusion proteins in a native conformation. Fluorescent scFv fusion proteins directed against a series of cluster of differentiation (CD) antigens were efficiently secreted by transiently transfected mammalian cells and insect cells infected with baculoviral expression constructs. Yields of the secreted proteins varied from 100 microg/l to 3 mg/l. The purified proteins were functionally active in flow cytometry, immunofluorescent microscopy, and competition binding experiments performed to delineate the epitopes recognized by different monoclonal antibodies against the same polypeptide. The use of two different scFv fragments fused with red and green fluorescent proteins and reacting with T- and B-cell lineage markers (CD7 and CD19), respectively, allowed a simplified quantitation of both subsets in two-color flow cytometry experiments with mixed populations of T- and B-lymphoid cells. Due to the lack of Fc domains in the scFv proteins, the fluorescent fusion proteins showed more than 20-fold reduced background fluorescence compared with whole antibodies of the same specificity in experiments with effector cells expressing the high affinity FcgammaRI receptor CD64. Thus, for a number of analytical applications, fluorescent scFv fusion proteins offer advantages over the use of complete primary antibodies and chemically labeled fluorescent secondary antibodies.     

In vivo selection of sFv from phage display libraries

The development of phage display technology has facilitated the development of many new and sometimes novel antibody based reagents for scientific research. However, present methods for selection from phage-sFv display libraries are limited to selection against purified antigens or ex vivo cells of known origin and phenotype. Existing methods therefore preclude the isolation of sFv against unknown molecules in their natural environment, where expression is complex and subject to diverse control mechanisms. Since such a complex environment is difficult to mimic in vitro, the development of an in vivo selection procedure would greatly enhance the selection from phage display antibody libraries and lead to the development of reagents against cell surface molecules in their natural environment. This would be particularly advantageous for isolation of sFv against vascular endothelium which can readily change phenotype when cultured and is believed to express molecules in a tissue specific manner and in response to different stimuli. We describe here the development of an in vivo selection procedure in the mouse and demonstrate its potential for the selection of sFv from a phage-sFv library. The target antigen for one sFv is expressed solely on the thymic endothelium, while the second, a 165-170 kDa molecule in present on both thymic endothelium and the perivascular epithelium.     

Immunotoxins and cytokine toxin fusion proteins

Paul Ehrlich first suggested the simple and elegant concept of creating specific cell toxins or 'magic bullets' through the fusion of cell specific antibodies and toxins. In practice it has proven difficult to create safe and effective 'magic bullets'. In the past several years, several immunotoxins have been applied to clinical testing. These immunotoxins have been created by the biochemical coupling of cell or lineage specific monoclonal antibodies to plant toxins or fragments thereof. These immunotoxins have been used to treat bone marrow transplant recipients and patients with autoimmune disorders. In recent years, another strategy has also been pursued to create hybrid toxins. Rather than use antibodies as the targeting moiety, cytokines have been used to target a select population of cells bearing a high copy number of receptors for the specific cytokine. Rather than biochemically couple a cytokine to the toxin, the cytokine and toxin are fused by a peptide bond established via genetic engineering. A prototype IL-2 diphtheria toxin-related fusion protein is now being tested in the clinic for treatment of hematopoietic malignancies and autoimmune disorders.     

Monoclonal antibodies and fusion proteins in medicine

Humanized antibodies and decoy receptors have been introduced in clinical practice to treat malignancies and systemic autoimmune disease because they ablate specific cells or disrupt pathogenic processes at distinct points. Reported clinical responses offer hope to treatment-resistant patients, particularly those with lymphomas and rheumatic diseases. Side effects from the use of biologic agents include lymphokine release syndrome, reactivation of tuberculosis, and immunosuppression. Further insights are needed regarding limitation of adverse effects, correct use in conjunction with existing drugs, and treatment of patients in whom resistance develops.     

差异表达蛋白质的分离鉴定及其应用

蛋白质组（proteome）的概念由Wilkins和Williams于1994年首先提出。它源于蛋白质（protein）与基因组（genome）两个词的组合,指一个基因组（genOME）或一个细胞、组织表达的所有蛋白质（PROTein）。蛋白质组学（proteomics）是以蛋白质组为研究对象,阐释所有蛋白质在不同时空的表达谱和功能谱,全景式地揭示生命活动的本质,特别是人体健康与疾病的机制。     

Inhibition of osteoblast mineralization by phosphorylated phage-derived apatite-specific peptide

Functionalization of biomaterials with material- and cell-specific peptide sequences allows for better control of their surface properties and communication with the surrounding environment. Using a combinatorial phage display approach, we previously identified the peptide VTKHLNQISQSY (VTK) with specific affinity to biomimetic apatite. Phosphorylation of the serine residues of the peptide (pVTK) caused a significant increase in binding to apatite, as well as a dose-dependent inhibition of osteoblast mineralization. In this study, we investigated the mechanisms behind pVTK mediated inhibition of mineralization using MC3T3 cells and testing the hypothesis that mineralization is inhibited via alteration of the Enpp1–TNAP–Ank axis. Inhibition of mineralization was not due to disruption of collagen deposition or calcium chelation by the negatively charged pVTK. The timing of peptide administration was important in inhibiting mineralization – pVTK had a greater effect at later stages of osteogenic differentiation (days 7–12 of culture corresponding to matrix maturation and mineralization), and could prevent progression of mineralization once it had started. pVTK treatment resulted in a significant decrease in ectonucleotide pyrophosphatase/phosphodiesterase 1 (Enpp1) enzyme activity and gene expression. The expression of ankylosis protein (Ank), osteopontin (OPN) and Pit-1 genes was also significantly reduced with peptide treatment, while tissue non-specific alkaline phosphatase (TNAP), bone sialoprotein (BSP), and Runx2 gene expression was significantly higher. The ability of pVTK to inhibit mineralization can potentially be translated into therapeutics against pathological calcification seen in cardiovascular disease, osteoarthritis or craniosynostosis, or be used to prevent failure of biomaterials due to calcification, such as bioprosthetic heart valves.     

Class II virus membrane fusion proteins

Enveloped animal viruses fuse their membrane with a host cell membrane, thus delivering the virus genetic material into the cytoplasm and initiating infection. This critical membrane fusion reaction is mediated by a virus transmembrane protein known as the fusion protein, which inserts its hydrophobic fusion peptide into the cell membrane and refolds to drive the fusion reaction. This review describes recent advances in our understanding of the structure and function of the class II fusion proteins of the alphaviruses and flaviviruses. Inhibition of the fusion protein refolding reaction confirms its importance in fusion and suggests new antiviral strategies for these medically important viruses.     

高免疫原性骨桥蛋白的构建、表达及鉴定

目的重组及表达高免疫原性骨桥蛋白（OPN）的融合蛋白。方法使用Lasergene软件分析OPN基因,选取出免疫原性高的区域作为目的片段。提取总RNA,应用逆转录、PCR等技术扩增OPN基因目的片段,将其分别与带有His标签的pET-32a及GST标签的pGEX-4T-1载体连接。构建表达载体,诱导表达OPN融合蛋白（分别含有His,GST—Tag）,并进行SDS—PAGE及Western blotting鉴定。结果扩增后获得一条351bp的DNA片段,经测序鉴定为目的片段序列,并实现了可溶性高表达,经Western blotting鉴定为高免疫原性OPN融合蛋白。结论采用原核表达经纯化后可获得高纯度高免疫原性的OPN融合蛋白,为进一步开发OPN诊断试剂打下基础。     

Construction and expression of antibody-tumor necrosis factor fusion proteins

The construction, expression and secretion of two genetically engineered antibody-cytokine hybrid fusion proteins is described. To target tumor necrosis factor (TNF) to tumor cells, recombinant antibody techniques were used to generate F(ab')2-like antibody-TNF fusion proteins. At the gene level, an antitransferrin receptor antibody heavy chain gene was linked to a synthetic gene coding for human TNF. The chimeric heavy chain-TNF genes were introduced into a light chain secreting transfectoma cell line, which was producing the light chain of the same antibody. Cell lines were isolated which secreted antibody-TNF fusion proteins of expected size and composition. Culture supernatant of these cell lines contained TNF cytotoxic activity towards murine L929 cells and human MCF-7 cells, indicating that TNF is still active in the fusion protein constructs. These results illustrate the feasibility of the antibody engineering technology to create and produce chimeric mouse-human immunotoxin-like molecules. Furthermore, they demonstrate the ability of mammalian (myeloma) cells to express and secrete antibody-cytokine hybrid molecules with potential use in anticancer therapy.     

Antibody-cytokine fusion proteins for the therapy of cancer

Advances in genetic engineering and expression systems have led to rapid progress in the development of antibodies fused to other proteins. These 'antibody fusion proteins' have novel properties and include antibodies with specificity for tumor associated antigens fused to cytokines such as interleukin-2 (IL2), granulocyte/macrophage colony-stimulating factor (GM-CSF), and interleukin-12 (IL12). The goal of this approach to cancer therapy is to concentrate the cytokine in the tumor microenvironment and in so doing directly enhance the tumoricidal effect of the antibody and/or enhance the host immune response (T-cell, B-cell or NK) against the tumor. In the past decade, multiple antibody-cytokine fusion proteins have been developed with different specificities targeting a broad variety of tumors. These novel molecules retain both antibody and cytokine associated functions. In addition, in animals bearing tumors, antibody-cytokine fusion proteins are able to target the tumor and to elicit a significant anti-tumor response that in some cases results in a complete elimination of the tumor. These results suggest that antibody-cytokine fusion proteins have potential for use in the treatment of human cancer. In the present review, we describe strategies for construction of antibody-cytokine fusion proteins and discuss the properties of several antibody-cytokine fusion proteins with IgG genetically fused to the cytokines IL2, GM-CSF or IL12.     

信息融合技术及其在医疗监护系统中的应用

信息融合技术是信息处理领域新的有力工具 ,近年来它在医学数据的采集和处理中也得到了广泛的应用。本文综述了信息融合技术的发展及其在神经手术监护、ICU、创伤评估和社区医疗系统中的应用     

Fluobodies: green fluorescent single-chain Fv fusion proteins

An expression system (pSKGFP), which permits the expression of single-chain variable fragments as fusion proteins with modified green fluorescent proteins, was designed. This expression system is comparable to frequently used phage display vectors and allows single-step characterization of the selected recombinant antibodies by flow cytometry or fluorescent cell staining. Two different single-chain variable fragment antibodies, both directed against the lipopolysaccharide of the bacterium Ralstonia solanacearum have been genetically fused to a red-shifted green fluorescent protein and the produced fusion protein tested for usefulness. These fluobodies can be produced in cultures of bacterial cells and purified using immobilized metal affinity chromatography. They function well in flow cytometry and immunofluorescent cell staining, are specific for their target antigens and, unlike FITC-conjugated antibodies, they do not fade upon illumination.     

Epitope analysis of GAD65Ab using fusion proteins and rFab

The identification of disease-specific autoantibodies to the 65-kDa isoform of glutamate decarboxylase (GAD65Ab) epitopes in type 1 diabetes has been hampered by their conformational nature. Here, we compared two methods of GAD65Ab epitope analysis: GAD65/67 fusion proteins and competition assays using GAD65-specific recombinant fraction antigen binding (rFab). Sera from newly diagnosed type 1 diabetes patients (n=61) were studied using both approaches. Competition of GAD65 binding by an rFab to a specific epitope did not correlate with binding to the fusion protein that represented this epitope. Conversely, samples that bound to specific fusion proteins were not necessarily competed with rFab specific to determinants in the same region. We conclude that epitopes of different characteristics are detected by fusion proteins and by competition with rFab. Fusion proteins allow the definition of large epitope regions; however, some conformational GAD65Ab epitopes, especially those residing in the middle region, are destroyed or distorted in the fusion proteins. Competition studies using rFab allow the identification of conformational epitopes. However, monoclonal rFab may only reflect a limited proportion of the epitopes recognized by polyclonal sera. A combined analysis using both approaches may therefore be necessary to gain best understanding of autoantibody characteristics and affinity maturation.     

Myristylation of gag-onc fusion proteins in mammalian transforming retroviruses

Four cell lines producing transforming proteins encoded by three mammalian oncogenes (fes, abl, and ras) were investigated for incorporation of [3H]myristate into gag-onc fusion proteins. Using 5-min pulse-labelings, fusion proteins of Abelson murine leukemia virus, Gardner-Arnstein strain of feline sarcoma virus (FeSV), and Snyder-Theilen strain of FeSV were shown to be myristylated. In a 4-hr pulse, p29gag-ras of rat sarcoma virus (RaSV) was also shown to incorporate radiolabel. The fatty acid was recovered from this labeled protein by acid hydrolysis, and identified by reverse-phase thin-layer chromatography to be [3H]myristic acid. The results indicate that substitution of viral gag sequences by cellular oncogene sequences does not abolish their ability to become post-translationally modified by this long chain fatty acid (A. Schultz and S. Oroszlan, J. Virol. 46, 355-361). It is assumed that in the fusion proteins the myristyl moiety is linked through an amide linkage to the amino-terminal glycine as previously found for several retroviral gag precursor polyproteins (L. E. Henderson, H. C. Krutzsch, and S. Oroszlan, Proc. Natl. Acad. Sci. USA 80, 339-343). The possible role of myristylation of transforming proteins is discussed.     

Human anti-HIV-1 tat sFv intrabodies for gene therapy of advanced HIV-1-infection and AIDS

The early successes of highly active anti-retroviral therapies (HAART) for the treatment of HIV-1-infection and AIDS have raised the question as to whether there is a legitimate role for gene therapy in the treatment of this chronic infectious disease. However, in many patients the profound suppression of viral replication is short lived, particularly if patients have been treated with sequential monotherapies in the past, have been infected with a highly drug resistant isolate of HIV-1, or have temporarily discontinued therapy as a "holiday" or because of drug intolerance. In addition, life-long adherence to maintenance HAART will probably be required even in responding patients with undetectable viremia because of the reservoirs of latently infected cells that can persist for years. Gene therapy through the introduction of anti-retroviral "resistance" genes into CD4(+) T cells is one approach that could give long term protection to these HIV-1 susceptible cells in vivo. We have explored this approach by developing intrabodies to the critical HIV-1 transactivator protein, Tat that is absolutely required for HIV-1 replication. This provocative treatment approach, that will be tested in a clinical gene therapy trial, sets the groundwork for determining if anti-Tat intrabody gene therapy together with HAART can provide a treatment strategy for the immune reconstitution of HIV-1-infected patients with advanced disease.