红细胞带Ⅲ蛋白C1肽链的纯化及其功能的初步研究

目的：研究红细胞带Ⅲ蛋白Ｃ１肽链（带Ⅲ蛋白Ａｌａ８９３－Ｖａ１９１１）的特性及其功能。方法：以１００ｍｍｏｌ／ＬＮａＯＨ预处理红细胞膜血影，再用胰蛋白酶消化，采用高效液相色谱（ＨＰＬＣ）及氨基酸测序仪分析检定了从红细胞血影中释放的多肽，并纯化了带Ⅲ蛋白Ｃ１肽链，以不同浓度的Ｃ１肽链与新鲜的红细胞血影进行温浴。结果：Ｃ１肽链与红细胞内的新蛋白酶活性有关。     

Monoclonal antibodies against dengue NS2B and NS3 proteins for the study of protein interactions in the flaviviral replication complex

The replication of dengue virus (DENV) RNA requires at least two viral non-structural (NS) proteins, NS3 and NS5. To facilitate the study of the DENV replication complex, human monoclonal IgG that are specific for NS proteins have been generated and characterised. The anti-NS3 IgG, 3F8, binds a conserved epitope (aa526-531) in the NS3 helicase domain, and cross-reacts with NS3 from all four DENV serotypes and the related yellow fever virus. The anti-NS2B IgG, 3F10, binds aa49-66 of NS2B (CF18), which forms part of the 47 aa hydrophilic cofactor region required for NS3 protease activity. The specificity of the IgG for their respective non-structural proteins has been demonstrated by immunofluorescence of cells infected with DENV and Western blotting. 3F8 is able to co-immunoprecipitate NS3 and NS5 from BHK-21 cells infected with DENV2, and 3F10 is able to detect an interaction between recombinant NS2B_CF18NS3 full-length protein and the NS5 RNA-dependent RNA polymerase (RdRp) domain in an ELISA-based binding assay. The assay is specific and highly reproducible, with a clear binding curve seen when RdRp is incubated with increasing amounts of full-length NS3, but not the NS3 protease domain. The NS3 helicase domain competes with NS3 full-length for NS5 RdRp binding, with a K_d. of 2.5 μM. Since NS3 and NS5 are required for DENV replication, this fascile assay could be used to screen for non-nucleoside, allosteric inhibitors that disrupt the interaction between the two proteins.     

The identification and characterization of a monoclonal antibody to the vaccinia virus E3 protein

Monoclonal antibodies with reactivity to vaccinia virus specific proteins are useful reagents to study the proteins as well as to help understand aspects of the poxvirus life cycle. Using a vaccinia virus proteomics microarray, we found a hybridoma (MAb 3015B2) from a vaccinia virus vaccinated mouse that reacted with the product of the E3L gene. The specificity to the E3 protein was confirmed by Western blotting and immunofluorescence of cells infected with either wild-type vaccinia virus or a mutant virus with the E3L gene deleted. Antibody reactivity with E3 was also seen in cells transfected with a plasmid expressing the E3 protein. A panel of mutated vaccinia viruses with truncations in the E3L gene revealed that while MAb 3015B2 reacted with E3 lacking the C-terminal 7 amino acids, it lost reactivity with a mutant E3 lacking the C-terminal 26 amino acids. This indicates that the antigenic site recognized by 3015B2 is on the C-terminus, somewhere between amino acids 164 through 183. The antibody also recognizes the E3 protein encoded by other orthopoxviruses. This antibody will be useful for further investigations of the E3 protein as well as a useful reagent to indicate vaccinia virus early protein expression.     

Molecular genetics of the glycophorin gene family,the antigens for MNSs blood groups: Multiple gene rearrangements and modulation of splice site usage result in extensive diversification

The purpose of the review is to describe a system of human erythrocyte membrane glycoproteinsexhibiting extensive diversity. Glycophorins A and B (GPA and GPB) are the antigens of the MNSsblood groups; thus individuals bearing variant glycophorins can be readily identified by serologicaltyping. Examination of the wide array of variants of these antigens showed that they include manyforms, possibly made evident by lack of constraints due to the apparent dispensability of the parentmolecules. This article reviews the molecular genetics of 25 variants of the glycophorin gene family, whose common denominator is that they arise from unequal gene recombinations or gene conversionscoupled to splice-site mutations. Most rearrangements occurred within a 2-kb region mainly within GPA and GPB of the gene family and only rarely within the third member, GPE. The key feature isthe shuffling of sequences within two specific exons (one of which is silent), homologous in the twoparent genes. This has resulted in expression of a mosaic of sequences within this region, leading topolymorphism. The common pattern of recombinations coupled to pre-mRNA splicing was the predominant mechanism of the origin of glycophorin diversity. Thus far this mechanism appears to be unique amonghuman gene families. It could have occurred by chance rearrangements among closely linked genes andbeen driven by a biological advantage, not as yet identified. This remains to be established. Nevertheless, gene rearrangements observed here are akin to those reported for the major histocompatibilitycomplex (MHC). In the glycophorin family the small size of the region within which gene interactionshave occurred and the participation of essentially only two alleles makes this relatively simpler systemmore focused and easier to dissect and describe molecularly. © 1995 Wiley-Liss, Inc.     

Identification of a neutralization epitope in the VP1 capsid protein of SV40

Three SV40 escape mutants were identified by selection in the presence of monoclonal antibodies with neutralizing activity. The VP1 amino acid alterations in these mutants were: (1) K73 → E (in loop BC); (2) D77 → E (in loop BC); (3) K171 → R (in loop EF); and (4) Q175 → H (in loop EF). These residues are clustered in close proximity to each other on the surface of the native capsid protein, strongly suggesting that they form a conformational epitope directly recognized by the neutralizing antibody. To our knowledge, the present study represents the first experimental mapping of a neutralization epitope of a polyomavirus family member. Structural information regarding the neutralization epitope should be useful for clarifying the extent of cross-reactivity exhibited by the humoral immune response towards related primate polyomaviruses (e.g., SV40, BKV, and JCV).     

HCV NS3蛋白单克隆抗体的制备及其识别区域的分析

目的 制备针对丙型肝炎病毒（HCV）非结构区NS3全长蛋白的单克隆抗体（MAb）,并分析获得的单抗识别表位所在区域,为建立以NS3蛋白为靶位的抗HCV研究提供抗体工具。方法 用原核表达的HCV非结构区NS3全长蛋白作为免疫原,采用小鼠腹股沟皮下NC膜包埋法免疫小鼠,按常规杂交瘤细胞的制备方法,经细胞融合、克隆化制备抗NS3蛋白的MAb。用间接免疫荧光法和Westem blot鉴定其特异性。分别构建NS3丝氨酸蛋白酶（NS3蛋白的N末端1／3）编码基因的真核表达质粒pcDNA3．1（-）-ns3p、NS3解旋酶（NS3蛋白的C末端2/3）编码基因的真核表达质粒pcDNA3．1（-）-ns3A,将其瞬时转染COS-7细胞后,以获得的单克隆抗体作为一抗,通过免疫荧光分析获得单抗识别表位所在的区域。结果 获得了2株抗NS3蛋白的单克隆抗体,这2株MAbs均特异识别NS3蛋白,并确定了它们的结合区域。结论 获得了针对NS3蛋白的单克隆抗体,并对其单抗识别表位所在的区域进行了分析,为下一步进行以NS3蛋白为靶位的抗HCV研究奠定了良好的基础。     

Pathogenic autoantibodies in the NZB mouse are specific for erythrocyte Band 3 protein

NZB mice spontaneously develop autoimmune hemolytic anemia (AIHA). The red blood cell (RBC) autoantigen bound by pathogenic IgG autoantibodies, previously designated “X”, was identified by immunoprecipitation. Autoantibody eluted from the RBC of AIHA-positive NZB mice precipitated a 105-kDa antigen that was identical in apparent molecular mass to Band 3, the RBC anion channel protein. Furthermore, the immunoblotted antigen also reacted specifically with BRIC 132, a monoclonal antibody against Band 3. The results, therefore, demonstrate that Band 3 bears autoantigenic epitopes that are important in the pathogenesis of AIHA in the NZB mouse.     

Identification of a B-cell antigenic epitope at the N-terminus of SARS-CoV M protein and characterization of monoclonal antibody against the protein

To identify the potential B-cell antigenic epitopes within the N-terminus of SARS-CoV (SARS-associated coronavirus, SARS-CoV) M protein and characterize monoclonal antibody (MAb) against the protein as well as its recognizing region, we expressed and purified a portion of SARS-CoV M protein (amino acid 1–43) in Escherichia coli (E. coli). By using Western blot and enzyme-linked immunosorbent assay (ELISA), we showed that the purified recombinant M protein could be recognized by four SARS-CoV-positive human sera even when those sera were 12,800-fold diluted. Furthermore, we characterized one representative IgG2 MAb, 3H9, which exhibited a strong immunoreaction to both recombinant M protein and native viral protein of SARS-CoV. We found a B-cell antigenic epitope located between amino acid 1–15 and defined the MAb recognizing region within amino acid 16–28 of M. These findings not only suggest that both recombinant M protein and its specific MAbs may be used as the diagnostic reagents for SARS, but also provide a potential target site for the design of an epitope-based vaccine against SARS. Chao Qian and Di Qin contributed equally to this work.     

FU-3 monoclonal antibody: a specific marker for malignant fibrous histiocytoma? An analysis of 32 malignant soft tissue and bone sarcomas

An immunohistochemical study on frozen sections was carried out on 51 malignant tumours of soft tissue and bone using the FU-3 monoclonal antibody. This antibody is claimed to be specific for malignant fibrous histiocytoma (MFH) and liposarcoma and for normal and tumour cells located in perivascular fields. The results show a lack of specificity in MFH staining: several malignant tumours such as synovial sarcoma, fibrosarcoma, rhabdomyosarcoma, osteogenic sarcoma, and including an anaplastic malignant melanoma, presented positive staining somewhat similar to that found in MFH. The value of this antibody in the differential diagnosis of MFH is doubtful. It might be useful to recognize a common pathway of terminal differentiation expressed by several pleomorphic sarcomatous neoplasms.This paper was presented in a short form at the XIX International Congress of the International Academy of Pathology, Madrid (18–23 October 1992).     

Production and characterization of monoclonal antibodies specific for the murine T cell receptor ζ chain

The T cell receptor (TCR) comprises an antigen-specific β heterodimer non-covalently associated with the CD3 γδε and TCR ζ subunits. Both the CD3 and TCR ζ subunits are proposed to be responsible for the intracellular signal-transduction events. We report here the production of eight monoclonal antibodies (mAbs) that bind in an ELISA assay to a 113 amino acid synthetic peptide corresponding to the cytoplasmic domain of TCR ζ. Western blot analysis of anti-CD8 precipitates of lysates of transfectants expressing chimeric CD8/ζ constructs encoding increasing COOH-terminal truncations of TCR ζ indicates that four of these mAbs recognized the region of TCR ζ chain comprising the last 29 COOH-terminal residues. Thus, this region of TCR ζ may encode an immunodominant epitope. Furthermore, one of these mAbs, G3, is capable of precipitating both non-phosphorylated and tyrosine phosphorylated TCR ζ. The G3 mAb should be useful for elucidiating the structural and signalling characteristics of the TCR ζ chain.     

Efficient generation of monoclonal antibodies for specific protein domains using recombinant immunoglobulin fusion proteins: pitfalls and solutions

Monoclonal antibody production (mAb) first requires the availability of large amounts of pure immunogen for animal immunisation and fusion screening procedures. To overcome this obstacle, we have developed a simple method for rapid generation of pure antigen by generation of recombinant protein containing the antigen of interest fused to the hinge and Fc domains of human immunoglobulin (Ig). The Fc domain forms a convenient 'tag' to enable detection of the protein in supernatant of transfected cells and for purification of immunogen by protein A affinity chromatography. The only requirement for immunogen preparation using this methodology is that a DNA sequence encoding a portion of the molecule of interest is known and that a suitable PCR template is available. Antibody production can be tailored to specific protein domains, for example functional domains, by expressing solely those domains in the fusion protein. We illustrate the technique with two different fusions used to raise antibodies against the porcine and human analogues of a complement (C) regulatory protein, decay accelerating factor (DAF) (CD55). Use of the specific Ig-fusion protein and a control protein facilitated screening of fusions by ELISA. We demonstrate two expression systems used to generate Ig fusion proteins, the first utilised a commercial vector to incorporate an amino terminal leader sequence and carboxy terminal Ig domains. Low levels of expression required subcloning into a high expression vector and resulted in yields of fusion protein at between 2 and 10 mg per litre of supernatant. The second expression system utilised the high expression vector directly, Ig domains of the chosen immunoglobulin isotype were amplified from peripheral blood mononuclear cell (PBMC) RNA and ligated into the vector in frame with DNA encoding the antigen. We describe potential pitfalls that may be encountered while using Ig fusion proteins as immunogen and demonstrate ways in which to tailor their design for optimal mAb production.     

Use of monoclonal antibodies to study interleukin -1β-converting enzyme expression: only precursor forms are detected in interleukin-1β-secreting cells

We have generated a series of monoclonal antibodies (mAb) using recombinant interleukin (IL)-1β-converting enzyme (ICE) p20 and p10 subunits as immunogens. The mAb have been selected for further study based on their reactivity with ICE in transfected COS cells and their lack of cross-reactivity with TX, the closest ICE homolog known to date. Two anti-p20 and one anti-p10 mAb have been used to study ICE expression by Western blotting and immunodetection. In ICE-transfected COS cells, the mAb recognize the p45 ICE precursor and the maturation products (p20 or p10 subunits) for which they are specific. In monocytes and cell lines expressing ICE, only precursor forms are detected and intracellular immunostaining followed by confocal microscopy shows that they are located in the cytoplasm. Quantification experiments show that THP1 cells express approximately 67000 molecules of ICE precursor per cell, with an estimated precursor to mature ratio of at least 100. In these cells as well as in monocytes, lipopolysaccharide stimulation did not change the pattern of ICE expression, although efficient secretion of mature IL-1β was measured. However, upon cell disruption, precursor maturation was observed. Our results, therefore, show that ICE is present in cells as a large pool of intracytoplasmic precursor, and that very limited amounts of mature ICE protein are present, but nevertheless sufficient to allow efficient IL-1β cleavage. Altogether, these observations suggest that post-translational maturation of the precursor protein could represent a specific step in the regulation of ICE enzymatic activity.     

Identification of antigenic differences between the phosphorylated and nonphosphorylated forms of the E7 protein of human papillomavirus type 16

To analyze the antigenic properties of the human papillomavirus type 16 E7 oncoprotein, two monoclonal antibodies, VD6 and IB10, that have different reactivities to the E7 protein were generated. While the VD6 antibody reacted strongly with E7 protein in CaSki cell extracts, the other antibody, IB10, showed much weaker reactivity with E7. This reactivity increased in a dose-dependent manner in the presence of the casein kinase II-specific inhibitor DRB (5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole). Antigenic site estimation and an in vitro phosphorylation assay, using bacterially expressed E7 protein, demonstrated that the weak reactivity of IB10 was related to the phosphorylation status of the E7 protein. Phosphorylation of E7 reduced considerably the reactivity of IB10 but did not affect the reactivity of VD6, which reacts with the N-terminal portion of E7. In immunoprecipitation (IP) assays, IB10 precipitated weakly the E7 protein from CaSki cell extracts. Together, these data suggest that unphosphorylated E7 protein shows distinct antigenic character compared to its phosphorylated form under denaturing conditions; however, under native conditions, the phosphorylated and nonphosphorylated E7 proteins have some antigenic cross-reactivity. J. Med. Virol. 54:129–134, 1998. © 1998 Wiley-Liss,Inc.     

CDw50 and ICAM-3: Two names for the same molecule

CDw50 differentiation antigen is a molecule broadly expressed on hematopoetic cells but not on other cells. Previous experiments showed that CDw50 monoclonal antibodies (mAb) inhibited primary mixed lymphocyte culture (MLC). To understand the function of CDw50 better, we purified it and obtained peptide sequence. At the same time, intercellular adhesion molecule (ICAM)-3, the third Iigand of lymphocyte function-associated molecule 1, was described by mAb and subsequent cDNA cloning. Immunochemical, functional, and protein sequencing studies show that ICAM-3 and CDw50 are the same glycoprotein, a 120-kDa surface molecule with presumably an important role in the immune responses.     

Identification of a common conserved neutralizing linear B-cell epitope in the VP3 protein of waterfowl parvoviruses

ABSTRACT

Waterfowl parvoviruses (WPVs) including goose parvovirus (GPV), novel GPV-related virus (NGPV) and Muscovy duck parvovirus (MDPV) cause significant economic losses and epizootic threat to the waterfowl industries, and little is known about the B-cell epitopes of WPVs. In this study, a monoclonal antibody (mAb) 5B5 against the VP3 protein of NGPV was used to identify the possible epitope in the three kinds of WPVs. The mAb 5B5 had neutralizing activities to the three viruses, and reacted with the conserved linear B-cell epitopes of ⁴³⁸LHNPPP⁴⁴³ in VP3 protein of GPV, NGPV and MDPV. To the authors’ best knowledge, this is the first report on identification of the common conserved neutralizing linear B-cell epitope on VP3 protein of three different WPVs, which would facilitate the development of a novel immunodiagnostic assay for rapid detection of WPV infection.     

Ca/calmodulin-dependent protein kinase II in the rat cerebellum: An immunohistochemical study with monoclonal antibodies specific to either α or β subunit

Monoclonal antibodies specific to either α or β subunit of Ca²⁺/calmodulin-dependent protein kinase II (CaM kinase II) of the rat brain were produced and the distribution of each subunit in the rat cerebellum was examined immunohistochemically. Each antibody detected solely the corresponding subunit in immunoblot analysis of crude homogenates of the rat forebrain and cerebellum, and purified CaM kinase II from the rat forebrain. Immunoreactivity for α subunit was present selectively in Purkinje cells: perikarya, dendrites with their spines, axons and their terminal-like structures in the cerebellar cortex, cerebellar nuclei and lateral vestibular nucleus. Many of these α subunit-immunoreactive axons from the cerebellum were traced only through the inferior cerebellar peduncle. β Subunit was detected in perikarya and dendrites of a limited number of Purkinje cells, many granule cells and neurons in the cerebellar nuclei. Thus, different distributions of α and β subunits of CaM kinase II in the cerebellum were demonstrated.