JC病毒衣壳蛋白VP1多克隆抗体的制备

目的制备pET-32a(+)-VP1蛋白的多克隆抗体。方法用纯化后的VP1蛋白分4次免疫兔子,颈动脉插管法取血,制得多克隆抗体。结果用ELISA法和Western blot鉴定多克隆抗体的效价得1?320000。该抗体可以用Western blot法检测出59 kD左右的VP1蛋白。结论成功制备高效价的JC病毒衣壳蛋白VP1多克隆抗体。     

Application of VP1 protein to develop monoclonal antibody against foot-and-mouth disease virus Asia1 type

In order to develop an anti-FMDV Asia1 type monoclonal antibody (mAb), BABL/c mice were immunized with recombinant FMDV VP1 protein. Three mAbs, 1B8, 5E1 and 5E2, were then further optimized. The result indicated that prepared anti-FMDV Asia1 mAbs had no cross-reactivity with Swine vesicular disease (SVD) and FMDV O, A and C type antigen. Their titers in abdomen liquor were 1:5×10⁶, 1:2×10⁶ and 1:5×10⁶, respectively. 1B8 was found to be of IgG₁ subtype, 5E1 and 5E2 belonged to IgG_2b subtype. In this study, the prepared mAbs are specific for detecting FMDV type Asia1, and is potentially useful for pen-side diagnosis. Foudation items: The National high Technology Research and Development Program of China (No.2006AA10A204); The National science & Technology Pillar Program (No. 2006BAD06A17)     

Preparation and characterization of monoclonal antibodies against VP1 protein of foot-and-mouth disease virus O/China99

Monoclonal antibodies (McAbs) 1A9 and 9F12 against Foot-and-mouth disease virus (FMDV) serotype O were produced by fusing SP2/0 myeloma cells with splenocyte from the mouse immunized with O/China99. Both McAbs reacted with O/China99 but not with Asia 1, as determined by immunohistochemistry assay. The microneutralization titer of the McAbs 1A9 and 9F12 were 640 and 1 280, respectively. Both McAbs contain kappa light chains, but the McAbs 1A9 and 9F12 were IgG1 and IgM, respectively. In order to define the McAbs binding epitopes, the reactivity of these McAbs against VP1, P20 and P14 were examined using indirect ELISA, the result showed that both McAbs reacted with VP1 and P20. McAbs may be used for further studies of vaccine, diagnostic methods, prophylaxis, etiological and immunological researches on FMDV.     

一株抗蓝舌病病毒8型VP2蛋白单克隆抗体识别的线性抗原表位的鉴定

为研究本实验室制备的一株抗蓝舌病病毒8型(BTV-8)VP2蛋白的单克隆抗体(MAb)3G11识别的B细胞抗原表位,利用噬菌体肽库展示技术对3G11识别的抗原表位进行筛选并鉴定。经过4轮淘选后挑取蓝斑测序,测序结果经分析后获得KLLAT序列,与BTV-8 VP2蛋白氨基酸序列比对后获得共同的短肽序列为283LL284;合成4种短肽序列:KLLAA、KALAT、KLAAT和KLLAT,与3G11细胞上清和腹水分别进行间接ELISA鉴定,结果表明,短肽KLLAA和KLLAT与3G11细胞上清及腹水具有较强的结合能力;与24种BTV标准阳性血清反应结果表明,这两种短肽都可与BTV-8阳性血清发生特异性反应;序列分析结果可见,该表位的氨基酸序列283LL284在不同来源的BTV-8毒株间保守,确定283LL284为MAb3G11识别抗原表位的关键氨基酸。本研究为建立8型BTV特异性的免疫学检测方法和相关病毒蛋白的功能研究奠定了基础。     

Establishment of a monoclonal antibody against human Toll-like receptor 3 that blocks double-stranded RNA-mediated signaling

A monoclonal antibody (mAb) against human Toll-like receptor (TLR) 3 was established and its effect on TLR3-mediated responses was tested using human fibroblast cell lines expressing TLR3 on the cell surface. Fibroblasts are known to produce IFN-beta upon viral infection or treatment with double-stranded RNA (dsRNA) through distinct signaling pathways. Here, we show the mAb to TLR3 suppressed poly(I):poly(C)-mediated IFN-beta production by human fibroblasts naturally expressing TLR3 on their surface. By reporter gene assay using HEK293 cells transfected with a human TLR3 expression vector, TLR3 recognized dsRNA to activate NF-kappaB and the IFN-beta promoter. TLR3 signaling was not elicited by either single-stranded RNA (ssRNA) or dsDNA. Thus, specific recognition of dsRNA by extracellular TLR3 is essential for induction of type I IFN: the interassociation between dsRNA and TLR3, regardless of direct or indirect binding, should be disrupted by mAb being attached to TLR3. The mAb against TLR3 reported herein may serve as a regulator for virus-mediated immune response via an alternative pathway involving the dsRNA-TLR3 recognition which might occur on host cells.     

抑制T细胞增殖的抗CD98重链单克隆抗体的建立

目的寻找能调节T细胞功能的相关分子,进行与T细胞介导的自身免疫性疾病相关的研究。方法收集BALB/c小鼠脾细胞,免疫Wistar大鼠,进行细胞融合,建立杂交瘤细胞系。筛选得到43株能调节T细胞功能的杂交瘤细胞系,对其中一株最能抑制T细胞增殖的杂交瘤细胞系进行了进一步的深入研究。结果显示其目标分子是CD98重链,同时后续实验显示抗CD98单克隆抗体能抑制纤连蛋白介导的细胞分布,但不影响氨基酸转运。而且混合淋巴细胞反应显示该抗体能显著抑制T细胞增殖反应。结论抗CD98单克隆抗体能有效抑制T细胞增殖,有望将本抗体用于T细胞介导的自身免疫性疾病的相关预防及治疗中。     

抑制T细胞增殖的抗CD45单克隆抗体的建立

目的寻找能调节T细胞功能的相关分子,进行与T细胞介导的自身免疫性疾病相关的研究。方法从BALB/c小鼠骨髓中收集树突状细胞,免疫Wistar大鼠,进行细胞融合,建立杂交瘤细胞系。筛选得到很多株能调节T细胞功能的杂交瘤细胞系,对其中一株最能抑制T细胞增殖的杂交瘤细胞系进行了进一步的深入研究。结果显示其目标分子是CD45,同时增殖实验结果显示该抗体能显著抑制T细胞增殖反应。结论抗CD45单克隆抗体能有效抑制T细胞增殖,有望将本抗体用于T细胞介导的自身免疫性疾病的相关预防及治疗中。     

灰飞虱内生病毒HiPV外壳蛋白VP1多克隆抗体的制备及在病毒检测中的应用

【目的】前期发现水稻条纹病毒(rice stripe virus, RSV)可与介体灰飞虱Laodelphax striatellus体内的HiPV病毒(Himetobi P virus, HiPV)互作。本研究旨在制备HiPV外壳蛋白VP1的多克隆抗体,并评估其在HiPV病毒检测中的可用性,以为深入研究HiPV-RSV和HiPV-灰飞虱的互作机制提供技术支持。【方法】以RT-PCR方法从灰飞虱成虫体内扩增HiPV主要外壳蛋白基因VP1,然后将VP1基因亚克隆至原核表达载体pET-32a中,构建表达载体pET-VP1。将重组质粒转化大肠杆菌Escherichia coli BL21 (DE3),经IPTG诱导、Ni²⁺-NTA亲和层析纯化,获得重组蛋白,免疫新西兰大白兔,制备抗体。【结果】从灰飞虱体内克隆到774 bp的HiPV外壳蛋白基因VP1,经原核表达、纯化,获得分子量约47.5 kD的融合蛋白,免疫新西兰大白兔后获得VP1多克隆抗体。该抗体间接ELISA效价达1∶819 200,与HiPV外壳蛋白VP1有特异性反应,而与灰飞虱蛋白无交叉反应。利用该多克隆抗体建立了检测单头灰飞虱成虫体内HiPV的Western blot和免疫捕获RT-PCR方法,检测结果显示HiPV在携带和不携带RSV的灰飞虱高亲和性群体内均广泛存在。【结论】利用制备的HiPV的VP1多克隆抗体可特异性检测灰飞虱体内HiPV。本研究为HiPV病毒的快速检测以及HiPV-RSV互作、HiPV-灰飞虱互作研究提供了技术支持。     

Generation of recombinant monoclonal antibodies to study structure-function of envelope protein VP28 of white spot syndrome virus from shrimp

White spot syndrome virus (WSSV) is a major pathogen in shrimp aquaculture. VP28 is one of the most important envelope proteins of WSSV. In this study, a recombinant antibody library, as single-chain fragment variable (scFv) format, displayed on phage was constructed using mRNA from spleen cells of mice immunized with full-length VP28 expressed in Escherichia coli. After several rounds of panning, six scFv antibodies specifically binding to the epitopes in the N-terminal, middle, and C-terminal regions of VP28, respectively, were isolated from the library. Using these scFv antibodies as tools, the epitopes in VP28 were located on the envelope of the virion by immuno-electron microscopy. Neutralization assay with these antibodies in vitro suggested that these epitopes may not be the attachment site of WSSV to host cell receptor. This study provides a new way to investigate the structure and function of the envelope proteins of WSSV.     

Detection of circulating Asian H5N1 viruses by a newly established monoclonal antibody

Monoclonal antibodies (MAbs) against the recently emerged Asian H5N1 virus (A/crow/Kyoto/53/2004) were generated. From five anti-hemagglutinin (HA) MAbs, four antibodies (3C11, 4C12, 3H12, and 3H4) broadly in vitro recognized and neutralized H5 subtypes, including H5N1. By contrast, the 4G6 MAb specifically reacted with H5N1-HA and not with H5N2- or H5N3-HAs from previous epidemics. The 4G6 MAb was useful for immunofluorescence assays but not for immunoblotting, suggesting that this antibody recognizes a conformational epitope of the H5N1-HA protein. An intensive epitope-mapping analysis demonstrated that the 4G6 MAb recognizes Asp59, which is highly conserved among currently circulating H5N1 lineages. Further, a 4G6-based antigen capture enzyme-linked immunosorbent assay detected H5N1 even that derived from clade 2.2 (A/chicken/Egypt/CL-61/2007) from infected chicken lung before virus isolation. Taken together, these results suggest that the established MAbs, especially 4G6, are useful for rapid and specific detection of Asian H5N1 viruses.     

Screening of phage-displayed peptide libraries with a monoclonal antibody raised against the F protein of the bovine respiratory syncytial virus

Summary Most of the monoclonal antibodies (MAbs) raised against the fusion (F) protein of the bovine respiratory syncytial virus recognize discontinuous epitopes on the protein. In order to find mimotopes of these epitopes, phage-displayed peptide libraries were screened with MAbs. The results obtained with MAb AL11C2 are described here. After four or five pannings, colony immunoscreening with AL11C2 allowed the isolation of positive clones that are specific for this monoclonal antibody. Four different sequences were determined on isolated phages, three of which are cysteine-constrained peptides in fusion with PVIII and one is a hexapeptide in fusion with PIII. In the case of the peptides containing two cysteines, the binding to AL11C2 was shown to be dependent on the presence of a disulfide bridge. The recombinant phages were also shown to inhibit the binding of AL11C2 to its natural antigen in a competitive ELISA assay.     

Characterization and application of a murine monoclonal antibody that reacts specifically with the serogroup D1Salmonella

A murine hybridoma cell line that produces monoclonal antibody (mAb) against the serogroup D1 Salmonella lipopolysaccharide (LPS) antigen was established. The trisaccharide tyvelose alpha 1----3 mannose alpha 1----4 rhamnose was shown to be involved in the reactive epitope of the mAb since this mAb reacted strongly with strains of serogroup D1 Salmonella but not with Salmonella strains from the O serogroups of A, B, and D2, and sodium meta-periodate was found to destroy the reactivity of the serogroup D1 O-antigen with the mAb. As such this mAb was found to be a useful serotyping reagent for the identification of serogroup D1 Salmonella, and for the differentiation of strains of serogroups D1 and D2 Salmonella which have identical flagellar H antigens.     

A new monoclonal antibody, 4-1a,that binds to the amino terminus of human lipoprotein lipase

Lipoprotein lipase (LPL) has been highly conserved through vertebrate evolution, making it challenging to generate useful antibodies. Some polyclonal antibodies against LPL have turned out to be nonspecific, and the available monoclonal antibodies (Mabs) against LPL, all of which bind to LPL's carboxyl terminus, have drawbacks for some purposes. We report a new LPL-specific monoclonal antibody, Mab 4-1a, which binds to the amino terminus of LPL (residues 5–25). Mab 4-1a binds human and bovine LPL avidly; it does not inhibit LPL catalytic activity nor does it interfere with the binding of LPL to heparin. Mab 4-1a does not bind to human hepatic lipase. Mab 4-1a binds to GPIHBP1-bound LPL and does not interfere with the ability of the LPL–GPIHBP1 complex to bind triglyceride-rich lipoproteins. Mab 4-1a will be a useful reagent for both biochemists and clinical laboratories.     

Generation and application of a monoclonal antibody that detects a wide variety of protein tyrosine kinases

To investigate expression profiles of the entire family of protein tyrosine kinases (PTKs), we attempted to generate an antibody that detects a variety of PTKs. For production of the antibody, antigenic peptides corresponding to amino acid sequences of a highly conserved region (subdomain VIB) of PTKs were synthesized and immunized to BALB/c mice. Among various antigens, a peptide with 11 amino acids, CYVHRDLRAAN, efficiently produced a polyclonal antibody with a broad cross-reactivity to PTKs. We established a hybridoma cell line producing a monoclonal antibody, YK34, which appeared to cross-react with at least 68 PTKs in the human genome, as evidenced by its reactivity with the recombinant Src tyrosine kinases whose subdomain VIB had been replaced by those of the other PTKs. When differentiation of HL-60 cells was induced by 12-O-tetradecanoylphorbol-13-acetate, on Western blotting we observed significant changes in immunoreactive bands with YK34 in HL-60 cell extracts along with changes in the morphology of the cells. These results suggest that the YK34 antibody will be a powerful tool for analysis of a variety of cellular PTKs.     

F(ab′)2 fragment of a gp41 NHR-trimer-induced IgM monoclonal antibody neutralizes HIV-1 infection and blocks viral fusion by targeting the conserved gp41 pocket

Using a recombinant protein N46FdFc that mimics the HIV-1 gp41 N-helix trimer to immunize mice, we identified the first IgM monoclonal antibody 18D3 that specifically bound to the conserved gp41 pocket. Its F(ab′)₂ fragment potently inhibited HIV-1 Env-mediated cell–cell fusion and neutralized infection by laboratory-adapted and primary HIV-1 isolates with different subtypes and tropism, including the T20-resistant variants. This F(ab′)₂ fragment can be used to develop a bispecific broad neutralizing monoclonal antibody or HIV-1 inactivator as a novel immunotherapeutic for treatment and prevention of HIV-1 infection.     

High expression of foot-and-mouth disease virus structural protein VP1 in tobacco chloroplasts

A tobacco chloroplast expression vector, pTRVP1, containing the foot-and-mouth disease virus (FMDV) VP1 gene and the selective marker aadA gene, was constructed and transferred to tobacco by biolistic method. Three resistant lines were obtained through spectinomycin selection, and each transgenic line was subjected to a second round of spectinomycin selection. PCR and PCR southern blot analysis revealed that the VP1 gene had integrated into the chloroplast genome. Western blot and quantification ELISA assays indicated that the VP1 gene was expressed in tobacco chloroplasts and accounted for 2–3% of total soluble protein. This suggested that plant chloroplasts were an efficient expression system for the potential production of recombinant antigens in plants.     

Effect of the DnaK chaperone on the conformational quality of JCV VP1 virus‐like particles produced in Escherichia coli

Protein nanoparticles such as virus‐like particles (VLPs) can be obtained by recombinant protein production of viral capsid proteins and spontaneous self‐assembling in cell factories. Contrarily to infective viral particles, VLPs lack infective viral genome while retaining important viral properties like cellular tropism and intracellular delivery of internalized molecules. These properties make VLPs promising and fully biocompatible nanovehicles for drug delivery. VLPs of human JC virus (hJCV) VP1 capsid protein produced in Escherichia coli elicit variable hemagglutination properties when incubated at different NaCl concentrations and pH conditions, being optimal at 200 mM NaCl and at pH range between 5.8 and 7.5. In addition, the presence or absence of chaperone DnaK in E. coli cells influence the solubility of recombinant VP1 and the conformational quality of this protein in the VLPs. The hemagglutination ability of hJCV VP1 VLPs contained in E. coli cell extracts can be modulated by buffer composition in the hemagglutination assay. It has been also determined that the production of recombinant hJCV VP1 in E. coli is favored by the absence of chaperone DnaK as observed by Western Blot analysis in different E. coli genetic backgrounds, indicating a proteolysis targeting role for DnaK. However, solubility is highly compromised in a DnaK^? E. coli strain suggesting an important role of this chaperone in reduction of protein aggregates. Finally, hemagglutination efficiency of recombinant VP1 is directly related to the presence of DnaK in the producing cells. © 2014 American Institute of Chemical Engineers Biotechnol. Prog., 30:744–748, 2014