B细胞表位定位的研究进展

B细胞抗原表位的研究对免疫原性多肽和新型疫苗分子的设计都起着指导作用,同时也有利于诊断试剂的开发以及临床疾病的诊断。本文综述了近年来实验确定和理论预测B细胞蛋白质抗原表位的常用方法,以及B细胞抗原表位分析的研究方法。     

Expression and characterization of hepatitis C virus core protein fused to hepatitis B virus core antigen

Recombinant plasmids were constructed by fusing the gene fragments encoding the full-length (1-191aa) and the truncated (1-40aa and 1-69aa) HCV core proteins (HCc) respectively to the core gene of HBV at the position of amino acid 144 and expressed in E. coli. The products were analyzed by ELISA, Western blotting as well as the immunization of the mice. The results showed that those fusion proteins (B144C191, B144C69, B144C40) possessed the dual antigenicity and immunogenicity of both hepatitis B virus core antigen (HBcAg) and hepatitis C virus core protein (HCc). Analysis by electron microscopy and CsCl density gradient ultra-centrifugation revealed that similar to the HBcAg itself, all fusion proteins were able to form particles. Comparison of the antigenicity and immunogenicity of those fusion proteins showed that the length of HCc gene fused to HBeAg had no much effect on the antigenicity and immunogenicity of HBcAg, however, B144C69 and B144C40 induced higher titres antibodies against HCc than B14d     

Fine epitope mapping of monoclonal antibodies against hemagglutinin of a highly pathogenic H5N1 influenza virus using yeast surface display

Highly pathogenic H5N1 avian influenza viruses pose a debilitating pandemic threat. Thus, understanding mechanisms of antibody-mediated viral inhibition and neutralization escape is critical. Here, a robust yeast display system for fine epitope mapping of viral surface hemagglutinin (HA)-specific antibodies is demonstrated. The full-length H5 subtype HA (HA0) was expressed on the yeast surface in a correctly folded conformation, determined by binding of a panel of extensively characterized neutralizing human monoclonal antibodies (mAbs). These mAbs target conformationally-dependent epitopes of influenza A HA, which are highly conserved across H5 clades and group 1 serotypes. By separately displaying HA1 and HA2 subunits on yeast, domain mapping of two anti-H5 mAbs, NR2728 and H5-2A, localized their epitopes to HA1. These anti-H5 mAb epitopes were further fine mapped by using a library of yeast-displayed HA1 mutants and selecting for loss of binding without prior knowledge of potential contact residues. By overlaying key mutant residues that impacted binding onto a crystal structure of HA, the NR2728 mAb was found to interact with a fully surface-exposed contiguous patch of residues at the receptor binding site (RBS), giving insight into the mechanism underlying its potent inhibition of virus binding. The non-neutralizing H5-2A mAb was similarly mapped to a highly conserved H5 strain-specific but poorly accessible location on a loop at the trimer HA interface. These data further augment our toolchest for studying HA antigenicity, epitope diversity and accessibility in response to natural and experimental influenza infection and vaccines.     

High throughput functional epitope mapping: Revisiting phage display platform to scan target antigen surface

Antibody engineering must be accompanied by mapping strategies focused on identifying the epitope recognized by each antibody to define its unique functional identity. High throughput fine specificity determination remains technically challenging. We review recent experiences aimed at revisiting the oldest and most extended display technology to develop a robust epitope mapping platform, based on the ability to manipulate target-derived molecules (ranging from the whole native antigen to antigen domains and smaller fragments) on filamentous phages. Single, multiple and combinatorial mutagenesis allowed comprehensive scanning of phage-displayed antigen surface that resulted in the identification of clusters of residues contributing to epitope formation. Functional pictures of the epitope(s) were thus delineated in the natural context. Successful mapping of antibodies against interleukin-2, epidermal growth factor and its receptor, and vascular endothelial growth factor showed the versatility of these procedures, which combine the accuracy of site-directed mutagenesis with the high throughput potential of phage display.     

High throughput functional epitope mapping: Revisiting phage display platform to scan target antigen surface

Antibody engineering must be accompanied by mapping strategies focused on identifying the epitope recognized by each antibody to define its unique functional identity. High throughput fine specificity determination remains technically challenging. We review recent experiences aimed at revisiting the oldest and most extended display technology to develop a robust epitope mapping platform, based on the ability to manipulate target-derived molecules (ranging from the whole native antigen to antigen domains and smaller fragments) on filamentous phages. Single, multiple and combinatorial mutagenesis allowed comprehensive scanning of phage-displayed antigen surface that resulted in the identification of clusters of residues contributing to epitope formation. Functional pictures of the epitope(s) were thus delineated in the natural context. Successful mapping of antibodies against interleukin-2, epidermal growth factor and its receptor, and vascular endothelial growth factor showed the versatility of these procedures, which combine the accuracy of site-directed mutagenesis with the high throughput potential of phage display.     

Cell surface display of Chi92 on Escherichia coli using ice nucleation protein for improved catalytic and antifungal activity

The gene encoding chitinase 92 (Chi92) from Aeromonas hydrophila JP10 has been displayed on the cell surface of Escherichia coli using the N-terminal region of ice nucleation proteins (INPN) as an anchoring motif. Immunofluorescence microscopy confirmed that Chi92 was anchored on the cell surface. Western blot analysis further identified the synthesis of INP derivatives containing the N-terminal domain INPN-Chi92 fusion protein of the expected size (112 kDa). Whole cell enzyme assay indicated that the displayed Chi92 showed enhanced catalytic activity toward colloidal chitin. In addition, the Chi92-displayed cells exhibited inhibitory effects on the mycelial growth of phytopathogenic fungi, including Fusarium decemcellulare, Sclerotium rolfsii, Rhizoctonia solani kuhn, and Fusarium oxysporum f.sp. melonis. This study suggested that the INP-based display systems can be used to express a large protein (90 kDa Chi92) on the cell surface of E. coli without growth inhibition. In addition, the display of chitinase on the cell surface may provide an attractive method for the development of biocontrol agents against phytopathogenic fungi.     

Fine epitope mapping of anti-epidermal growth factor receptor antibodies through random mutagenesis and yeast surface display

Fine epitope mapping of therapeutically relevant monoclonal antibodies (mAbs) specific for the epidermal growth factor receptor (EGFR) was accomplished through random mutagenesis and yeast surface display. Using this method, we have identified key residues energetically important for the binding of EGFR to the mAbs 806, 225, and 13A9. A yeast-displayed library of single point mutants of an EGFR ectodomain fragment (residues 273-621) was constructed by random mutagenesis and was screened for reduced binding to EGFR mAbs. If an EGFR mutant showed loss of binding to a mAb, this suggested that the mutated residue was potentially a contact residue. The mAb 806 binding epitope was localized to one face of a loop comprised of EGFR residues Cys287-Cys302, which is constrained by a disulfide bond and two salt bridges. The mAb 806 epitope as identified here is not fully accessible in the autoinhibited EGFR monomer conformation, which is consistent with the hypothesis that mAb 806 binds to a transitional form of EGFR as it changes from an autoinhibited to extended monomer. The amino acids Lys465 and Ile467 were identified as energetic hot spot residues for mAb 225 binding to EGFR. These residues are adjacent to the EGFR ligand-binding site, which is consistent with the ability of mAb 225 to block binding of epidermal growth factor (EGF) and transforming growth factor-alpha (TGF-alpha) ligands. Ser468 and Glu472 were identified as energetically important for mAb 13A9 binding to EGFR, and the location of this epitope suggests that mAb 13A9 mediates observed TGF-alpha blocking effects through conformational perturbation of EGFR domain III. Combinatorial library screening of yeast-displayed mutagenic proteins is a novel method to identify discontinuous and heat-denaturable mAb binding epitopes with residue-level resolution.     

A new epitope peptide derived from hepatitis C virus 1b possessing the capacity to induce cytotoxic T-lymphocytes in HCV1b-infected patients with HLA-A11, -A31, and -A33

Background Hepatitis C virus (HCV) frequently causes chronic hepatitis, cirrhosis, and hepatocellular carcinoma after long-term persistent infection. Among various genotypes of HCV, HCV1b is resistant to standard interferon therapy, and thus the development of new treatment modality is needed. Results To provide a scientific basis for specific immunotherapy for HCV1b, we investigated HCV1b-derived epitope peptides recognized by human leukocyte antigen (HLA)-A11, -A31, or -A33-restricted cytotoxic T-lymphocytes (CTLs), and report here three novel vaccine candidate peptides selected by both antibody screening and CTL-inducing capacity from among 46 peptides of conserved regions of HCV1b sequences with binding motifs to HLA-A11, -A31, and -A33. Significant levels of IgG reactive to each of the three peptides were detected in the plasma of more than 50% of the HCV1b⁺ patients. One peptide at positions 30–39 of the core protein induced peptide-specific CTLs from peripheral blood mononuclear cells (PBMCs) of HLA-A11⁺, -A31⁺, and -A33⁺ patients. The other two peptides at positions 35–43 of the core protein and at positions 918–926 of the non-structural protein 2 also induced peptide-specific CTLs from the PBMCs of HLA-A11⁺ and -A33⁺ patients. Conclusion Therefore, the peptide at positions 30–39 of the core protein could be an appropriate target molecule of specific immunotherapy for all HLA-A11⁺, -A31⁺, and -A33⁺ patients with HCV1b-related diseases.     

基于蛋白质跨外膜自转运系统的细菌细胞表面蛋白展示技术研究进展

革兰氏阴性菌Ⅴ型分泌系统是细菌病原蛋白分泌的主要途径之一,可分为Ⅴa-Ⅴe5个亚型,其中Ⅴa型(即经典的单体自转运蛋白)是细菌毒力和黏附因子向细胞外分泌的重要工具,其在内膜Sec易位子和外膜BAM蛋白复合体的协助下,通过2个连续的跨膜步骤介导蛋白质穿过阴性菌的内外膜.据信Va型是目前已知蛋白质跨膜转运时最简单的分泌途径...     

Antigenic epitope mapping of the M24 protein of Streptococcus pyogenes: implications for serodiagnosis of rheumatic fever

Abstract Rheumatic fever continues to be a significant problem in Australian Aboriginal communities and developing countries worldwide. Early diagnosis could facilitate the institution of penicillin prophylaxis resulting in the prevention of recurrences of rheumatic fever. An overlapping biotinylated peptide bank of 82 peptides, based on the known sequence of Streptococcus pyogenes M24 protein, was used in a standard enzyme immunoassay. A total of 82 sera were tested from both aboriginal and non-aboriginal subjects with clinically proven rheumatic fever, rheumatic heart disease and matched controls. Two peptides with significant sequence homology at the C-terminal end were found to be discriminatory between aboriginal cases and controls. It is proposed that these peptides could be the basis of a serological test for rheumatic fever.     

Protein cell surface display in Gram-positive bacteria: from single protein to macromolecular protein structure

In the course of evolution, Gram-positive bacteria, defined here as prokaryotes from the domain Bacteria with a cell envelope composed of one biological membrane (monodermita) and a cell wall composed at least of peptidoglycan and covalently linked teichoic acids, have developed several mechanisms permitting to a cytoplasmic synthesized protein to be present on the bacterial cell surface. Four major types of cell surface displayed proteins are currently recognized: (i) transmembrane proteins, (ii) lipoproteins, (iii) LPXTG-like proteins and (iv) cell wall binding proteins. The subset of proteins exposed on the bacterial cell surface, and thus interacting with extracellular milieu, constitutes the surfaceome. Here, we review exhaustively the current molecular mechanisms involved in protein attachment within the cell envelope of Gram-positive bacteria, from single protein to macromolecular protein structure.     

Mapping of antigenic determinants of hepatitis C virus proteins using phage display

Analysis of the properties for individual hepatitis C virus (HCV) proteins makes it possible to establish their molecular structure and conformation, to localize antigenic and immunogenic determinants, to identify protective epitopes, and to solve applied problems (e.g., design of diagnostic tests, vaccines, and drugs). Linear and conformational epitopes of HCV proteins were localized using the phage display technique, and the peptides exposed on the phages selected with monoclonal antibodies against HCV proteins were tested for immunogenicity. Of the 11 epitopes revealed, three were strongly linear; two depended on the secondary; and one on the tertiary structure of the corresponding protein (conformational epitopes). Amino acid sequences involved in the other epitopes were established. The results can be used to improve the diagnosis of hepatitis C, to study the effect of amino acid substitutions on the antigenic properties of HCV proteins, and to analyze the immune response in patients infected with genotypically different HCV. It was shown with the example of the NS5A epitope that phage particles with epitope-mimicking peptides (mimotopes) induce production of antibodies against the corresponding HCV proteins.     

Functional display of foreign protein on surface of Escherichia coli using N-terminal domain of ice nucleation protein

We investigated the ability of the N-terminal domain of InaK, an ice nucleation protein from Pseudomonas syringae KCTC1832, to act as an anchoring motif for the display of foreign proteins on the Escherichia coli cell surface. Total expression level and surface display efficiency of green fluorescent protein (GFP) was compared following their fusion with either the N-terminal domain of InaK (InaK-N), or with the known truncated InaK containing both N- and C-terminal domains (InaK-NC). We report that the InaK-N/GFP fusion protein showed a similar cell surface display efficiency ( approximately 50%) as InaK-NC/GFP, demonstrating that the InaK N-terminal region alone can direct translocation of foreign proteins to the cell surface and can be employed as a potential cell surface display motif. Moreover, InaK-N/GFP showed the highest levels of total expression and surface display based on unit cell density. InaK-N was also successful in directing cell surface display of organophosphorus hydrolase (OPH), confirming its ability to act as a display motif.     

Development of a whole-cell biosensor by cell surface display of a gold-binding polypeptide on the gold surface

Cell surface display was used as a strategy to display the gold-binding polypeptide (GBP) fusion protein on the surface of Escherichia coli , and consequently to immobilize the cells on the gold surface. The DNA encoding the GBP was fused to the truncated fadL gene and was expressed by the tac promoter. For the display of the core streptavidin (cSA) of Streptomyces avidinii , the cSA gene was fused to the truncated oprF gene. After the dual display of FadL–GBP and OprF–cSA on the surface of E. coli , binding of cells on the gold surface and the interaction of OprF–cSA with the biotin–horseradish peroxidase (HRP) were studied by surface plasmon resonance (SPR) analysis. Cells displaying the FadL–GBP fusion protein could be immobilized on the SPR sensor chip as shown by the SPR angle shift of 0.5°, which was stably bound at least for 60 h with a washing solution. When the FadL–GBP and OprF–cSA fusion proteins were displayed on the same cell surface, the former was used to immobilize the cells on the gold surface and the latter was used for the interaction studies with the biotin–HRP, which demonstrates that the strategy should be useful for developing whole-cell biosensor chips.     

丝状真菌表面展示技术研究进展

丝状真菌表面展示技术是将表达的目的蛋白固定在丝状真菌细胞表面的一项新兴基因工程技术。丝状真菌具有极强的蛋白质分泌能力和良好的蛋白质翻译后加工能力,因而越来越多的丝状真菌表面展示技术得到开发和应用。本文就丝状真菌表面展示系统的研发和应用进展进行综述,并介绍与该系统构建密切相关的丝状真菌的细胞壁组成、锚定蛋白和遗传转化方法等技术。     

Surface display of transglucosidase on Escherichia coli by using the ice nucleation protein of Xanthomonas campestris and its application in glucosylation of hydroquinone

A surface anchoring motif using the ice nucleation protein (INP) of Xanthomonas campestris pv. campestris BCRC 12,846 for display of transglucosidase has been developed. The transglucosidase gene from Xanthomonas campestris pv. campestris BCRC 12,608 was fused to the truncated ina gene. This truncated INP consisting of N- and C-terminal domains (INPNC) was able to direct the expressed transglucosidase fusion protein to the cell surface of E. coli with apparent high enzymatic activity. The localization of the truncated INPNC-transglucosidase fusion protein was examined by Western blot analysis and immunofluorescence labeling, and by whole-cell enzyme activity in the glucosylation of hydroquinone. The glucosylation reaction was carried out at 40 degrees C for 1 h, which gave 23 g/L of alpha-arbutin, and the molar conversion based on the amount of hydroquinone reached 83%. The use of whole-cells of the wild type strain resulted in an alpha-arbutin concentration of 4 g/L and a molar conversion of 16% only under the same conditions. The results suggested that E. coli displaying transglucosidase using truncated INPNC as an anchoring motif can be employed as a whole-cell biocatalyst in glucosylation.     

乙肝病毒核心蛋白病毒样颗粒表面抗原密度对抗体应答水平的影响

【目的】为了探究乙肝病毒核心蛋白(HepatitisBviruscoreprotein,HBc)病毒样颗粒(Virus-like particles,VLPs)表面抗原密度对免疫后抗体应答水平的影响,制备了不同抗原密度的HBc VLPs疫苗,并检测了其在小鼠体内的抗体应答水平。【方法】首先制备了N端带有3个甘氨酸的人巨细胞病毒重组抗原域AD-4作为模式抗原,接着通过Sortase A的介导将AD-4连接到HBc VLPs表面上。将系列浓度梯度AD-4抗原在SortaseA介导下分别与相同浓度的HBcVLPs发生反应,制备不同抗原密度的HBc-AD-4 VLPs。将其分别免疫6–8周龄BALB/c小鼠3次,每次免疫间隔2 w,间接ELISA法检测被免疫小鼠血清的抗体应答水平。【结果】结果表明,当HBc VLPs表面抗原密度为44.4%时,即HBc反应浓度∶AD-4反应浓度为1:0.5时,不足以引起高滴度的抗体产生;当HBc VLPs表面抗原密度为64.2%时,即HBc反应浓度∶AD-4反应浓度为1:1时,HBc-AD-4 VLPs诱导的AD-4特异性抗体滴度与100%抗原密度的HBc-AD-4VLPs所引起的抗体滴度相当;当HBcVLPs表面抗原密度大于64.2%时,引起的抗体应答水平不因抗原密度增加而进一步增强。【结论】发现了HBcVLPs表面抗原密度与免疫后抗体滴度呈正相关,然而免疫64.2%抗原密度的HBc VLPs所产生的抗体滴度可达峰值,抗原密度进一步增加,抗体应答水平不会进一步加强。