Objective To investigate antigen optimization of Shisa like protein 1 (SHISAL1) for preparing mouse anti-human SHISAL1 polyclonal antibody and to identify the specificity of the prepared antibody. Methods Bioinformatics was employed to predict the antigenic epitope region of SHISAL1 protein, and then a polypeptide composed of amino acid residues from the site of 28 to 97 of SHISAL1, termed SHISAL1-N, was selected as the antigen. The coding region of SHISAL1-N was cloned by molecular cloning technique, and then it was inserted into pET-28a to generate pET28a-SHISAL1-N recombinant plasmid. The two recombinant plasmids pET28a-SHISAL1-N and pET28a-SHISAL1 were transformed into BL21 (DE3) bacteria and induced to express by IPTG. The two proteins were purified and immunized to female Kunming mice, respectively. The specificities and sensitivities of the acquired antibodies were detected by Western blot analysis, immunoprecipitation and immunofluorescent cytochemical staining. Results pET28a-SHISAL1-N recombinant plasmid was successfully constructed, and the two fused proteins, SHISAL1 and SHISAL1-N, were induced to express. Moreover, two types of SHISAL1 mouse polyclonal antibodies, derived from SHISAL1-N and SHISAL1 antigens, were obtained. Western blot results showed that the antibody prepared from SHISAL1 antigen was less specific and sensitive compared with the antibody prepared from SHISAL1-N antigen which could specifically identify different endogenous SHISAL1 protein. Immunoprecipitation results showed that SHISAL1-N antibody could specifically pull down SHIISAL1 protein in hepatocellular carcinoma cells and immunofluorescence results demonstrated that SHISAL1-N antibody could specifically bind to SHISAL1 protein in the cytoplasm. Conclusion We have optimized the SHISAL1 antigen and prepared the mouse anti-human SHISAL1 polyclonal antibodies successfully, which can be used for Western blot analysis, immunoprecipitation and immunofluorescence cytochemical staining.
Animals ; Female ; Humans ; Mice ; Antibodies ; Antibody Specificity ; Blotting, Western ; Cloning, Molecular ; Epitopes/genetics*

Objective To investigate the preventive therapeutic effect and possible mechanism of single chain variable fragments chimeric protein (SD) of ovalbumin epitopes internalizing receptor DEC-205 antibody on food allergy in mice. Methods Mice were randomly divided to five groups (control, PBS, scFv DEC 100 μg, SD 50 μg, SD 100 μg) and treated for 24 hours before OVA administration. After challenge, the serum level of OVA-specific IgE, IgG1, IgG2a and IL-4 were detected by ELISA. Infiltration of eosinophils and mast cells in the jejunum was observed by HE staining and toluidine blue staining respectively. The bone marrow of tibia and femur was isolated and cultured to obtain immature dendritic cells(BMDCs), which were further treated with LPS (10 ng/mL), TSLP (50 ng/mL), scFv DEC protein (1000 ng/mL) and SD protein (10,100,1000)ng/mL for 24 hours, and the IL-10 level of supernatant was assayed by ELISA. Results Compared with PBS group, the number of SD-treated mice with diarrhea was markedly reduced. The difference in rectal temperature and the levels of serum OVA-specific IgE, IgG1, IgG2a and IL-4 decreased significantly after prophylactic administration of SD; The number of eosinophils and mast cells in jejunum also decreased significantly while the IL-10 level in the supernatant of BMDCs increased significantly after SD intervention. Conclusion SD mitigates experimental FA response by fosters the immune tolerance property of dendritic cells.
Mice ; Animals ; Ovalbumin ; Interleukin-10 ; Single-Chain Antibodies/genetics* ; Immunoglobulin E ; Epitopes/therapeutic use* ; Interleukin-4 ; Food Hypersensitivity/prevention & control* ; Immunoglobulin G ; Recombinant Fusion Proteins/genetics* ; Mice, Inbred BALB C ; Disease Models, Animal

Objectives To develop a multi-stage and multi-epitope vaccine, which consists of epitopes from the early secretory and latency-associated antigens of Mycobacterium tuberculosis (MTB). Methods The B-cell, cytotoxic T-lymphocyte (CTL) and helper T-lymphocyte (HTL) epitopes of 12 proteins were predicted using an immunoinformatics. The epitopes with antigenicity, without cytotoxicity and sensitization, were further screened to construct the multi-epitope vaccine. Furthermore, the proposed vaccine underwent physicochemical properties analysis and secondary structure prediction as well as 3D structure modeling, refinement and validation. Then the refined model was docked with TLR4. Finally, an immune simulation of the vaccine was carried out. Results The proposed vaccine, which consists of 12 B-cell, 11 CTL and 12 HTL epitopes, had a flexible and stable globular conformation as well as a thermostable and hydrophilic structure. A stable interaction of the vaccine with TLR4 was confirmed by molecular docking. The efficiency of the candidate vaccine to trigger effective cellular and humoral immune responses was assessed by immune simulation. Conclusion A multi-stage multi-epitope MTB vaccine construction strategy based on immunoinformatics is proposed, which is expected to prevent both active and latent MTB infection.
Mycobacterium tuberculosis/metabolism* ; Molecular Docking Simulation ; Toll-Like Receptor 4 ; Epitopes, T-Lymphocyte/chemistry* ; Epitopes, B-Lymphocyte/chemistry* ; Vaccines, Subunit/chemistry* ; Computational Biology/methods*

OBJECTIVE: To analyze the RNA binding protein of Toxoplasma gondii (TgDDX39) using bioinformatics technology, and to evaluate the immunogenicity of TgDDX39, so as to provide insights into development of toxoplasmosis vaccines. METHODS: The amino acid sequences of TgDDX39 were retrieved from the ToxoDB database, and the physicochemical properties, transmembrane structure domain, signal peptide sites, post-translational modification sites, coils, secondary and tertiary structures, hydrophobicity, and antigenic epitopes of the TgDDX39 protein were predicted using online bioinformatics tools, incluiding ProtParam, TMHMM 2.0, SignalP 5.0, NetPhos 3.1, COILS, SOPMA, Phyre2, ProtScale, ABCpred, SYFPEITHI and DNA-STAR. RESULTS: TgDDX39 protein was predicted to be an unstable hydrophilic protein with the molecular formula of C₂₁₇₃H₃₄₅₈N₅₉₈O₆₆₁S₁₈, which contained 434 amino acids and had an estimated molecular weight of 49.1 kDa and a theoretical isoelectric point of 5.55. The protein was predicted to have an extremely low possibility of signal peptides, without transmembrane regions, and contain 27 phosphorylation sites. The β turn and random coils accounted for 39.63% of the secondary structure of the TgDDX39 protein, and a coiled helix tended to produce in one site. In addition, the TgDDX39 protein contained multiple B and T cell antigenic epitopes. CONCLUSIONS Bioinformatics analyses predict that TgDDX39 protein has high immunogenicity and contains multiple antigenic epitopes. TgDDX39 protein is a potential candidate antigen for vaccine development.
Humans ; Toxoplasma/metabolism* ; Toxoplasmosis/prevention & control* ; Vaccines ; Epitopes, T-Lymphocyte ; Computational Biology ; Protozoan Proteins/chemistry*

New threats posed by the emerging circulating variants of SARS-CoV-2 highlight the need to find conserved neutralizing epitopes for therapeutic antibodies and efficient vaccine design. Here, we identified a receptor-binding domain (RBD)-binding antibody, XG014, which potently neutralizes β-coronavirus lineage B (β-CoV-B), including SARS-CoV-2, its circulating variants, SARS-CoV and bat SARSr-CoV WIV1. Interestingly, antibody family members competing with XG014 binding show reduced levels of cross-reactivity and induce antibody-dependent SARS-CoV-2 spike (S) protein-mediated cell-cell fusion, suggesting a unique mode of recognition by XG014. Structural analyses reveal that XG014 recognizes a conserved epitope outside the ACE2 binding site and completely locks RBD in the non-functional "down" conformation, while its family member XG005 directly competes with ACE2 binding and position the RBD "up". Single administration of XG014 is effective in protection against and therapy of SARS-CoV-2 infection in vivo. Our findings suggest the potential to develop XG014 as pan-β-CoV-B therapeutics and the importance of the XG014 conserved antigenic epitope for designing broadly protective vaccines against β-CoV-B and newly emerging SARS-CoV-2 variants of concern.
Angiotensin-Converting Enzyme 2 ; Antibodies, Neutralizing ; Antibodies, Viral ; COVID-19 ; Epitopes ; Humans ; SARS-CoV-2/genetics* ; Spike Glycoprotein, Coronavirus/genetics*

OBJECTIVE: KRAS gene is one of the most common mutations of proto-oncogenes in human tumors, G12V is one of the most common mutation types for KRAS. It's challenging to chemically acquire the targeted drug for this mutation. Recent studies reported that this mutation peptides can form a neoepitope for T cell recognition. Our study aims to clone the T cell receptor (TCR) which specifically recognizes the neoepitope for KRAS G12V mutation and constructs TCR engineered T cells (TCR-T), and to investigate if TCR-Ts have strong antitumor response ability. METHODS: In this study, tumor infiltrating lymphocytes were obtained from one colorectal cancer patient carrying KRAS G12V mutation. Tumor-reactive TCR was obtained by single-cell RT-5' rapid-amplification of cDNA ends PCR analysis and introduced into peripheral blood lymphocytes to generate TCR-Ts. RESULTS: We obtained a high-affinity TCR sequence that specifically recognized the HLA-A^*11:01-restricted KRAS G12V_8-16 epitope: KVA11-01. KVA11-01 TCR-T could significantly kill various tumor cells such as PANC-1, SW480 and HeLa (overexpressing HLA-A^*11:01 and KRAS G12V), and secreting high levels of interferon-γ (IFN-γ). Non-specific killing experiments suggested KVA11-01 specifically recognized tumor cells expressing both mutant KRAS G12V and HLA-A^*11:01. In vivo assay, tumor inhibition experiments demonstrated that infusion of approximately 1E7 KVA11-01 TCR-T could significantly inhibit the growth of subcuta-neously transplanted tumors of PANC-1 and HeLa (overexpressing HLA-A^*11:01 and KRAS G12V) cells in nude mice. No destruction of the morphologies of the liver, spleen and brain were observed. We also found that KVA11-01 TCR-T could significantly infiltrate into tumor tissue and had a better homing ability. CONCLUSION KVA11-01 TCR-T cells can effectively target a variety of malignant tumor cells carrying KRAS G12V mutation through in vitro and in vivo assay. KVA11-01 TCR-T cells have excellent biological activity, high specificity of target antigen and homing ability into solid tumor tissue. KVA11-01 TCR-T is expected to be an effective treatment for patients with KRAS G12V mutant solid malignancies.
Animals ; DNA, Complementary ; Epitopes ; HLA-A Antigens ; Humans ; Interferon-gamma ; Mice ; Mice, Nude ; Mutation ; Neoplasms ; Proto-Oncogene Proteins p21(ras)/genetics* ; Receptors, Antigen, T-Cell/genetics*

OBJECTIVE: To establish a new method for synthesizing Lewis blood group antigens, that is, the mimotopes of Lewis blood group antigens were screened by using an alpaca phage display nanobody library. METHODS: We selected mimotopes of the Lewis a (le^a) antigen by affinity panning of an alpaca phage display nanobody library using a monoclonal anti-le^a antibody. Enzyme-linked immunosorbent assay (ELISA) was used to test the affinity of the positive clones for the monoclonal anti-le^a antibody, and the high-affinity positive clones were selected for sequencing and synthesis. Finally, the sensitivity, specificity and reactivity of the synthesized le^a mimotope in clinical samples were verified by ELISA. RESULTS: A total of 96 phage clones were randomly selected, and 24 were positive. Fourteen positive clones with the highest affinity were selected for sequencing. The result showed that there were 5 different sequences, among which 3 sequences with the highest frequency, largest difference and highest affinity were selected for expression and synthesis. The sensitivity and specificity of le^a mimic antigen by ELISA showed that, the minimum detection limit of gel microcolumn assay (GMA) and ELISA method were 25 times different, and the le^a mimic antigen had no cross reacted with the other five unrelated monoclonal antibodies(P<0.001). Finally, 30 clinical plasma samples were analyzed. The mean absorbance of the 15 positive plasma samples was significantly higher than that of the 15 negative plasma samples (P=0.02). However, the positive signal values of the clinical samples were much lower than those of the monoclonal antibodies. CONCLUSION A new method of screening le^a mimic antigen by using alpaca phage nanoantibody library has been established, which is expected to realize the screening of le^a mimotopes, thus realizing the application of high-sensitivity detection methods such as ELISA and chemiluminescence in blood group antibody identification.
Animals ; Antibodies, Monoclonal ; Antineoplastic Agents, Immunological ; Bacteriophages ; Blood Group Antigens ; Camelids, New World ; Enzyme-Linked Immunosorbent Assay/methods* ; Epitopes ; Humans ; Lewis Blood Group Antigens ; Peptide Library

The aim of this study was to refold the OvisAries leukocyte antigen (OLA) class Ⅰ protein with peptides derived from sheeppox virus (SPPV) to identify SPPV T cell epitopes. Two pairs of primers were designed based on the published sequence of a sheep major histocompatibility complex Ⅰ to amplify the heavy chain gene of OLA Ⅰ α-BSP and the light chain gene of OLA Ⅰ-β₂m. Both genes were cloned into a pET-28a(+) expression vector, respectively, and induced with ITPG for protein expression. After purification, the heavy chain and light chain proteins as well as peptides derived from SPPV were refolded at a ratio of 1:1:1 using a gradual dilution method. Molecular exclusion chromatography was used to test whether these peptides bind to the OLA Ⅰ complex. T-cell responses were assessed using freshly isolated PBMCs from immunized sheep through IFN-γ ELISPOT with peptides derived from SPPV protein. The results showed that the cloned heavy chain and light chain expressed sufficiently, with a molecular weight of 36.3 kDa and 16.7 kDa, respectively. The protein separated via a Superdex^TM 200 increase 10/300 GL column was collected and verified by SDS-PAGE after refolding. One SPPV CTL epitope was identified after combined refolding and functional studies based on T-cell epitopes derived from SPPV. An OLA Ⅰ/peptide complex was refolded correctly, which is necessary for the structural characterization. This study may contribute to the development of sheep vaccine based on peptides.
Animals ; Capripoxvirus ; Epitopes, T-Lymphocyte/genetics* ; Peptides/genetics* ; Poxviridae Infections ; Sheep ; Sheep Diseases

The GapC protein of Streptococcus uberis located on the surface of bacteria is a protein with glyceraldehyde-3-phosphate dehydrogenase activity. It participates in cellular processes and exhibits a variety of biological activities. In addition, it has good antigenicity. The aim of this study was to predict the possible B-cell epitopes of the GapC protein and verify the immunogenicity of candidate epitope peptides. The gapC gene of S. uberis isolate RF5-1 was cloned into a recombinant expression plasmid pET-28a-GapC and inducibly expressed. The purified protein was used to immunize experimental rabbits to produce anti-GapC polyclonal antibodies. The three-dimensional structure and three-dimensional location of the GapC B-cell epitopes and the homology comparison of the GapC protein and its B-cell epitopes were carried out using bioinformatics softwares. The results showed that the 44-kDa GapC protein had a good immunological reactivity. Six linear and 3 conformational dominant B-cell epitopes against the GapC protein were selected and synthesized. Three dimensional analysis indicated that the selected peptides have better antigen epitope formation potential. Rabbit anti-GapC polyclonal antibodies were generated after immunized with the purified GapC protein, and the polyclonal antibodies were used to identify the epitope peptide by an indirect ELISA. The ELISA results showed that all of the 9 epitope peptides could react with anti-GapC polyclonal antibodies with varying titers. Among them, the epitope polypeptide ²⁶⁶AANDSYGYTEDPIVSSD²⁸² reacted with the polyclonal antibodies significantly stronger than with other epitope peptides. This study laid an experimental foundation for in-depth understanding of the immunological properties and utilizing effective epitopes of the GapC protein of S. uberis.
Animals ; Antigens, Bacterial/genetics* ; Bacterial Proteins/genetics* ; Epitopes, B-Lymphocyte/genetics* ; Mice ; Mice, Inbred BALB C ; Rabbits ; Streptococcus

OBJECTIVE: To conduct eukaryotic expression of the leucine-rich repeat containing 15 (LRRC15), a differentially expressed protein in excretory secretory antigens of Taenia solium cysticercus, and predict its antigen epitope. METHODS: The molecular weight, stability, amino acid sequence composition, isoelectric point and T lymphocyte epitope of the LRRC15 protein were predicted using the bioinformatics online softwares ExPASy-PortParam and Protean. The full-length splicing primers were designed using PCR-based accurate synthesis, and the LRRC15 gene was synthesized. The recombinant pcDNA3.4-LRRC15 plasmid was constructed and transfected into HEK293 cells to express the LRRC15 protein. In addition, the LRRC15 protein was characterized by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting. RESULTS: The recombinant pcDNA3.4-LRRC15 plasmid was successfully constructed, which expressed the target LRRC15 protein with an approximately molecular weight of 70 kDa. Bioinformatics prediction with the ExPASy-PortParam software showed that LRRC15 was a hydrophilic protein, which was consisted of 644 amino acids and had a molecular weight of 69.89 kDa and an isoelectric point of 5.6. The molecular formula of the LRRC15 protein was C₃₀₇₃H₄₉₄₂N₈₄₆O₉₅₃S₂₈ and had an instability coefficient is 50.3, indicating that LRRC15 was an instable protein. Bioinformatics prediction with the Protean software showed that the dominant T-cell antigen epitopes were located in 292 to 295, 353 to 361, 521 to 526 and 555 to 564 amino acids of the LRRC15 protein, and the T-cell antigen epitopes with a high hydrophilicity, good flexibility, high surface accessibility and high antigenicity index were found in 122 to 131, 216 to 233, 249 to 254, 333 to 343, 358 to 361, 368 to 372, 384 to 386, 407 to 412, 445 to 450, 469 to 481, 553 to 564, 588 to 594, 607 to 617 and 624 to 639 amino acids. Following transfection of the recombinant pcDNA3.4-LRRC15 plasmid into HEK293 cells, SDS-PAGE and Western blotting identified LRRC15 proteins in cell secretory culture media, cell lysis supernatants and sediments. The LRRC15-His fusion protein was purified from the cell culture medium, and SDS-PAGE identified a remarkable band at approximately 70 kDa, while Western blotting successfully recognized the band of the recombinant LRRC15 protein. CONCLUSIONS The eukaryotic expression and antigen epitope prediction of the LRRC15 protein in the excretory secretory antigens of T. solium cysticercus have been successfully performed, which provides insights into further understandings of its biological functions.
Amino Acids ; Animals ; Antigens, Helminth/genetics* ; Cysticercus/genetics* ; Epitopes/genetics* ; Eukaryota ; HEK293 Cells ; Humans ; Leucine-Rich Repeat Proteins ; Membrane Proteins ; Taenia solium/genetics*