In order to prepare human-mouse chimeric cytomegalovirus-immunoglobulin M (CMV-IgM) in vitro and study the effects of different signal peptides on the secretion of CMV-IgM, genes were amplified from hybridoma cell line using RLM-RACE to construct the expression vector of chimeric CMV-IgM. Then, the signal peptide of SigF itself was replaced by five different secreted signal peptides (SigA-SigE) by PCR method, and the CHO cell was chosen as host cell for in vitro expression. SDS-PAGE, SEC-HPLC and ELISA experiments were carried out to evaluate the protein expression level and immunoreactivity of the purified CMV-IgM. A 910 kDa recombinant protein was successfully prepared and signal peptides (SigA-SigE) had an increased expressed CMV-IgM, which were 6.72, 5.19, 1.44, 1.85 and 1.98 times higher than that of the CMV 6# cell signal peptide SigF. In summary, this work provides a theoretical basis for the development of human-mouse chimeric CMV-IgM, and a novel route to increase the expression level of CMV-IgM.
Animals ; Antibodies, Viral ; genetics ; immunology ; Cricetinae ; Cytomegalovirus ; immunology ; Enzyme-Linked Immunosorbent Assay ; Gene Expression ; Humans ; Immunoglobulin M ; immunology ; Mice ; Protein Sorting Signals ; Recombinant Fusion Proteins ; immunology

OBJECTIVETo eliminate the side effects of aluminum adjuvant and His-tag, we constructed chimeric VLPs displaying the epitope of EV71 (SP70) without His-tagged. Then evaluating whether the VLPs could efficiently evoke not only humoral but also cellular immune responses against EV71 without adjuvant.

METHODSThe fusion protein was constructed by inserting SP70 into the MIR of truncated HBcAg sequence, expressed in E. Coli, and purified through ion exchange chromatography and density gradient centrifugation. Mice were immunized with the VLPs and sera were collected afterwards. The specific antibody titers, IgG subtypes and neutralizing efficacy were detected by ELISA, neutralization assay, and EV71 lethal challenge. IFN-γ and IL-4 secreted by splenocytes were tested by ELISPOT assay.

RESULTSHBc-SP70 proteins can self-assemble into empty VLPs. After immunization with HBc-SP70 VLPs, the detectable anti-EV71 antibodies were effective in neutralizing EV71 and protected newborn mice from EV71 lethal challenge. There was no significant difference for the immune efficacy whether the aluminum adjuvant was added or not. The specific IgG subtypes were mainly IgG1 and IgG2b and splenocytes from the mice immunized produced high levels of IFN-γ and IL-4.

CONCLUSIONThe fusion proteins without His-tagged was expressed and purified as soluble chimeric HBc-SP70 VLPs without renaturation. In the absence of adjuvant, they were efficient to elicit high levels of Th1/Th2 mixed immune response as well as assisted by aluminum adjuvant. Furthermore, the chimeric VLPs have potential to prevent HBV and EV71 infection simultaneously.

Adjuvants, Immunologic ; Animals ; Antibodies, Neutralizing ; Antibodies, Viral ; blood ; Enterovirus A, Human ; genetics ; Enterovirus Infections ; immunology ; virology ; Epitopes ; immunology ; metabolism ; Escherichia coli ; metabolism ; Female ; Immunity, Cellular ; Immunity, Humoral ; Mice ; Recombinant Fusion Proteins ; immunology

Acinetobacter baumannii (A. Baumannii) is an emerging opportunistic pathogen responsible for hospital-acquired infections, and which now constitutes a sufficiently serious threat to public health to necessitate the development of an effective vaccine. In this study, a recombinant fused protein named OmpK/Omp22 and two individual proteins OmpK and Omp22 were obtained using recombinant expression and Ni-affinity purification. Groups of BALB/c mice were immunized with these proteins and challenged with a clinically isolated strain of A. baumannii. The bacterial load in the blood, pathological changes in the lung tissue and survival rates after challenge were evaluated. Mice immunized with OmpK/Omp22 fused protein provided significantly greater protection against A. baumannii challenge than those immunized with either of the two proteins individually. The results provide novel clues for future design of vaccines against A. baumannii.
Acinetobacter Infections ; pathology ; prevention & control ; Acinetobacter baumannii ; genetics ; immunology ; Animals ; Antibodies, Bacterial ; blood ; Bacterial Load ; Bacterial Outer Membrane Proteins ; genetics ; immunology ; Bacterial Vaccines ; immunology ; Disease Models, Animal ; Female ; Mice, Inbred BALB C ; Pneumonia, Bacterial ; pathology ; prevention & control ; Recombinant Fusion Proteins ; genetics ; immunology

Cytotoxic T lymphocytes (CTLs) and natural killer (NK) cells contribute to the body's immune defenses. Current chimeric antigen receptor (CAR)-modified T cell immunotherapy shows strong promise for treating various cancers and infectious diseases. Although CAR-modified NK cell immunotherapy is rapidly gaining attention, its clinical applications are mainly focused on preclinical investigations using the NK92 cell line. Despite recent advances in CAR-modified T cell immunotherapy, cost and severe toxicity have hindered its widespread use. To alleviate these disadvantages of CAR-modified T cell immunotherapy, additional cytotoxic cell-mediated immunotherapies are urgently needed. The unique biology of NK cells allows them to serve as a safe, effective, alternative immunotherapeutic strategy to CAR-modified T cells in the clinic. While the fundamental mechanisms underlying the cytotoxicity and side effects of CAR-modified T and NK cell immunotherapies remain poorly understood, the formation of the immunological synapse (IS) between CAR-modified T or NK cells and their susceptible target cells is known to be essential. The role of the IS in CAR T and NK cell immunotherapies will allow scientists to harness the power of CAR-modified T and NK cells to treat cancer and infectious diseases. In this review, we highlight the potential applications of CAR-modified NK cells to treat cancer and human immunodeficiency virus (HIV), and discuss the challenges and possible future directions of CAR-modified NK cell immunotherapy, as well as the importance of understanding the molecular mechanisms of CAR-modified T cell- or NK cell-mediated cytotoxicity and side effects, with a focus on the CAR-modified NK cell IS.
Animals ; HIV Infections ; immunology ; therapy ; HIV-1 ; immunology ; Humans ; Immunity, Cellular ; Immunological Synapses ; Immunotherapy ; Killer Cells, Natural ; transplantation ; Neoplasms ; immunology ; therapy ; Receptors, Antigen, T-Cell ; genetics ; immunology ; Recombinant Fusion Proteins ; genetics ; immunology ; T-Lymphocytes ; immunology ; transplantation

Entero virus 71 (EV71) causes hand, foot, and mouth disease (HFMD) and occasionally leads to severe neurological complications and even death. Scavenger receptor class B member 2 (SCARB2) is a functional receptor for EV71, that mediates viral attachment, internalization, and uncoating. However, the exact binding site of EV71 on SCARB2 is unknown. In this study, we generated a monoclonal antibody (mAb) that binds to human but not mouse SCARB2. It is named JL2, and it can effectively inhibit EV71 infection of target cells. Using a set of chimeras of human and mouse SCARB2, we identified that the region containing residues 77-113 of human SCARB2 contributes significantly to JL2 binding. The structure of the SCARB2-JL2 complex revealed that JL2 binds to the apical region of SCARB2 involving α-helices 2, 5, and 14. Our results provide new insights into the potential binding sites for EV71 on SCARB2 and the molecular mechanism of EV71 entry.
Amino Acid Sequence ; Animals ; Antibodies, Monoclonal ; chemistry ; genetics ; metabolism ; Binding Sites ; Cell Line ; Crystallography, X-Ray ; Enterovirus A, Human ; drug effects ; genetics ; growth & development ; immunology ; Fibroblasts ; drug effects ; virology ; Gene Expression ; HEK293 Cells ; Humans ; Immunoglobulin Fab Fragments ; chemistry ; genetics ; metabolism ; Lysosome-Associated Membrane Glycoproteins ; chemistry ; genetics ; immunology ; Mice ; Models, Molecular ; Protein Binding ; Protein Conformation, alpha-Helical ; Protein Conformation, beta-Strand ; Protein Interaction Domains and Motifs ; Receptors, Scavenger ; chemistry ; genetics ; immunology ; Receptors, Virus ; chemistry ; genetics ; immunology ; Recombinant Fusion Proteins ; chemistry ; genetics ; immunology ; Sequence Alignment ; Sequence Homology, Amino Acid ; Sf9 Cells ; Spodoptera ; Thermodynamics

The clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated 9 (CRISPR/Cas9) system, an RNA-guided DNA targeting technology, is triggering a revolution in the field of biology. CRISPR/Cas9 has demonstrated great potential for genetic manipulation. In this review, we discuss the current development of CRISPR/Cas9 technologies for therapeutic applications, especially chimeric antigen receptor (CAR) T cell-based adoptive immunotherapy. Different methods used to facilitate efficient CRISPR delivery and gene editing in T cells are compared. The potential of genetic manipulation using CRISPR/Cas9 system to generate universal CAR T cells and potent T cells that are resistant to exhaustion and inhibition is explored. We also address the safety concerns associated with the use of CRISPR/Cas9 gene editing and provide potential solutions and future directions of CRISPR application in the field of CAR T cell immunotherapy. As an integration-free gene insertion method, CRISPR/Cas9 holds great promise as an efficient gene knock-in platform. Given the tremendous progress that has been made in the past few years, we believe that the CRISPR/Cas9 technology holds immense promise for advancing immunotherapy.
Animals ; Clustered Regularly Interspaced Short Palindromic Repeats ; immunology ; Gene Editing ; methods ; Humans ; Immunotherapy ; methods ; Receptors, Antigen, T-Cell ; genetics ; immunology ; Recombinant Fusion Proteins ; genetics ; immunology

Chimeric antigen receptor (CAR) T cell therapy is a promising cancer treatment that has recently been undergoing rapid development. However, there are still some major challenges, including precise tumor targeting to avoid off-target or "on-target/off-tumor" toxicity, adequate T cell infiltration and migration to solid tumors and T cell proliferation and persistence across the physical and biochemical barriers of solid tumors. In this review, we focus on the primary challenges and strategies to design safe and effective CAR T cells, including using novel cutting-edge technologies for CAR and vector designs to increase both the safety and efficacy, further T cell modification to overcome the tumor-associated immune suppression, and using gene editing technologies to generate universal CAR T cells. All these efforts promote the development and evolution of CAR T cell therapy and move toward our ultimate goal-curing cancer with high safety, high efficacy, and low cost.
Cell Movement ; immunology ; Cell Proliferation ; Gene Expression ; Genetic Vectors ; chemistry ; metabolism ; Humans ; Immunotherapy, Adoptive ; methods ; Lymphocyte Activation ; Lymphocytes, Tumor-Infiltrating ; cytology ; immunology ; transplantation ; Neoplasms ; genetics ; immunology ; pathology ; therapy ; Patient Safety ; Receptors, Antigen, T-Cell ; chemistry ; genetics ; immunology ; Recombinant Fusion Proteins ; chemistry ; genetics ; immunology ; Signal Transduction ; Single-Chain Antibodies ; chemistry ; genetics ; T-Lymphocytes ; cytology ; immunology ; transplantation ; Treatment Outcome

IFT46 is one of the important components of intraflagellar transport complex B in Chlamydomonas reinhardtii, and plays important roles in the assembly, movement and perception of ciliary. To study its functional mechanism, a GST-tagged and an MBP-tagged prokaryotic expression plasmid, pGEX-2T-ift46 and pMAL-C2X-ift46 were constructed, respectively, by inserting ift46 into the pGEX-2T and pMAL-C2X vector, and then transformed into Escherichia coli BL21 (DE3) for protein expression. SDS-PAGE (15%) analysis results showed that the molecular weights of the fusion protein GST-IFT46 and MBP-IFT46 were 70 kDa and 86 kDa, respectively. We used the fusion protein GST-IFT46 purified by affinity adsorption purification (more than 95% purity) for immunity to New Zealand white rabbits. The 5th immune serum was collected and the antibody titer was determined to be 256 000 by ELISA. The antiserum was purified by Protein A affinity adsorption purification and immobilized MBP-IFT46 purification, and the specificity of polyclonal antibodies was evaluated by Western blotting and immunofluorescence. Results showed that the polyclonal antibody prepared could specifically and precisely bind IFT46 in C. reinhardtii, and IFT46 was mainly concentrated at basal body regions and few localized along the entire length of the flagellum as punctuated dots, which will make a foundation to further study the mechanism of IFT46 in cilia related diseases such as obesity, diabetes and polycystic kidney disease.
Algal Proteins ; biosynthesis ; immunology ; Animals ; Antibodies ; chemistry ; Blotting, Western ; Chlamydomonas reinhardtii ; chemistry ; genetics ; Electrophoresis, Polyacrylamide Gel ; Enzyme-Linked Immunosorbent Assay ; Escherichia coli ; Fluorescent Antibody Technique ; Intracellular Signaling Peptides and Proteins ; biosynthesis ; immunology ; Plasmids ; Rabbits ; Recombinant Fusion Proteins ; biosynthesis

Heat-shock protein (Hsp) 70 potentiates specific immune responses to some antigenic peptides fused to it. Here, the prokaryotic plasmids harboring the envelope glycoprotein E0 gene of classical swine fever virus (CSFV) and/or the Hsp70 gene of Haemophilus parasuis were constructed and expressed in Escherichia coli Rosseta 2(R2). The fusion proteins were then purified. Groups of Balb/c mice were immunized with these fusion proteins, respectively, and sera collected 7 days after the third immunization. Immune effects were determined via an enzyme-linked immunosorbent assay and flow cytometric analyses. E0-Hsp70 fusion protein and E0+Hsp70 mixture significantly improved the titer of E-specific antibody, levels of CD4+ and CD8+ T cells, and release of interferon-γ. These findings suggested that Hsp70 can significantly enhance the immune effects of the envelope glycoprotein E0 of CSFV, thereby laying the foundation of further application in pigs.
Animals ; Antibodies, Viral ; blood ; CD4-Positive T-Lymphocytes ; cytology ; immunology ; CD8-Positive T-Lymphocytes ; cytology ; immunology ; Cell Proliferation ; Classical swine fever virus ; genetics ; Female ; HSP70 Heat-Shock Proteins ; genetics ; immunology ; Haemophilus parasuis ; genetics ; Immunization ; Interferon-gamma ; metabolism ; Mice ; Mice, Inbred BALB C ; Plasmids ; genetics ; Recombinant Fusion Proteins ; genetics ; immunology ; Viral Envelope Proteins ; genetics

OBJECTIVETo construct, express and purify human fusion proteins composed of a single-chain antibody fragment scFv that recognizes the prostate specific membrane antigen (PSMA) protein, Fdt, HA2 and tp, and to analyze the binding activity of the expressed fusion proteins.

METHODSThe fusion protein genes scFv, scFv-tp, and scFv-Fdt-HA2-tp were amplified by PCR, and the genes obtained were then cloned into the expression vector pET28 and expressed in E. coli BL21. The expressed products were identified by SDS-PAGE and Western blot and purified with Ni(2+)-NTA chelating agarose. The antigen-binding activity of the fusion proteins was determined by ELISA.

RESULTSThe human anti-PSMA fusion gene was successfully constructed and expressed in M15 as the inclusion body after induced with IPTG. All the target proteins expressed could bind the PSMA antigen.

CONCLUSIONFusion proteins can specifically bind the PSMA antigen. This finding contributes to the study of the targeted delivery of siRNA.

Antigens, Surface ; immunology ; Cloning, Molecular ; Electrophoresis, Polyacrylamide Gel ; Escherichia coli ; genetics ; immunology ; Glutamate Carboxypeptidase II ; immunology ; Humans ; Male ; Polymerase Chain Reaction ; RNA, Small Interfering ; administration & dosage ; immunology ; Recombinant Fusion Proteins ; genetics ; immunology ; Single-Chain Antibodies ; genetics ; immunology