To achieve uniform soluble expression of multiple proteins in the same Escherichia coli strain, and simplify the process steps of antigen production in genetic engineering subunit multivalent vaccine, we co-expressed three avian virus proteins including the fowl adenovirus serotype 4 (FAdV-4) Fiber-2 protein, infectious bursal disease virus (IBDV) VP2 protein and egg-drop syndrome virus (EDSV) Fiber protein in E. coli BL21(DE3) cells after optimization of gene codon, promoter, and tandem expression order. The purified proteins were analyzed by Western blotting and agar gel precipitation (AGP). The content of the three proteins were well-proportioned after co-expression and the purity of the purified proteins were more than 80%. Western blotting analysis and AGP experiment results show that all the three co-expression proteins had immunoreactivity and antigenicity. It is the first time to achieve the three different avian virus antigens co-expression and co-purification, which simplified the process of antigen production and laid a foundation for the development of genetic engineering subunit multivalent vaccine.
Animals ; Antigens, Viral/genetics* ; Biological Assay ; Chickens/immunology* ; Escherichia coli/genetics* ; Infectious bursal disease virus/immunology* ; Poultry Diseases ; Vaccines, Synthetic/isolation & purification* ; Viral Structural Proteins/immunology* ; Viral Vaccines/immunology*

To improve the specific recognition and presentation of virus-like particle (VLPs), and to develop immune-targeted VLPs vaccine, the gene fragment encoding OVA₂₅₇₋₂₆₄ peptide was inserted into the VP3 gene of foot-and-mouth disease virus (FMDV) between the 171th and 172th amino acids (aa) or 173th and 174th aa by reverse PCR. The recombinant proteins were expressed by using Escherichia coli and assembled into chimeric VLP (VLP(OVA)) in vitro after purification. The VLP(OVA) was measured by dynamic light scattering and transmission electron microscopy. The recombinant protein and the assembled VLPs were evaluated by Western blotting, enzyme-linked immunosorbent assay and laser scanning confocal microscopy to confirm the insertion of OVA₂₅₇₋₂₆₄ peptide into VP3 and its location. The results show that insertion of OVA₂₅₇₋₂₆₄ into the 173th and 174th aa of FMDV VP3 did not affect the assembly of VLPs. The VLP(OVA) in size was larger than VLPs, and the OVA₂₅₇₋₂₆₄ peptide was located on the surface of VLP(OVA).
Animals ; Escherichia coli ; genetics ; Foot-and-Mouth Disease ; virology ; Foot-and-Mouth Disease Virus ; genetics ; Recombinant Proteins ; genetics ; metabolism ; Vaccines, Virus-Like Particle

Porcine epidemic diarrhea (PED) is a highly contagious enteric swine disease. The large economic impact of PED on the swine industry worldwide has made the development of an effective PED vaccine a necessity. S0, a truncated region of the porcine epidemic diarrhea virus (PEDV) spike protein, has been suggested as a candidate antigen for PED subunit vaccines; however, poor solubility problems when the protein is expressed in Escherichia coli, and the inherent problems of subunit vaccines, such as low immunogenicity, remain. Flagellin has been widely used as a fusion partner to enhance the immunogenicity and solubility of many difficult-to-express proteins; however, the conjugation effect of flagellin varies depending on the target antigen or the position of the fusion placement. Here, we conjugated flagellin, Vibrio vulnificus FlaB, to the N- and C-termini of S0 and evaluated the ability of the fusion to enhance the solubility and immunogenicity of S0. Flagellin conjugation in the presence of the trigger factor chaperone tig greatly improved the solubility of the fusion protein (up to 99%) regardless of its conjugation position. Of importance, flagellin conjugated to the N-terminus of S0 significantly enhanced S0-specific humoral immune responses compared to other recombinant antigens in Balb/c mice. The mechanism of this phenomenon was investigated through in vitro and in vivo studies. These findings provide important information for the development of a novel PED vaccine and flagellin-based immunotherapeutics.
Animals ; Diarrhea ; Escherichia coli ; Flagellin ; Immunity, Humoral ; In Vitro Techniques ; Mice ; Porcine epidemic diarrhea virus ; Solubility ; Swine ; Swine Diseases ; Vaccines, Subunit ; Vibrio vulnificus ; Vibrio

Vaccination is one of the most successful strategies to prevent diseases caused by pathogens. Although various expression systems including Escherichia coli, yeast, insect, and mammalian cells are currently used for producing many of vaccines, these conventional platforms have the limitation of post-translational modification, high cost, and expensive scalability. In this respect, the plant-based expression system has been considered as an attractive platform to produce recombinant vaccines due to fast, cost-effective and scalable production as well as safety. This review discusses the development of plant-derived vaccines and the current stage of plant-based expression system.
Antibodies ; Efficiency ; Escherichia coli ; Humans ; Insects ; Plants ; Plants, Genetically Modified ; Protein Processing, Post-Translational ; Vaccination ; Vaccines ; Vaccines, Synthetic ; Yeasts

BACKGROUND: Meningitis is a clinically important disease because of its high mortality and morbidity. The epidemiology of this disease has changed remarkably due to the introduction of pneumococcal vaccines and Haemophilus influenzae type b (Hib) conjugate vaccine. Therefore, it is required to continuously monitor and research the organisms isolated from cerebrospinal fluid (CSF) cultures. METHODS: We analyzed trends of bacteria and fungi isolates obtained from CSF cultures between 1997 and 2016 in a tertiary care hospital according to year, month, gender, and age. RESULTS: Out of a total of 38,450 samples, we identified 504 (1.3%) isolates. The isolation rate in the first tested decade (1997–2006) ranged from 1.3% to 3.1%, while that in the second decade (2007–2016) ranged from 0.4% to 1.5%. The most common organisms was coagulase-negative staphylococci (CoNS) (31.9%), followed by Staphylococcus aureus (9.5%), Streptococcus pneumoniae (7.5%), Acinetobacter baumannii (5.8%), and Mycobacterium tuberculosis (5.8%). Monthly isolation rates were highest in May and July and lowest in February and December. Male to female ratio was 1.5:1. The isolation rates of S. pneumoniae, Enterococcus faecium, and Escherichia coli were similar in children and adults, but those of S. aureus, E. faecalis, A. baumannii, Pseudomonas aeruginosa, M. tuberculosis, and Cryptococcus neoformans were higher in adults than in children. CONCLUSION: During the last two decades, the isolation rate of CSF culture per year has decreased, with monthly isolation rates being highest in May and July. CoNS, S. aureus, and S. pneumoniae were most common in males, whereas CoNS, S. pneumoniae, and M. tuberculosis were most common in females. While Group B Streptococcus was most common in infants younger than 1 year, S. aureus and C. neoformans were more common in adults.
Acinetobacter baumannii ; Adult ; Bacteria* ; Cerebrospinal Fluid* ; Child ; Cryptococcus neoformans ; Enterococcus faecium ; Epidemiology ; Escherichia coli ; Female ; Fungi* ; Haemophilus influenzae type b ; Humans ; Infant ; Male ; Meningitis ; Mortality ; Mycobacterium tuberculosis ; Pneumococcal Vaccines ; Pneumonia ; Pseudomonas aeruginosa ; Staphylococcus aureus ; Streptococcus ; Streptococcus pneumoniae ; Tertiary Healthcare* ; Tuberculosis

HPV16 L2E7 is a fusion protein used for therapeutical vaccine targeting HPV virus. To increase its expression in Escherichia coli, we optimized the codon usage of HPV16 l2e7 gene based on its codon usage bias. The optimized gene of HPV16 sl2e7 was cloned into three different vectors: pGEX-5X-1, pQE30, ET41a, and expressed in JM109, JM109 (DE3) and BL21 (DE3) lines separately. A high expression line was selected with pET41a vector in BL21 (DE3) cells. After optimization of the growth condition, including inoculation amount, IPTG concentration, induction time and temperature, the expression level of HPV16 L2E7 was increased from less than 10% to about 28% of total protein. HPV16 L2E7 protein was then purified from 15 L culture by means of SP Sepharose Fast Flow, Q Sepharose Fast Flow and Superdex 200 pg. After renaturing, HPV16 L2E7 protein with ≥ 95% purity was achieved, which was confirmed via SDS-PAGE gel and Western blotting. The combined use of purified HPV16 L2E7 and CpG helper has shown clear inhibition of tumor growth in mice injected with tumor cells, with six out of eight mice shown no sign of tumor. This study lays a solid foundation for a new pipeline of large-scale vaccine production.
Animals ; Capsid Proteins ; biosynthesis ; Codon ; Electrophoresis, Polyacrylamide Gel ; Escherichia coli ; Genetic Vectors ; Human papillomavirus 16 ; Mice ; Neoplasms, Experimental ; prevention & control ; Oncogene Proteins, Viral ; biosynthesis ; Papillomavirus E7 Proteins ; biosynthesis ; Papillomavirus Vaccines ; therapeutic use ; Recombinant Fusion Proteins ; biosynthesis

To construct an HSV-1 vector vaccine carrying HIV-1 antigens, HIV-1 gp160, gag, protease and the expression elements were chained together, and then inserted into the internal inverted repeat sequence region of HSV-1 by bacterial artificial chromosome technology. Firstly, HIV-1 gp160 (including type B and C), gag and protease genes were cloned into pcDNA3 in series to generate the pcDNA/gBgp and pcDNA/gCgp, then the recombinant plasmids were transfected into 293FT cells, and HIV-1 antigen was detected from transfected cells by Western blotting. Then the expression cassettes from pcDNA/gBgp and pcDNA/gCgp, comprising HIV-1 antigen genes and expression elements, were cloned into pKO5/BN to generate the shuttle plasmids pKO5/BN/gBgp and pKO5/BN/gCgp. The shuttle plasmids were electroporated into E. coli cells that harbor an HSV-BAC, the recombinant bacteria were screened, and the recombinant DNA was extracted and transfected into Vero cells. The recombinant virus was purified through picking plaques, the virus' DNAs were identified by Southern blotting; HIV-1 antigen was detected from the recombinant HSV-1 infected cells by Western blotting, and the virus' replication competent was analyzed. As the results, gp160 and gag proteins were detected from 293FT cells transfected with pcDNA/gBgp and pcDNA/gCgp by Western blotting. The recombinant bacteria were generated from the E. coli electroporated with pKO5/BN/gBgp or pKO5/BN/gCgp. The recombinant HSV was purified from the Vero cells transfected with the recombinant DNA, the unique DNA fragment was detected from the genome of recombination HSV by Southern blotting; gp120 and gp41 were detected from the infected cells by Western blotting, and the recombinant HSV retained replication competent in mammalian cells. The results indicate that the recombinant HSV carrying HIV-1 gp160, gag and protease genes was generated, the virus retains replication competent in mammalian cells, and could be used as a replicated viral vector vaccine.
Animals ; Cercopithecus aethiops ; Chromosomes, Artificial, Bacterial ; DNA, Recombinant ; genetics ; DNA, Viral ; genetics ; Escherichia coli ; HIV Antigens ; genetics ; immunology ; HIV Envelope Protein gp160 ; genetics ; immunology ; HIV Protease ; genetics ; immunology ; Herpes Simplex Virus Vaccines ; immunology ; Herpesvirus 1, Human ; physiology ; Plasmids ; Transfection ; Vero Cells ; Virus Replication ; gag Gene Products, Human Immunodeficiency Virus ; genetics ; immunology

OBJECTIVETo construct a vector encoding T-cell epitopes of major allergen group 1 of Dermatophagoides pteronyssinus as a vaccine delivered by MHC class II pathway.

METHODSThe nucleotide sequences of the 3 target genes were synthesized, including TAT, IhC and the recombinant fragment of Der p 1 encoding 3 T-cell epitopes. After amplification of the 3 target fragments by PCR and digestion with corresponding restriction endonucleases, the recombinant gene TAT-IhC-Der p 1-3T was ligated using T4 DNA ligase and inserted into the prokaryotic expression vector pET28a(+) to construct the recombinant plasmid pET-28a(+)-TAT-IhC-Der p 1-3T, which was confirmed by digestion with restriction endonucleases and sequencing. The recombinant vector was transformed into E. coli strain BL21 (DE3) and induced with IPTG, and the induced protein TAT-IhC-Der p 1-3T was detected by SDS-PAGE. After purification, the recombinant protein was confirmed by Western blotting and its allergenicity tested using IgE-binding assay.

RESULTSThe recombinant plasmid pET-28a-TAT-IhC-Der p 1-3T was successfully constructed as confirmed by restriction endonuclease digestion and sequencing and the expression of the recombinant protein TAT-IhC-Der p 1-3T was induced in E. coli. Western blotting verified successfull purification of the target protein, which showed a stronger IgE-binding ability than Der p 1.

CONCLUSIONWe successfully constructed a recombinant expression vector pET-28a-TAT-IhC-Der p 1-3T expressing a T-cell epitope vaccine delivered by MHC II pathway with strong IgE-binding ability, which provides a basis for further study on specific immunotherapy via MHC class II pathway.

Allergens ; immunology ; Animals ; Antigens, Dermatophagoides ; immunology ; Arthropod Proteins ; immunology ; Base Sequence ; Cloning, Molecular ; Cysteine Endopeptidases ; immunology ; Dermatophagoides pteronyssinus ; Epitopes, T-Lymphocyte ; Escherichia coli ; Gene Expression ; Genes, MHC Class II ; Genetic Vectors ; Plasmids ; Polymerase Chain Reaction ; Recombinant Proteins ; immunology ; Vaccines ; immunology

PURPOSE: Protein cages are promising nanoplatform candidates for efficient delivery systems due to their homogenous size and structure with high biocompatibility and biodegradability. In this study, we investigate the potential of lumazine synthase protein cage as an antigen delivery system to dendritic cells (DCs), which induce antigen-specific T cell proliferation. MATERIALS AND METHODS: Ovalbumin (OVA) peptides OT-1 (SIINFEKL) and OT-2 (ISQAVHAAHAEINEAGR) were genetically inserted to lumazine synthase and each protein cage was over-expressed in Escherichia coli as a soluble protein. The efficiency of antigen delivery and the resulting antigen-specific T cell proliferation by DCs was examined in vitro as well as in vivo. RESULTS: We successfully generated and characterized OVA peptides carrying lumazine synthase protein cages. The OT-1 and OT-2 peptides carried by lumazine synthases were efficiently delivered and processed by DCs in vitro as well as in vivo, and induced proliferation of OT-1-specific CD8+T cells and OT-2-specific CD4+T cells. CONCLUSION: Our data demonstrate the potential of lumazine synthase protein cage being used as a novel antigen delivery system for DC-based vaccine development in future clinical applications.
Antigen Presentation ; Cell Proliferation ; Dendritic Cells ; Escherichia coli ; Nanoparticles* ; Ovalbumin ; Ovum ; Peptides ; Vaccines

PURPOSE: FimH (the adhesion fragment of type 1 fimbriae) is implicated in uropathogenic Escherichia coli (UPEC) attachment to epithelial cells through interaction with mannose. Recently, some studies have found that UPEC can thrive intracellularly causing recurrent urinary tract infection (UTI). Almost all vaccines have been designed to induce antibodies against UPEC. Yet, the humoral immune response is not potent enough to overcome neither the primary UTI nor recurrent infections. However, DNA vaccines offer the possibility of inducing cell mediated immune responses and may be a promising preventive tool. MATERIALS AND METHODS: In this study, we employed two different open reading frames within mammalian (mam) and wild type (wt) codons of fimH gene. Optimized fragments were cloned in pVAX-1. Expression of the protein in COS-7 was confirmed by western blot analysis after assessing pVAX/fimH(mam) and pVAX/fimH(wt). The constructs were injected to BALB/c mice at plantar surface of feet followed by electroporation. RESULTS: The mice immunized with both constructs following booster injection with recombinant FimH showed increased interferon-gamma and interleukin-12 responses significantly higher than non-immunized ones (p<0.05). The immunized mice were challenged with UPEC and then the number of bacteria recovered from the immunized mice was compared with the non-immunized ones. Decreased colony count in immunized mice along with cytokine responses confirmed the promising immune response by the DNA vaccines developed in this study. CONCLUSION: In conclusion, DNA vaccines of UPEC proteins may confer some levels of protection which can be improved by multiple constructs or boosters.
Animals ; Antibodies ; Bacteria ; Blotting, Western ; Clone Cells ; Codon* ; DNA* ; Electroporation ; Epithelial Cells ; Foot ; Immunity, Cellular* ; Immunity, Humoral ; Interferon-gamma ; Interleukin-12 ; Mannose ; Mice ; Open Reading Frames ; Urinary Tract Infections* ; Uropathogenic Escherichia coli ; Vaccines ; Vaccines, DNA