Hepatitis B virus core protein can self-assemble into icosahedral symmetrical viral-like particles (VLPs) in vitro, and display exogenous sequences repeatedly and densely on the surface. VLPs also have strong immunogenicity and biological activity. When the nanoparticles enter the body, they quickly induce specific humoral and cellular immune responses to exogenous antigens. In this study, we designed an HBc-VLPs that can be coupled with antigens at specific sites, and developed a set of efficient methods to prepare HBc-VLPs. Through site-specific mutation technology, the 80th amino acid of peptide was changed from Ala to Cys, a specific cross-linking site was inserted into the main immunodominant region of HBc-VLPs, and the prokaryotic expression vector pET28a(+)-hbc was constructed. After expression and purification, high purity HBc(A80C) monomer protein was assembled into HBc-VLPs nanoparticles in Phosphate Buffer. The results of particle size analysis show that the average particle size of nanoparticles was 29.8 nm. Transmission electron microscopy (TEM) showed that HBc-VLPs formed spherical particles with a particle size of about 30 nm, and its morphology was similar to that of natural HBV particles. The influenza virus antigen M2e peptide as model antigen was connected to Cys residue of HBc-VLPs by Sulfo-SMCC, an amino sulfhydryl bifunctional cross-linking agent, and M2e-HBc-VLPs model vaccine was prepared. The integrity of HBc-VLPs structure and the correct cross-linking of M2e were verified by cell fluorescence tracing. Animal immune experiments showed that the vaccine can effectively stimulate the production of antigen-specific IgG antibody in mice, which verified the effectiveness of the vaccine carrier HBc-VLPs. This study lays a foundation for the research of HBc-VLPs as vaccine vector, and help to promote the development of HBc-VLPs vaccine and the application of HBc-VLPs in other fields.
Animals ; Hepatitis B Core Antigens ; genetics ; immunology ; Immunity, Cellular ; immunology ; Immunoglobulin G ; blood ; Mice ; Mice, Inbred BALB C ; Vaccines, Virus-Like Particle ; genetics ; immunology

Characterization of the colon cancer immunome and its autoantibody signature from differentially-reactive antigens (DIRAGs) could provide insights into aberrant cellular mechanisms or enriched networks associated with diseases. The purpose of this study was to characterize the antibody profile of plasma samples from 32 colorectal cancer (CRC) patients and 32 controls using proteins isolated from 15,417 human cDNA expression clones on microarrays. 671 unique DIRAGs were identified and 632 were more highly reactive in CRC samples. Bioinformatics analyses reveal that compared to control samples, the immunoproteomic IgG profiling of CRC samples is mainly associated with cell death, survival, and proliferation pathways, especially proteins involved in EIF2 and mTOR signaling. Ribosomal proteins (e.g., RPL7, RPL22, and RPL27A) and CRC-related genes such as APC, AXIN1, E2F4, MSH2, PMS2, and TP53 were highly enriched. In addition, differential pathways were observed between the CRC and control samples. Furthermore, 103 DIRAGs were reported in the SEREX antigen database, demonstrating our ability to identify known and new reactive antigens. We also found an overlap of 7 antigens with 48 "CRC genes." These data indicate that immunomics profiling on protein microarrays is able to reveal the complexity of immune responses in cancerous diseases and faithfully reflects the underlying pathology.
Adult ; Aged ; Aged, 80 and over ; Biomarkers, Tumor ; genetics ; immunology ; metabolism ; Case-Control Studies ; Colonic Neoplasms ; immunology ; metabolism ; Computational Biology ; methods ; Computer Simulation ; Female ; Gene Expression Profiling ; Gene Expression Regulation, Neoplastic ; Humans ; Immunoglobulin G ; immunology ; Male ; Middle Aged ; Protein Array Analysis ; methods

OBJECTIVES: The seroprevalence of hepatitis E virus (HEV) among high-risk groups overseas is high, but studies in these groups are rare in South Korea. We conducted the present study from April to November 2012 to obtain data on the seroprevalence and associated risk factors for HEV among slaughterhouse workers in South Korea. METHODS: Slaughterhouse workers from 80 workplaces nationwide were surveyed in South Korea in 2012. The subjects comprised 1848 cases: 1434 slaughter workers and 414 residual products handlers. By visiting 80 slaughterhouses, which were mixed with 75 of which also performed residual products handling, we conducted a questionnaire survey for risk factors and obtained blood samples in order to determine the seropositivity and seroprevalence of HEV. Anti-HEV IgG and IgM were measured using HEV IgG and IgM enzyme-linked immunospecific assay kits and HEV antigen was measured by reverse transcription polymerase chain reaction (RT-PCR). RESULTS: The seropositivity of anti-HEV IgG was 33.5% (slaughter workers 32.8% and residual products handlers 36.2%), and among the seropositive individuals the seroprevalence of anti-HEV IgM was 0.5% (slaughter workers 0.5%, residual products handlers 0.7%). The response rate of HEV-antigen as measured by RT-PCR was 0.2%. Risk factors significantly related to anti-HEV IgG seropositivity were age, sex , and working duration (slaughter workers only). CONCLUSIONS: There were significant risk factors (sex, age, and working duration) for HEV identified in our study. All three positive cases for HEV-antigen by RT-PCR were related to pig slaughter but without statistical significance. To prevent HEV, an educational program and working guidelines may be needed for high risk groups.
Abattoirs ; Adult ; Aged ; Enzyme-Linked Immunosorbent Assay ; Female ; Hepatitis Antibodies/blood ; Hepatitis E/*diagnosis/epidemiology/virology ; Hepatitis E virus/genetics/*immunology/metabolism ; Humans ; Immunoglobulin G/blood ; Immunoglobulin M/blood ; Male ; Middle Aged ; Multivariate Analysis ; Prevalence ; Republic of Korea/epidemiology ; Reverse Transcriptase Polymerase Chain Reaction ; Risk Factors ; Workplace

To obtain human antibodies against the Gn protein of Severe fever with thrombocytopenia syndrome virus (SFTSV) with phage display technology, this study aimed to screen anti-Gn protein antibodies from an anti-SFTSV Fab human phage display library. Antibody genes were identified by sequence analysis and the specificity of antibodies was confirmed by ELISA. The Fab antibody genes were cloned into the HL51-14 vector and expressed in a mammalian cell expression system. IgG antibodies were then purified by protein A affinity chromatography,and the results were further confirmed by ELISA,IFA,western blotting assays and micro-neutralization tests. The results showed that, after three rounds of panning, there were 390 human Fab antibodies against SFTSV particles, of which 364 were specific for nucleoprotein. Coated with the Gn protein, eight different Fab antibodies specific for Gn protein were obtained after the determination of the subtype and subclass of antibodies by gene sequencing; five of these antibodies were from the Lambda library and three were from the Kappa library. The eight IgG antibodies could specifically bind to Gn protein according to the ELISA, IFA and Western blotting assays. The micro-neutralization test showed that these eight antibodies had no neutralizing activity,but they could still provide a reference for research in human monoclonal antibodies against SFTSV.
Antibodies ; genetics ; immunology ; Bunyaviridae Infections ; genetics ; immunology ; virology ; Cell Line ; Cloning, Molecular ; Humans ; Immunoglobulin Fab Fragments ; genetics ; immunology ; Immunoglobulin G ; genetics ; immunology ; Neutralization Tests ; Phlebovirus ; genetics ; immunology ; Viral Proteins ; genetics ; immunology

To identify SJCHGC01743 gene of Schistosoma japonicum and evaluate the potential of the recombinant protein as a new vaccine candidate for schistosomiasis, polymerase chain reaction (PCR) technique was used to amplify the cDNA of the gene and real-time RT-PCR was used to analyze the transcription profiles of SJCHGC01743 at different development stages. Recombinant plasmid was successfully constructed and transformed into competent Escherichia coli BL21 (DE3). Then the recombinant protein was expressed, purified and emulsified with ISA206 adjuvant to immunize BALB/c mice for three times. The immunogenicity was confirmed by Western blotting and tissue localization was detected by indirect immunofluorescent assay. The specific antibody level was detected by ELISA. The immunoprotection of rSjOST48 was evaluated by the reduction in worm and egg counts in mice. A cDNA with 1 248 nucleotides was isolated from 28-day-old schistosomes cDNAs by PCR. Sequence analysis revealed that SJCHGC01743 was a 48-kDa subunit of the oligosaccharyltransferase complex (OST48) and named as SjOST48. Real-time PCR analysis indicated that this gene was expressed in all investigated stages and had the highest expression level in 28 d worms, the level of gene transcription in female worms was significantly higher than that of male worms. Then recombinant plasmid pET28a(+)-SjOST48 was successfully constructed and expressed in E. coli BL21 (DE3). Western blotting analysis showed that rSjOST48 had good immunogenicity. Indirect immunofluorescent analysis revealed that SjOST48 was mainly distributed on the tegument of the worms. The result of ELISA indicated that the rSjOST48 vaccinated group could induce a significant increase in the level of specific IgG, IgG1 and IgG2a. An immunoprotection experiment showed that the vaccination of rSjOST48 in mice induced 32.62% (P < 0.05) reduction in the numbers of worms and 57.61% (P < 0.01) in eggs in liver, compared with that of the control group. This study provides the foundation for proceeding further research on the biological function of SjOST48 and screening new vaccine candidates for schistosomiasis.
Animals ; Antibodies, Helminth ; blood ; Cloning, Molecular ; DNA, Complementary ; Escherichia coli ; Female ; Genes, Helminth ; Helminth Proteins ; genetics ; immunology ; Immunoglobulin G ; blood ; Male ; Mice ; Mice, Inbred BALB C ; Real-Time Polymerase Chain Reaction ; Recombinant Proteins ; immunology ; Schistosoma japonicum ; genetics ; Schistosomiasis japonica ; prevention & control ; Vaccination

Diisocyanate (DI) is the most common cause of occupational asthma (OA) in Korea. Mannose-binding lectin (MBL) initiates the lectin complement activation pathway following oxidative stress and plays an important role in the regulation of inflammatory processes. To determine whether there is a genetic association between MBL2 polymorphisms and DI-OA, 99 patients with DI-OA, 99 asymptomatic exposed controls (AECs) and 144 unexposed normal controls were enrolled in this study. Three polymorphisms (-554 G>C, - 431A>C and - 225 G>C) in the MBL2 promoter were genotyped, and serum MBL levels were determined by enzyme-linked immunosorbent assay. Functional variabilities in the promoter polymorphisms were analyzed by a luciferase reporter assay and electrophoretic mobility shift assay (EMSA). A significantly higher frequency of haplotype (ht) 2 [CAG] was noted in the DI-OA group compared with the AEC group (P=0.044). The patients with DI-OA carrying ht2 [CAG] had significantly lower PC20 methacholine levels (P<0.001) than the non-carriers. The serum MBL levels were significantly higher in the DI-exposed subjects (both the DI-OA patients and AECs) carrying ht1 [GAG] (P=0.028). Luciferase activity was significantly enhanced in ht1 [GAG] compared with ht2 [CAG] in human hepatocarcinoma cells (Hep3B) (P=0.002). The EMSA showed that a - 554G probe produced a specific shifted band compared with the - 554C probe. These findings suggest that decreased serum MBL levels due to polymorphisms of the MBL2 gene may increase susceptibility to the development of DI-OA in DI-exposed individuals.
Adult ; Alleles ; Asthma, Occupational/diagnosis/*etiology ; Cell Line ; Female ; Forced Expiratory Volume ; Gene Frequency ; Genotype ; Haplotypes ; Humans ; Immunoglobulin E/immunology ; Immunoglobulin G/immunology ; Isocyanates/*adverse effects/immunology ; Male ; Mannose-Binding Lectin/blood/*genetics ; Middle Aged ; *Polymorphism, Genetic ; Polymorphism, Single Nucleotide ; Protein Binding ; Transcriptional Activation ; Young Adult

The aim of this cross sectional case control study was to examine the serofrequency and serointensity of Toxoplasma gondii (Tg) IgG, IgM, and DNA among patients with schizophrenia. A total of 101 patients with schizophrenia and 55 healthy controls from Sungai Buloh Hospital, Selangor, Malaysia and University Malaya Medical Center (UMMC) were included in this study. The diagnosis of schizophrenia was made based on the Diagnostic and Statistical Manual of Mental Disorders, Fourth Edition (DSM-IV). The presence of Tg infection was examined using both indirect (ELISA) and direct (quantitative real-time PCR) detection methods by measuring Tg IgG and IgM and DNA, respectively. The serofrequency of Tg IgG antibodies (51.5%, 52/101) and DNA (32.67%, 33/101) among patients with schizophrenia was significantly higher than IgG (18.2%, 10/55) and DNA (3.64%, 2/55) of the controls (IgG, P=0.000, OD=4.8, CI=2.2-10.5; DNA, P=0.000, OD=12.9, CI=2.17-10.51). However, the Tg IgM antibody between patients with schizophrenia and controls was not significant (P>0.005). There was no significant difference (P>0.005) in both serointensity of Tg IgG and DNA between patients with schizophrenia and controls. These findings have further demonstrated the strong association between the active Tg infection and schizophrenia.
Adolescent ; Adult ; Aged ; Antibodies, Protozoan/*blood ; Case-Control Studies ; Cross-Sectional Studies ; DNA, Protozoan/*blood ; Enzyme-Linked Immunosorbent Assay ; Female ; Humans ; Immunoglobulin G/blood ; Immunoglobulin M/blood ; Malaysia ; Male ; Middle Aged ; Real-Time Polymerase Chain Reaction ; Schizophrenia/*complications ; Seroepidemiologic Studies ; Toxoplasma/classification/genetics/immunology/*isolation & purification ; Toxoplasmosis/*epidemiology/immunology/*parasitology ; Young Adult

The aim of this cross sectional case control study was to examine the serofrequency and serointensity of Toxoplasma gondii (Tg) IgG, IgM, and DNA among patients with schizophrenia. A total of 101 patients with schizophrenia and 55 healthy controls from Sungai Buloh Hospital, Selangor, Malaysia and University Malaya Medical Center (UMMC) were included in this study. The diagnosis of schizophrenia was made based on the Diagnostic and Statistical Manual of Mental Disorders, Fourth Edition (DSM-IV). The presence of Tg infection was examined using both indirect (ELISA) and direct (quantitative real-time PCR) detection methods by measuring Tg IgG and IgM and DNA, respectively. The serofrequency of Tg IgG antibodies (51.5%, 52/101) and DNA (32.67%, 33/101) among patients with schizophrenia was significantly higher than IgG (18.2%, 10/55) and DNA (3.64%, 2/55) of the controls (IgG, P=0.000, OD=4.8, CI=2.2-10.5; DNA, P=0.000, OD=12.9, CI=2.17-10.51). However, the Tg IgM antibody between patients with schizophrenia and controls was not significant (P>0.005). There was no significant difference (P>0.005) in both serointensity of Tg IgG and DNA between patients with schizophrenia and controls. These findings have further demonstrated the strong association between the active Tg infection and schizophrenia.
Adolescent ; Adult ; Aged ; Antibodies, Protozoan/*blood ; Case-Control Studies ; Cross-Sectional Studies ; DNA, Protozoan/*blood ; Enzyme-Linked Immunosorbent Assay ; Female ; Humans ; Immunoglobulin G/blood ; Immunoglobulin M/blood ; Malaysia ; Male ; Middle Aged ; Real-Time Polymerase Chain Reaction ; Schizophrenia/*complications ; Seroepidemiologic Studies ; Toxoplasma/classification/genetics/immunology/*isolation & purification ; Toxoplasmosis/*epidemiology/immunology/*parasitology ; Young Adult

Recombinant antigenic proteins of Toxoplasma gondii are alternative source of antigens which are easily obtainable for serodiagnosis of toxoplasmosis. In this study, highly antigenic secretory organellar proteins, dense granular GRA2 and GRA3, rhoptrial ROP2, and micronemal MIC2, were analyzed by bioinformatics approach to express as water-soluble forms of antigenic domains. The transmembrane region and disorder tendency of 4 secretory proteins were predicted to clone the genes into pGEX-4T-1 vector. Recombinant plasmids were transformed into BL21 (DE3) pLysS E. coli, and GST fusion proteins were expressed with IPTG. As a result, GST fusion proteins with GRA225-105, GRA339-138, ROP2324-561, and MIC21-284 domains had respectively higher value of IgG avidity. The rGST-GRA225-105 and rGST-GRA339-138 were soluble, while rGST-ROP2324-561 and rGST-MIC21-284 were not. GRA231-71, intrinsically unstructured domain (IUD) of GRA2, was used as a linker to enhance the solubility. The rGST-GRA231-71-ROP2324-561, a chimeric protein, appeared to be soluble. Moreover, rGST-GRA231-71-MIC21-284 was also soluble and had higher IgG avidity comparing to rGST-MIC21-284. These 4 highly expressed and water-soluble recombinant antigenic proteins may be promising candidates to improve the serodiagnosis of toxoplasmosis in addition to the major surface antigen of SAG1.
Animals ; Antibodies, Protozoan/immunology ; Antibody Affinity ; Antigens, Protozoan/chemistry/*diagnostic use/genetics/immunology ; *Gene Expression ; Immunoglobulin G/blood/immunology ; Mice, Inbred BALB C ; Recombinant Proteins/chemistry/*diagnostic use/genetics/immunology ; Serologic Tests/methods ; Solubility ; Toxoplasma/genetics/immunology/*metabolism ; Toxoplasmosis/diagnosis

Serologic tests are widely accepted for diagnosing Toxoplasma gondii but purification and standardization of antigen needs to be improved. Recently, surface tachyzoite and bradyzoite antigens have become more attractive for this purpose. In this study, diagnostic usefulness of 3 recombinant antigens (SAG1, SAG2, and SAG3) were evaluated, and their efficacy was compared with the available commercial ELISA. The recombinant plasmids were transformed to JM109 strain of Escherichia coli, and the recombinants were expressed and purified. Recombinant SAG1, SAG2, and SAG3 antigens were evaluated using different groups of sera in an ELISA system, and the results were compared to those of a commercial IgG and IgM ELISA kit. The sensitivity and specificity of recombinant surface antigens for detection of anti-Toxoplasma IgG in comparison with commercially available ELISA were as follows: SAG1 (93.6% and 92.9%), SAG2 (100.0% and 89.4%), and SAG3 (95.4% and 91.2%), respectively. A high degree of agreement (96.9%) was observed between recombinant SAG2 and commercial ELISA in terms of detecting IgG anti-Toxoplasma antibodies. P22 had the best performance in detecting anti-Toxoplasma IgM in comparison with the other 2 recombinant antigens. Recombinant SAG1, SAG2, and SAG3 could all be used for diagnosis of IgG-specific antibodies against T. gondii.
Antibodies, Protozoan/*blood ; Antigens, Protozoan/diagnostic use/*genetics ; Enzyme-Linked Immunosorbent Assay ; Humans ; Immunoglobulin G/blood ; Immunoglobulin M/blood ; Membrane Glycoproteins/*genetics ; Protozoan Proteins/*genetics ; Recombinant Proteins/diagnostic use/immunology ; Sensitivity and Specificity ; Toxoplasma/immunology ; Toxoplasmosis/blood/*diagnosis