Animals ; Antibodies, Neutralizing/biosynthesis* ; Antibodies, Viral/biosynthesis* ; Body Weight/immunology* ; COVID-19/virology* ; Disease Models, Animal ; Disease Progression ; Humans ; Immunohistochemistry ; Lung/virology* ; Male ; Mesocricetus ; Nasal Cavity/virology* ; RNA, Viral/immunology* ; SARS-CoV-2/pathogenicity* ; Severity of Illness Index ; Viral Load

Influenza A virus is an enveloped virus that belongs to the Orthomyxoviridae family. It has 8 negative RNA segments that encode 16 viral proteins. The viral polymerase consists of 3 proteins (PB 1, PB2 and PA) which plays an important role in the transcription and replication of the influenza A virus. Polymerase basic protein 1 (PB 1) is a critical member of viral polymerase complex. In order to further study the function of PB1, we need to prepare the PB1 antibody with good quality. Therefore, we amplified PB1 conserved region (nt1648-2265) by PCR and cloned it into pET-30a vector, and transformed into Escherichia coli BL2 1. The expression of His tagged PB 1 protein was induced by IPTG, and His-PB 1 proteins were purified by Ni-NTA resin. For preparation of PB 1 protein antiserum, rabbits were immunized with His-PB 1 fusion protein 3 times. Then the titer of PB 1 polyclonal antibody was measured by indirect ELISA. The antibody was purified by membrane affinity purification and subjected to immunoblotting analysis. Data showed that PB1 antibody can recognize PB 1 protein from WSN virus infected or pCMV FLAG-PB 1 transfected cells. Meanwhile, PB 1 antibody can also recognize specifically other subtype strains of influenza A virus such as H9N2 and H3N2. PB 1 polyclonal antibody we generated will be a useful tool to study the biological function of PB1.
Animals ; Antibodies, Viral ; biosynthesis ; Cloning, Molecular ; Enzyme-Linked Immunosorbent Assay ; Escherichia coli ; metabolism ; Genetic Vectors ; Influenza A Virus, H3N2 Subtype ; Influenza A Virus, H9N2 Subtype ; Plasmids ; Rabbits ; Viral Proteins ; immunology

To increase detection sensitivity and specificity on hepatitis C virus (HCV) is vital for prevention and controlling of the disease. To establish a more reliable detection method for HCV diagnosis, the full gene fragment of ns3 (non-structural protein of HCV) from recombinant plasmid of J6/JFH1 2a was amplified and then connected into the pET-28a prokaryotic expression vector, and the latter was subsequently transformed into Escherichia coli BL21 (DE3) to have the target protein expression. As a result, a protein with a molecular weight of 72 kDa was obtained and visualized in 10% SDS-PAGE. The purified NS3 protein was used as immunogen to inoculate BALB/c mice and the sera was collected after the fourth immunization. The antibody titer of serum is determined to be about 1:256000 with ELISA. Western blotting and indirect immunofluorescence analysis showed that the mouse polyclonal antibody could react specifically with the native NS3 protein in Huh 7.5.1 cells infected with HCV. These findings may provide basis for further preparation of monoclonal antibodies against NS3 and the development of related detection kit.
Animals ; Antibodies, Viral ; immunology ; Enzyme-Linked Immunosorbent Assay ; Escherichia coli ; Hepacivirus ; Mice ; Mice, Inbred BALB C ; Plasmids ; Viral Nonstructural Proteins ; biosynthesis ; immunology

In order to develop monoclonal antibodies (McAbs) against the gp90 protein of reticuloendotheliosis virus (REV), the His-tagged gp90 protein of REV was used to immunize BALB/c mice. Hybridomas were generated by fusing mouse myeloma cells SP2/0 with the splenocytes from the immunized mice. After screening and 3 rounds of cloning process, 3 hybridomas (3G5-B8, 3G5-A10 and 1G12) that stably secreted McAbs against the REV-gp90 were obtained. The isotypes of the McAbs were determined to be IgG1, IgG1 and IgG2b. The McAbs specifically bound to gp90 in REV-infected DF-1 cells, as demonstrated by Western blotting and indirect immunofluorescence assay. The recognition regions on gp90 that were recognized by 3G5-B8/3G5-A10 and 1G12 were located between amino acids 200 to 245 and 230 to 235, respectively, as demonstrated by Western blotting analysis. These McAbs will be useful in the diagnosis and pathogenesis study of REV.
Animals ; Antibodies, Monoclonal ; biosynthesis ; Antibody Specificity ; Blotting, Western ; Epitope Mapping ; Hybridomas ; Immunoglobulin G ; biosynthesis ; Mice ; Mice, Inbred BALB C ; Reticuloendotheliosis virus ; immunology ; Viral Envelope Proteins ; immunology

BACKGROUND: Without appropriate culture systems for hepatitis E virus (HEV), sufficient natural viral proteins are difficult to generate for use in serological tests. Therefore, it is important to produce large amounts of HEV recombinant proteins in an economical way. The present study developed ELISAs using 2 truncated forms of the HEV open reading frame (ORF) 2 protein in order to detect anti-HEV IgG in serum samples. METHODS: Two truncated forms of the ORF2 protein were expressed in Escherichia coli and were purified by Ni2+-chelate-affinity chromatography (Qiagen, Germany). Two ELISAs were developed using these proteins and were compared with DIA.PRO HEV IgG ELISA kit (DIA.PRO. Italy) in 220 serum samples. RESULTS: High yields of the target proteins were obtained through codon optimization. The concentration and purity of the proteins were improved with Amicon filters (EMD Millipore, USA). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting analysis of the resultant proteins showed a protein band of approximately 60 kDa corresponding to ORF2.1 (amino acids 112-660) and a protein band of approximately 55 kDa corresponding to ORF2.2 (amino acids 112-607). Positive agreement, negative agreement, and concordance of the 2 in-house ELISAs compared with DIA.PRO HEV IgG ELISA kit were 87%, 99.5%, and 98.1%, respectively (kappa=0.899, P=0.625). CONCLUSIONS: The newly developed ELISAs are useful for detecting anti-HEV IgG in serum samples and are highly concordant with DIA.PRO HEV IgG ELISA kit.
Amino Acid Sequence ; Antibodies/*blood ; *Enzyme-Linked Immunosorbent Assay ; Escherichia coli/metabolism ; Hepatitis E virus/*metabolism ; Humans ; Immunoglobulin G/*blood ; Molecular Sequence Data ; Recombinant Proteins/biosynthesis/immunology/isolation & purification ; Sequence Alignment ; Viral Proteins/chemistry/*immunology/metabolism

Infectious bursal disease virus (IBDV) VP4 plays an important role in immunosuppression of host. In order to develop monoclonal antibodies (McAbs) against VP4, we vaccinated BALB/c mice with His-VP4, screened and subcloned positive clones. We established 4 hybridoma cell lines that stably secreted McAbs against VP4 and named these cell lines 3B3, 3H11, 4C8 and 4G6, respectively. We tested the dissociation constant (Kd) of these McAbs, and found that their K(d)s were 4.61 x 10(-11), 1.71 x 10(-10), 4.26 x 10(-11), 5.02 x 10(-11), respectively. The isotypes of these McAbs were determined to be IgG1, IgG1, IgG2b and IgG1. These McAbs specifically bound to VP4 in IBDV infected DF-1 cells as demonstrated by Western blotting analysis and fluorescence antibody assay. These McAbs would help to detect IBDV infection and to analyze the biological activities of IBDV VP4.
Animals ; Antibodies, Monoclonal ; biosynthesis ; Blotting, Western ; Cell Line ; Fluorescent Antibody Technique ; Hybridomas ; Infectious bursal disease virus ; Mice ; Mice, Inbred BALB C ; Viral Structural Proteins ; immunology

Antibodies, Monoclonal ; chemistry ; immunology ; Antigens, Viral ; chemistry ; genetics ; immunology ; Binding Sites ; Capsid Proteins ; chemistry ; genetics ; immunology ; Epitopes ; chemistry ; genetics ; immunology ; Escherichia coli ; genetics ; metabolism ; Gene Expression ; Hepatitis E ; immunology ; prevention & control ; virology ; Hepatitis E virus ; chemistry ; immunology ; Humans ; Molecular Docking Simulation ; Mutagenesis, Site-Directed ; Peptide Mapping ; Protein Binding ; Recombinant Proteins ; chemistry ; genetics ; immunology ; Viral Hepatitis Vaccines ; administration & dosage ; biosynthesis

To observe how anti-group A rotavirus antibody seropositivity rates and levels have changed in the western region of Gyeongnam Province, 2,030 serum samples collected at four collection periods (1989-1990, 1994-1995, 1999-2000, and 2004-2005) were tested by Enzyme-Linked Immunosorbent Assay for IgG, and IgA antibodies reacting to recombinant VP6 protein. The seroprevalences exhibit no regular patterns over a 16-yr period. For all four collection periods, the anti-rVP6 IgG levels rose steadily during the first 5 months of life, after which they remained high. However, the 2-9 yr and 10-39 yr groups had significantly higher IgG levels in 1999-2000 and 2004-2005, respectively, than in the other collection periods. The 1-5 mo, 40- > or = 60 yr, and 4-29 yr groups had significantly higher IgA levels in 1989-1990, 1999-2000, and 2004-2005, respectively. The 4 yr (25.0%), 5-9 yr (18.8%), 10-14 yr (41.1%), 20-29 yr (35.0%), and 30-39 yr (20.0%) groups in 2004-2005 had significant higher IgA seropositivity rate compared to the other three collection periods. These observations suggest that in the western region of Gyeongnam Province since the late 1990s, rotavirus reinfection has occurred more frequently than previously, with all ages being at risk.
Adult ; Aged ; Antibodies, Viral/*blood ; Antigens, Viral/genetics/immunology/metabolism ; Capsid Proteins/genetics/immunology/metabolism ; Child ; Child, Preschool ; Enzyme-Linked Immunosorbent Assay ; Female ; Humans ; Immunoglobulin A/blood ; Immunoglobulin G/blood ; Infant ; Infant, Newborn ; Male ; Middle Aged ; Recombinant Proteins/biosynthesis/genetics/immunology ; Republic of Korea/epidemiology ; Rotavirus/isolation & purification/*metabolism ; Rotavirus Infections/*epidemiology/virology ; Seroepidemiologic Studies ; Time Factors ; Young Adult

To construct a recombinant replication-defective human adenovirus type 5 expressing Cap protein of PCV2 and test the immunological efficacy in mice. In this study, the recombinant replication-defective human adenovirus type 5, named as rAd5-Cap (wt-rAd5), was constructed through homologous recombination internally in the HEK293AD cells after co-transfection of the Pac I-linearized backbone plasmid and the shuttle plasmid pacAd5CMV-Cap containing the open reading frame (ORF2) of the porcine circovirus type 2 (PCV2) cap protein or pacAd5CMV without inserted fragment. Furthermore, the rAd5-Cap could induce the expression of PCV2 cap protein in the HEK293AD cells with high efficacy evaluated by the RT-PCR and indirect immunofluorescence assay (IFA). The virus titer of rAd5-Cap could reach up to 10(8.5) TCID50/mL similarly to that of wt-rAd5, indicating that there was little affect on the virus proliferation after the insertion of PCV2 cap protein gene. The humeral immune responses could be activated and detected 14 days after the inoculation of the mice with 10(7) TCID50 rAd5-Cap intramuscularly, and constantly in crease in another 14 days. These molecular biological and animal experiments results demonstrated that the PCV2 cap protein could be efficiently expressed by the recombinant adenovirus rAd5-Cap in eukaryotic cells and induce robust immune responses in mice, which laid a good foundation for the development of new type vaccine against porcine circovirus.
Adenoviruses, Human ; genetics ; Animals ; Antibodies, Viral ; blood ; Capsid Proteins ; genetics ; immunology ; Circovirus ; immunology ; Defective Viruses ; genetics ; HEK293 Cells ; Humans ; Mice ; Recombinant Proteins ; biosynthesis ; immunology ; Virus Replication

To develop a method based on immunoreactions for detection of Ectropis obliqua Nucleopolyhedrovirus (EoNPV), the polyhedra of the virus were purified and used to immunize the mouse BALB/c. The spleen cells from the immunized mice were then fused with the myeloma cell line Sp2/0. A hybridoma cell line which can stably secrete the monoclonal antibody against EoNPV was achieved by using indirect ELISA screening and cloning methods, and was named as 7D3. Meanwhile, the polyhedrin gene was cloned from EoNPV and expressed in E. coli. Western blotting analysis showed that the monoclonal antibody prepared from 7D3 could specifically react with the recombinant polyhedrin. An indirect ELISA method based on this monoclonal antibody for detecting EoNPV in infected tea looper was developed.
Animals ; Antibodies, Monoclonal ; biosynthesis ; genetics ; immunology ; Antibody Specificity ; Cloning, Molecular ; Enzyme-Linked Immunosorbent Assay ; methods ; Escherichia coli ; genetics ; metabolism ; Genetic Vectors ; genetics ; Hybridomas ; secretion ; Lepidoptera ; growth & development ; virology ; Mice ; Mice, Inbred BALB C ; Recombinant Proteins ; biosynthesis ; genetics ; immunology ; Viral Structural Proteins ; biosynthesis ; genetics ; immunology