Antigens, Viral ; genetics ; Humans ; Mutation ; Orthomyxoviridae ; classification ; genetics ; immunology

E2 gene of classical swine fever virus (CSFV) was cloned into secretory pPIC9K Pichia pastoris expression vector. After being linearized by digestion, the vector was transformed into Pichia pastoris by electroporation to integrate with the genome, the transformants with high copies were screened by G418 and were induced to express with methonal. The results of SDS-PAGE and Western blot demonstrated that the supernatant of the induced P. pastoris culture contained protein E2. The results of the study on the immunological activity indicated that the protein E2 expressed in P. pastoris can elicit animal bodies to produce antibodies against protein E2.
Animals ; Antibodies, Viral ; immunology ; Antigens, Viral ; genetics ; immunology ; Classical swine fever virus ; genetics ; immunology ; Cloning, Molecular ; Gene Expression ; Pichia ; Rabbits ; Swine ; Viral Envelope Proteins ; genetics ; immunology

BACKGROUNDTo determine the antigenicity of HGV NS5 recombinant proteins expressed in E.coli.

METHODSHGV NS5a,NS5b and core/NS5b fusion genes were cloned into pThioC vector. Three expression plasmids were transformed into JM109(DE3) competent cells then expressed with induction by IPTG. Western blot and ELISA were used to determine the antigenicity after the three recombinant proteins were purified.

RESULTSAfter identification by restriction enzyme and sequencing, it was confirmed that the expressed was target proteins espected. Purified expression proteins were found strongly immunoreactive among anti HGV positive sera by Western blot and ELISA. Compared with mixed recombinant antigen (including core, NS5a synthetic peptide and NS3 recombinant proteins), in the 22 positive sera detected with mixed antigen, 68%(15/22), 90%(20/22) and 73%(16/22) were positive by P5a,P5b and Pc?5b antigens; In the 70 negative samples with mixed antigen, 7%(5/70), 1%(1/70) and 6%(4/70) were positive by P5a, P5b and Pc?5b antigens. The positive alone was found among RTPCR positive specimen using these recombinant antigens.

CONCLUSIONSNS5 gene expressed in E.coli?which couldn't be covered with other regions of antigens was one of the essential epitopes to HGV immunologic diagnosis.

Antibodies, Viral ; blood ; Antigens, Viral ; blood ; Epitopes ; immunology ; GB virus C ; genetics ; immunology ; Humans ; Plasmids ; genetics ; Recombinant Proteins ; biosynthesis ; immunology ; Viral Nonstructural Proteins ; genetics ; immunology

This article aimed to study the antigenicity of nucleocapsid proteins (NPs) in six pathogenic phleboviruses and to provide theoretical evidence for the development of serological diagnostic reagents. NPs of six pathogenic phleboviruses were expressed and purified using a prokaryotic expression system and rabbits were immunized with individual recombinant NPs. Cross-reactions among NPs and rabbit sera were determined by both indirect ELISA and Western blotting analyses, and the sera titer was determined by indirect ELISA. Furthermore, sera from SFTS patients were also detected by each recombinant NP as a coating antigen using indirect ELISA. The cross-reactions and the sera titer were subsequently determined. Both the concentration and purity of recombinant NPs of six pathogenic phleboviruses met the standards for immunization and detection. The results of indirect ELISA and Western blotting showed that each anti-phlebovirus NP rabbit immune serum had potential serological cross-reactivity with the other five virus NP antigens. Furthermore, the sera from SFTS patients also had cross-reactivity with the other five NP antigens to a certain extent. Our preliminary study evaluated the antigenicity and immune reactivity of six pathogenic phleboviruses NPs and laid the foundation for the development of diagnostic reagents.
Animals ; Antibodies, Viral ; immunology ; Antigens, Viral ; genetics ; immunology ; Cross Reactions ; Humans ; Nucleocapsid Proteins ; genetics ; immunology ; Phlebotomus Fever ; diagnosis ; immunology ; virology ; Phlebovirus ; classification ; genetics ; immunology ; isolation & purification ; Rabbits

To understand the effectiveness of prokaryotic expression of fusion protein (F) of human metapneumovirus (hMPV) and its application as antigen, F proteins from different genotypes of hMPV were expressed in prokaryotic expression system and purified by Ni-NTA affinity chromatography column. According to the hydrophobicity, antigen index and surface probability of F protein, the subunit 1 (F1) region of F protein was generated and expressed in E. Coil. BL21(DE3). The 6-His-F1 proteins with molecular weight of approximately 37 kD generated from hMPV of two genotypes were expressed efficiently mainly in inclusion body. The antigenicity and specificity of the expressed proteins were tested and confirmed by Western Blot using polyclonal antibody against hMPV and one serum specimen from a patient with confirmed hMPV acute infection,and polyclonal antibodies against human respiratory syncytial virus and parainfluenza virus 2 and 3. The results of preliminary use of the expressed proteins for detecting antibodies against hMPV in 457 serum specimens collected from different age groups in Beijing indicated that 66%-67% of sera in all age groups were positive. The positive rate of antibodies declined in children in age groups from birth to 2-year-old and then rose along with the increase in age, in which the lowest was in age group from 1 to 2-year-old and the highest in newborn and people older than 60 years. The data indicated the existence of maternal transferred antibodies against hMPV in infants and the risk of hMPV infections in children younger than 2 years old.
Adult ; Antibodies, Viral ; immunology ; Antigens, Viral ; genetics ; immunology ; Escherichia coli ; genetics ; Gene Expression ; Humans ; Immunoglobulin G ; blood ; immunology ; Infant ; Infant, Newborn ; Metapneumovirus ; genetics ; Middle Aged ; Plasmids ; genetics ; Protein Engineering ; Protein Subunits ; genetics ; immunology ; Viral Fusion Proteins ; genetics ; immunology

To enhance the immuogenicity of DNA vaccines expressing the GP5 protein of Porcine Reproductive and Respiratory Syndrome Virus (PRRSV), the tegument protein VP22 (encoded by VP22 gene) of Bovine Herpesvirus 1 (BHV-1), which has been demonstrated to exhibit the unusual protein transduction property, was fused to N-terminus of GP5 of DNA vaccine construct pCI-ORF5M, resulting in pCI-VP22-ORF5M expressing VP22-GP5 fusion protein. The expression of VP22-GP5 fusion protein was confirmed by both indirect immunofluorescence assay (IFA) and Western blot. To investigate its immunogenicity, BALB/c mice were immunized with the fusion expression plasmid pCI-VP22-ORF5M and non-fusion expression plasmid pCI-ORF5M, respectively. The GP5-specific ELISA antibodies, neutralizing antibodies and lymphocyte proliferative responses were evaluated at various time points after primary immunization. The results showed that GP5-specific ELISA antibodies, neutralizing antibodies, and lymphocyte proliferative responses induced by DNA vaccine pCI-VP22-ORF5M were higher significantly than those of DNA vaccine pCI-ORF5M, indicating that fusion expression with BHV-1 VP22 significantly enhances the immuogenicity of DNA vaccine expressing the PRRSV GP5 protein, and that this strategy may also be useful to develop more efficient DNA vaccines against other pathogens.
Animals ; Antigens, Viral ; genetics ; immunology ; Artificial Gene Fusion ; Female ; Mice ; Mice, Inbred BALB C ; Porcine Reproductive and Respiratory Syndrome ; prevention & control ; Random Allocation ; Vaccines, DNA ; genetics ; immunology ; Viral Envelope Proteins ; genetics ; immunology ; Viral Structural Proteins ; genetics ; Viral Vaccines ; genetics ; immunology

Taking the genome DNA of Infectious Bovine Rhinotracheitis Virus (IBRV) as the template, the gG gene was amplified with PCR and cloned into the T cloning vector pMD18-T. After being identified by restriction digestion and DNA sequencing, the insert was subcloned into the expression vector pGEX-KG. Sodium docecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot assay showed that this gene was expressed as both soluble form and inclusion body by the transformed E. coli BL21 strain (DE3). The fusion protein was purified and used as the coating antigen to develop the indirect Enzyme-Linked Immunosorbent Assay (ELISA). Comparison between this gG-ELISA and commercial IBRV gB-ELISA Kit (IDEXX) was made in the detection of 380 cow serum samples. The results demonstrated an agreement of 92%. By using this novel gG-ELISA, 1248 cow serum samples were tested and the average positive rate of IBRV antibodies for imported cows is 21.7%, while the positive rate ranged greatly from 0.0%-41.5% for Hubei local Chinese Black and White Dairy Cows.
Animals ; Antibodies, Viral ; blood ; immunology ; Antigens, Viral ; genetics ; immunology ; Cattle ; Cloning, Molecular ; Enzyme-Linked Immunosorbent Assay ; methods ; Escherichia coli ; genetics ; metabolism ; Female ; Herpesvirus 1, Bovine ; genetics ; immunology ; Male ; Recombinant Proteins ; biosynthesis ; genetics ; immunology ; Sensitivity and Specificity ; Viral Proteins ; biosynthesis ; genetics ; immunology

Monovalent antisera of 3 vaccine strains and 7 representative field isolates were prepared based on the comparison of genetic diversity of the hypervariable region I of S1 gene (HVR I from 3 infectious bronchitis (IB) vaccine strains (H120, Ma5 and 4/91) ,one reference strain M41 and 26 IB field isolates. These 30 strains were classified in 7 different genotypes, respectively. Virus-neutralizing test on tracheal organ cultures (TOC) with chicken embryo were used to evaluate relatedness values of the antigenicity based on the antibody titer, to analyze the antigenic relationships between the isolates and vaccine strains, as well as to determine the serotypes of 26 IB viruses isolated from the field in Guangxi between 1985 and 2008. The results showed 30 strains were classified into 7 distinct serotypes and there were two predominant serotypes within the 26 isolates, serotypes 1 (totally 13 isolates) and serotype 2 (totally 5 isolates), respectively. In addition, there were some differences observed between the results of serotyping and the genotyping (including the S1, N, M and 3'UTR). The results of the study demonstrated that there were different predominant serotypes and multiple serotypes of IBV circulated in Guangxi in recent years, antigenic variation existed between Guangxi field isolates and vaccine strains.
Animals ; Antibodies, Viral ; immunology ; Antigens, Viral ; genetics ; immunology ; Chick Embryo ; Chickens ; China ; Coronavirus Infections ; immunology ; veterinary ; virology ; Genetic Variation ; Genotype ; Infectious bronchitis virus ; classification ; genetics ; immunology ; isolation & purification ; Phylogeny ; Poultry Diseases ; immunology ; virology ; Viral Envelope Proteins ; genetics ; immunology

OBJECTIVETo construct a replication-defective recombinant adenovirus expressing the ORF2 (112-660aa) antigen of hepatitis E virus (HEV) and evaluate its immunization effect in BALB/c mice by mucosal inoculation.

METHODSThe HEV ORF2 gene encoding for 112-660aa was amplified from plasmid pUC-HEV and inserted into the transfer vector pTrack-CMV. The recombinant plasmid and adenoviral backbone plasmid pAdEasy-1 were co-transformed into E. coli strain BJ5183. Taking the advantage of the high efficient homologous recombination machinery presented in bacteria, the recombinant adenovirus backbone plasmid was generated in BJ5183, and then was transfected into 293 cells. Recombinant Adenoviruses were propagated in 293 cells with high titers. 8-week-old BALB/c mice were inoculated intraperitoneally and intranasally with 10(7) pfu recombinant adenovirus each on weeks 0, 3, 5, 7, 10.

RESULTSBoth groups of mice induced humoral IgG immune response with the highest titers 1:1,000 and 1:10,000 each. Only the group inoculated intranasally could induce mucosal IgA immune response.

CONCLUSIONSThe adenoviral recombinant can stimulate specific humoral and mucosal immune response in mice and is potentially to be used as a candidate vaccine for the treatment of HEV infection.

Adenoviruses, Human ; genetics ; Animals ; Hepatitis Antigens ; genetics ; immunology ; Immunoglobulin A ; immunology ; Immunoglobulin G ; immunology ; Male ; Mice ; Mice, Inbred BALB C ; Nasal Mucosa ; immunology ; Peritoneum ; immunology ; Recombinant Proteins ; biosynthesis ; genetics ; immunology ; Viral Hepatitis Vaccines ; Viral Proteins ; biosynthesis ; genetics ; immunology

Human cytomegalovirus (HCMV) gB is known to play important roles in cell surface attachment, virion penetration, spread of infection from cell to cell, and provocation of neutralizing antibody. This study was performed to determine the role of anti-HCMV gB antibody in overall neutralizing response in patients with HCMV infection and healthy control with past infection. HCMV gB was stably expressed in 293 cells. With the stable cell line expressing gB as a specific immunosorbent, anti-gB antibody was removed from the current and past HCMV-infected sera and the remaining neutralizing activity was measured by plaque assay. It was shown that 19-50% of the total virus-neutralizing activity of sera with past HCMV infections was derived from anti-gB antibody, but anti-gB antibody had little effect on the total serum virus-neutralizing activity in patients currently infected with HCMV. This result suggests that neutralizing antibody to HCMV gB may reflect disease status.
Adult ; Antibodies, Monoclonal ; Antibodies, Viral/immunology* ; Antibodies, Viral/blood ; Antigens, Viral/immunology ; Antigens, Viral/genetics ; Cells, Cultured ; Cytomegalovirus/immunology* ; Cytomegalovirus Infections/prevention & control ; Cytomegalovirus Infections/immunology* ; Female ; Fetus/cytology ; Fibroblasts/cytology ; Gene Expression Regulation, Viral/immunology ; Human ; Immunosorbents ; Lung/cytology ; Male ; Middle Age ; Neutralization Tests ; Recombinant Proteins/genetics ; Viral Envelope Proteins/immunology* ; Viral Vaccines