Rubella virus (RV), one of pathogens in TORCH syndrome, can lead to anisotropy of the fetus, and therefore it is of great significance to screen RV infection among women at child-bearing age. At the present, the screening of RV infection is mainly based on the ELISA method, the specificity of RV antigens is very important for ELISA tests. The antigenicity of RV is closely associated with its structural proteins, including capsid protein C, and envelope glycoproteins E1 and E2, which are important surface antigens of RV. This paper reviews the advances in the studies on the structure features, immunogenicity and recombinant proteins of the three structural proteins.
Antibodies, Viral ; analysis ; Enzyme-Linked Immunosorbent Assay ; Recombinant Proteins ; immunology ; Rubella virus ; genetics ; immunology ; Viral Core Proteins ; genetics ; immunology ; Viral Envelope Proteins ; genetics ; immunology ; Viral Structural 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

The human cytomegalovirus(HCMV) gene encoding the protein reactive with the sera of HCMV-infected patient was cloned and characterized. A reactive phage clone was screened from a lambda gt11 expression library of cDNA of HCMV AD169 strain using HCMV-infected patient sera. The recombinant protein was expressed as 138 kDa-fusion protein with beta-galactosidase, which was reactive with IgM or IgG HCMV antibody-positive sera, but not with anti-HCMV antibody-negative sera. A homology search of the DNA sequence of the cloned gene with HCMV AD169 sequences revealed that it was composed of 709 base pairs spanning between 0.174 and 0.177 map units of the UL32 region of the HCMV AD169 strain genome. This position corresponded to a part of the gene encoding 150 kDa phosphoprotein-(pp150), a major tegument protein, which was reported as an immunogenic protein which evoked strong and longstanding antibody response and had no sequence homology with the proteins of other herpesviruses. These results suggested that pp150 was an immunogenic protein in natural HCMV infection and therefore this clone was regarded as a useful candidate for developing an antigen for the serodiagnosis of HCMV.
Antibodies, Viral/*blood/immunology ; Antigens, Viral/*genetics/immunology ; Cloning, Molecular ; Cytomegalovirus/genetics/*immunology ; Cytomegalovirus Infections/blood/*immunology/virology ; DNA, Complementary/genetics ; DNA, Viral/genetics ; Gene Library ; *Genes, Structural, Viral ; Human ; Recombinant Fusion Proteins/biosynthesis/immunology ; Sequence Homology, Nucleic Acid ; Support, Non-U.S. Gov't ; Viral Matrix Proteins/*genetics/immunology

Six recombinant plasmids co-expressing the wild-type GP5 gene or the codon-optimized GP5 gene (containing pan-DR epitope) of porcine reproductive and respiratory syndrome virus (PRRSV) and the E2 gene of classical swine fever virus (CSFV) or the E2 fused with the UL49 of pseudorabies virus (PrV) were constructed based on the suicidal DNA vaccine pSFV1CS-E2 described previously. Expression of GP5 and E2 was confirmed by indirect immunofluorescence assay. The immunogenicity of six plasmids was evaluated in BALB/c mouse model. For the six plasmids, low-level of E2 and GP5 protein specific antibodies could be detected in the sera of the immunized mice. Specific lymphoproliferative responses to the PRRSV or CSFV stimulation were induced in the splenocytes of the immunized mice as demonstrated by CFSE staining assay and WST-8 assay. Antigen specific IFN-gamma and L-4 secretion was detected in the splenocytes of some immunized mice by cytokine ELSIA. Fusion with the PrV UL49 in the suicidal vaccines induced significantly higher lymphoproliferative responses and cytokine secretion. Taken together, the suicidal DNA vaccines co-expressing GP5 and E2 could induce PRRSV and CSFV specific humoral and cell-mediated immune responses.
Animals ; Antibodies, Viral ; blood ; Antibody Formation ; Cytokines ; blood ; Female ; Immunity, Cellular ; Lymphocytes ; immunology ; Mice ; Mice, Inbred BALB C ; Random Allocation ; Vaccines, DNA ; biosynthesis ; immunology ; Viral Envelope Proteins ; genetics ; immunology ; Viral Structural Proteins ; genetics ; immunology ; Viral Vaccines ; biosynthesis ; immunology

In order to research immunogenicity of the recombinant rVP2-IL-2 fusion protein, we obtained the rVP2-IL-2 fusion protein using Pichia pastoris expression system, and then evaluated its potential to induce immune responses in chicken. The effect was determined in the form of protective anti-IBDV VP2 titers, antibodies (IgG1 and IgG2a), lymphocyte proliferation, the levels of interferon-gamma and interleukin-4 cytokines, and challenge experiment. Antibody titers and proliferation lymphocyte level suggested that the fusion protein could elicit specific humoral immune and cellular immune responses, antibody sub-type results indicated that the rVP2-IL-2 fusion protein induced secretion both of IgG1 and IgG2a. The seem result elicited from cytokines ELISA test, secretion of both of Th1 (gamma-IFN) and Th2 (IL-4) were induced by the rVP2-IL-2 fusion protein. Challenge experiment result shown that chicken immunized the rVP2-IL-2 fusion protein obtained 85% protection. These results confirm that the fusion protein enhances the protection against IBDV through both humoral and cell-mediated immunity, and thus could serve as a candidate for the development of IBDV subunit vaccine.
Animals ; Antibodies, Viral ; biosynthesis ; blood ; Chickens ; immunology ; Immunoglobulin G ; blood ; Interleukin-2 ; biosynthesis ; genetics ; Pichia ; genetics ; metabolism ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; immunology ; Th1 Cells ; immunology ; Th2 Cells ; immunology ; Vaccines, Subunit ; immunology ; Viral Structural Proteins ; biosynthesis ; genetics ; Viral Vaccines ; immunology

The newly emerged Middle East respiratory syndrome coronavirus (MERS-CoV) is a highly pathogenic respiratory virus with pathogenic mechanisms that may be driven by innate immune pathways. The goal of this study is to characterize the expression of the structural (S, E, M, N) and accessory (ORF 3, ORF 4a, ORF 4b, ORF 5) proteins of MERS-CoV and to determine whether any of these proteins acts as an interferon antagonist. Individual structural and accessory protein-coding plasmids with an N-terminal HA tag were constructed and transiently transfected into cells, and their native expression and subcellular localization were assessed using Wes tern blotting and indirect immunofluorescence. While ORF 4b demonstrated majorly nuclear localization, all of the other proteins demonstrated cytoplasmic localization. In addition, for the first time, our experiments revealed that the M, ORF 4a, ORF 4b, and ORF 5 proteins are potent interferon antagonists. Further examination revealed that the ORF 4a protein of MERS-CoV has the most potential to counteract the antiviral effects of IFN via the inhibition of both the interferon production (IFN-β promoter activity, IRF-3/7 and NF-κB activation) and ISRE promoter element signaling pathways. Together, our results provide new insights into the function and pathogenic role of the structural and accessory proteins of MERS-CoV.
Cell Line ; Coronavirus ; genetics ; pathogenicity ; Genes, Viral ; Humans ; Interferons ; antagonists & inhibitors ; Open Reading Frames ; Recombinant Proteins ; genetics ; metabolism ; Viral Matrix Proteins ; genetics ; metabolism ; Viral Regulatory and Accessory Proteins ; genetics ; metabolism ; Viral Structural Proteins ; genetics ; metabolism

Genes encoding nucleocaspid (N) and membrane (M) protein of SARS coronavirus were obtained by RT-PCR and were cloned into expression vector pET22b and pBV222. DNA sequencing showed that the genes cloned from a patient in Beijing were identical to the gene sequences from reported Toronto strain. The genes were over-expressed in E. coli either as inclusion body or as soluble form. The recombinant proteins were purified by ion-exchange, or ion-exchange followed by metal chelate affinity chromatography. The recombinant N protein was demonstrated highly antigenic and could be employed as antigen to detect SARS antibodies in ELISA system for SARS diagnosis.
Chromatography, Affinity ; Chromatography, Ion Exchange ; Enzyme-Linked Immunosorbent Assay ; Escherichia coli ; genetics ; metabolism ; Nucleocapsid Proteins ; genetics ; isolation & purification ; metabolism ; Reverse Transcriptase Polymerase Chain Reaction ; SARS Virus ; genetics ; metabolism ; Viral Structural Proteins ; genetics ; isolation & purification ; metabolism

Based on the infectious clone of JEV vaccine strain SA14-14-2, prM-E genes and C-prM-E genes were cloned into pCDNA3.1 vector. The recombinant plasmid pCJE-ME was transfected into BHK-21 cells, the expressed proteins were toxic to the cell growth and accelerated the cell death. But when transfected with the plasmid pCJE-CME, the cell lines continuously expressing structural proteins could be selected with G418. And the expression products of pCJE-CME vector could be detected by ELISA, Western Blot and IFA assay. It showed that the JEV capsid protein could enhance the stability of the cell lines expressing the structural proteins. The established cell lines can make the acquirement of the virus-like particles much easier.
Animals ; Apoptosis ; CHO Cells ; Capsid Proteins ; genetics ; metabolism ; Cell Line ; cytology ; virology ; Cricetinae ; Cricetulus ; Encephalitis Virus, Japanese ; genetics ; metabolism ; Gene Expression ; Genetic Vectors ; Viral Structural Proteins ; genetics ; metabolism

Active Transport, Cell Nucleus ; Capsid Proteins ; physiology ; Heparan Sulfate Proteoglycans ; physiology ; Humans ; Oncogene Proteins, Viral ; physiology ; Papillomaviridae ; genetics ; physiology ; Papillomavirus Infections ; etiology ; Viral Structural Proteins ; physiology ; Virion ; physiology ; Virus Assembly

To develop a recombinant lentivirus co-expressing structural protein of hepatitis C virus (HCV) and secreted Gaussia Luciferase (Gluc), we first constructed an expression vector that encoded HCV structural protein (C, E1, E2) and GLuc named pCSGluc2aCE1E2. The expression of HCV proteins and Gluc was confirmed by an immunofluorescence assay (IFA) and the detection of luciferase activity. Recombinant lentivirus (VSVpp-HCV) was developed by the co-transfection of pCSGluc2aCE1E2 into 293T cells with pHR'CMVA8.2 and pVSVG. The infectivity of VSVpp-HCV was confirmed by luciferase activity detection, IFA and western blotting. Virus-like particles were identified using electron microscopy after concentration. The results showed that the level of luciferase activity correlated with the expression of HCV protein after the infection of cells with lentivirus VSVpp-HCV. Therefore, the expression level of HCV proteins could be evaluated by detecting the luciferase activity of Gluc. In conclusion, this research pave a way for the development of transgenic mice that express HCV proteins and Gluc, which enable the evaluation of anti-HCV therapy and vaccine in vivo.
Animals ; Copepoda ; Genes, Reporter ; Genetic Vectors ; genetics ; metabolism ; Hepacivirus ; genetics ; metabolism ; Hepatitis C ; virology ; Humans ; Lentivirus ; genetics ; metabolism ; Luciferases ; genetics ; metabolism ; Recombinant Fusion Proteins ; genetics ; metabolism ; Viral Structural Proteins ; genetics ; metabolism