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

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

Protective immune response of the available IBD vaccine is insufficient to fully protect against the prevailing strain of the infectious bursal disease virus (IBDV). Such a vaccination escape IBDV field isolate idenfied from Anhui province of China in December 2007, where IBD broke out at 2 weeks post vaccination. The IBDV vp2 gene was cloned into pFastBacHTA donor plasmid, followed by generation of the recombinant bacmid DNA pBac-VP2. The latter was used to transfect insect cell Sf9 with Lipofectamine to produce recombinant baculovirus vBac-VP2. The Sf9 cells infected with vBac-VP2 were stained positive against IBDV antibody using the indirect immunofluorescence assay (IFA), which was also confirmed by the detection of IBDV Vp2 protein in the infected Sf9 cells by IBDV sandwich ELISA. Western blotting revealed that the calculated protein of approximately 53 kDa was in the expressed in the insect cells. Moreover, virus-like particles (VLPs) and "inclusion body-like"structure in the infected Sf9 cells were observed under electron microscopy. We further developed an indirect ELISA for the detection of the IBDV antibodies, which was specific and sensitive. In addition, the lysates of vBac-VP2 infected cells was used to immunize 2-week-old SPF chickens, followed by challenging with the virulent IBDV, the survival rate was 30% at 14 days post primary immunization, however, the survival rate was 100% at 14 d after the booster vaccination. The ELISA antibody titers was up to 3.2 x 10(3) and neutralization antibody titer was 2536, significantly higher than those of one-shot vaccination, 8 x 10(2) and 1106, respectively. The immunized chickens did not show any clinical signs and histopathological changes of infection in 7-days trial time. The bursa/body-weight ratios were higher than those of the unimmunized control (P < 0.05). The virus-like-particle recombinant Vp2 protein expressed in insect cells promises to be a novel subunit vaccine and diagnostic reagent candidate for IBDV.
Animals ; Baculoviridae ; genetics ; Cell Line ; Chickens ; Infectious bursal disease virus ; immunology ; Insecta ; genetics ; metabolism ; Poultry Diseases ; prevention & control ; Recombinant Proteins ; biosynthesis ; genetics ; immunology ; Viral Structural Proteins ; biosynthesis ; genetics ; Viral Vaccines ; 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

A transfer plasmid vector pUC18-US10-VP2 was first constructed by inserting the gene of the enhancer green fluorescent protein(eGFP) fused to the VP2 gene of very virulent Infectious bursal disease virus (IBDV) JS strain into the US10 fragment of the Marek's disease virus (MDV) CV1988/Rispens. The recombinant virus, designated as rMDV, was developed by co-transfecting CEF with the transfer plasmid vector and simultaneously infecting with the CVI988/Rispens virus. The PCR and IFA results indicated that the rMDV is stable after 31 passages. Chickens vaccinated with rMDV were protected from challenge with 100LD50 of IBDV JS. The protection ratio of the chickens vaccinated with the 1000PFU, 2000PFU, 5000PFU of the rMDV were 50%, 60%, and 80% respectively. It is interesting that the average histopathology BF lesion scores of chicken group immunized with 5000PFU of rMDV by one-time vaccination was close to that of chicken group vaccinated with IBDV live vaccine NF8 strain for twice (2.0/1.5). There is no difference in protection between the groups (P > 0.05) but significent difference between groups immunized with 5000 PFU of rMDV and with normal MDV. This demonstrated that rMDV expressing VP2 fusion protein was effective vaccine against IBDV in SPF chickens.
Animals ; Birnaviridae Infections ; prevention & control ; veterinary ; Chickens ; Genetic Vectors ; Green Fluorescent Proteins ; genetics ; Infectious bursal disease virus ; genetics ; immunology ; Mardivirus ; genetics ; metabolism ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; immunology ; Recombination, Genetic ; Transfection ; Vaccination ; Vaccines, DNA ; genetics ; immunology ; Viral Structural Proteins ; biosynthesis ; genetics ; immunology ; Viral Vaccines ; genetics ; immunology

The ability of a heat-inactivated whole virus from a highly virulent infectious bursal disease virus (hvIBDV) and VP2 protein from hvIBDV expressed in E. coli provided protection against a hvIBDV challenge in specificpathogen- free (SPF) chickens. Six out of seven chickens that were injected three times with crude VP2 protein developed significant antibody titer against IBDV. However, only four out of the seven chickens survived the hvIBDV challenge. Despite showing low antibody titer profiles, all chickens immunized with the heat-inactivated whole virus also survived the challenged with hvIBDV. However, all of these chickens had bursal atrophy and mild to moderate depletion of lymphocytes. Thus, antibodies raised against IBDV VP2 protein expressed in E. coli and denatured IBDV proteins induced some degree of protection against mortality but not against bursal damage following challenge with hvIBDV.
Animals ; Antibodies, Viral/blood ; Birnaviridae Infections/immunology/prevention & control/*veterinary/virology ; Chickens ; Enzyme-Linked Immunosorbent Assay/veterinary ; Escherichia coli/genetics ; Immunization/standards/*veterinary ; Infectious bursal disease virus/genetics/*immunology/pathogenicity ; Poultry Diseases/*immunology/prevention&control/virology ; Recombinant Proteins/genetics/*immunology ; Specific Pathogen-Free Organisms ; Vaccines, Attenuated/immunology/pharmacology ; Vaccines, Synthetic/immunology/pharmacology ; Viral Structural Proteins/biosynthesis/genetics/*immunology ; Viral Vaccines/*immunology/pharmacology

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