RESUMO
Type 2 porcine circovirus (PCV2) is associated with post-weaning multisystemic wasting syndrome in pigs. In this study, three monoclonal antibodies (mAbs) against the capsid protein (Cap) of PCV2, eight mAbs to Cap of type 1 porcine circovirus (PCV1) and five mAbs specific for Cap of both PCV1 and PCV2, were generated and used to finely map the antigenic sites of PCV1 and PCV2, and to identify the antigenic phenotype of PCV2 with different length of genome. Five linear B-cell epitopes, including the residues 231-233 and 195-202 specific for PCV2, residues 92-103 specific for PCV1, and residues 156-162 and 175-192 shared between PCV1 and PCV2, were finely defined with synthetic peptides, and the critical residue in epitope 231-233 and 156-162 was located at residues 233 ((233)Proline) and 156 ((156)Tyrosine), respectively. The conformational epitopes recognized by mAbs with neutralizing activity against both PCV1 and PCV2 were detected in transfected PK-15 and the residues 231-233 also participated in the formation of conformational epitopes. Analysis of antigenic diversity on these epitopes exhibited three antigenic phenotypes of PCV2, (1766)PCV2, (1767)PCV2 and (1768)PCV2 using mAbs. The results from this study first demonstrated the different antigenic phenotype between PCV2 isolates.
Assuntos
Antígenos Virais/imunologia , Proteínas do Capsídeo/química , Proteínas do Capsídeo/imunologia , Circovirus/química , Circovirus/imunologia , Mapeamento de Epitopos , Epitopos/imunologia , Sequência de Aminoácidos , Animais , Anticorpos Monoclonais/imunologia , Formação de Anticorpos , Antígenos Virais/química , Linhagem Celular , Infecções por Circoviridae/imunologia , Circovirus/isolamento & purificação , Ensaio de Imunoadsorção Enzimática , Epitopos/química , Genoma , Cinética , Camundongos , Dados de Sequência Molecular , Testes de Neutralização , Fenótipo , Conformação Proteica , Sus scrofa , TransfecçãoRESUMO
Porcine circovirus type 2 (PCV2) has been recently associated with a number of disease syndromes, especially postweaning multisystemic wasting disease (PMWS). Herein, an alternative indirect enzyme-linked immunosorbent assay (ELISA) for detection of PCV2 antibody was developed using nuclear localization signal-truncated capsid protein of PCV2 produced in Escherichia coli (CAP ELISA). This assay was validated by comparison with an indirect immunofluorescence assay (IIF) and a PCV2-based ELISA. The diagnostic sensitivity (DSN), specificity (DSP) and accuracy of the CAP ELISA were 95.3%, 93.9% and 95.1%, compared with IIF on 1080 field serum samples, and 93.3%, 84.2% and 91.1%, compared with the PCV2-based ELISA on 79 field sera, respectively. Cross-reactivity assay showed that this assay was PCV2-specific. Repeatability tests revealed that the coefficients of variation of positive sera within and between runs were less than 15%. This ELISA is simpler to produce and perform, time-saving and suitable for large scale surveys of PCV2 infection at low cost and the evaluation of the efficiency of various vaccines against PCV2.
Assuntos
Anticorpos Antivirais/sangue , Proteínas do Capsídeo , Circovirus/imunologia , Ensaio de Imunoadsorção Enzimática/veterinária , Doenças dos Suínos/diagnóstico , Animais , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Suínos , Doenças dos Suínos/sangue , Doenças dos Suínos/imunologiaRESUMO
The nuclear localization signal (NLS)-defected capsid protein gene (dCap) of porcine circovirus type 2 (PCV2) was expressed firstly in Escherichia coli as a fusion protein with glutathione S-transferase (rGST-dCap protein). The purified rGST-dCap protein and NLS-defected Cap protein of PCV2 (rdCap protein) from the purified rGST-dCap protein reacted specifically with swine antiserum to PCV2. Furthermore, the obtained monoclonal antibodies (mAbs) to rdCap protein were shown to bind to PCV2 particles replicated in PK15 cell. MAbs to rdCap protein also revealed the neutralizing ability to PCV2 particles. These results demonstrate that rGST-dCap protein expressed in E. coli is folded correctly or at least partly, and all mAbs to rdCap protein possess the binding epitopes of PCV2 particle whereas mAbs 4C4,3F6 and 2G7 to rdCap protein keep the neutralization epitopes of PCV2 particle, showing a potential of rGST-dCap protein as a vaccine antigen or serodiagnostic reagent.
Assuntos
Proteínas do Capsídeo/imunologia , Circovirus/imunologia , Proteínas Recombinantes de Fusão/imunologia , Animais , Anticorpos Monoclonais/imunologia , Proteínas do Capsídeo/biossíntese , Proteínas do Capsídeo/genética , Linhagem Celular , Circovirus/genética , Mapeamento de Epitopos , Escherichia coli/genética , Glutationa Transferase/genética , Camundongos , Camundongos Endogâmicos BALB C , Sinais de Localização Nuclear , Proteínas Recombinantes de Fusão/biossíntese , SuínosRESUMO
We expressed firstly the Capsid protein gene defecting the nuclear localization signal (NLS) of Porcine circovirus type II (PCV2) in Escherichia coli as a fusion protein with glutathione S-transferase (rGST-dCap protein). The purified rGST-dCap protein and the recombinant NLS-defected Cap protein of PCV2 (rdCap protein) from the purified rGST-dCap protein reacted specifically with swine antiserum to PCV2. Furthermore, the obtained monoclonal antibodies (mAbs) to rdCap protein were shown to bind to PCV2 particles replicated in PK15 cell and capsid protein (Cap protein) of PCV2 expressed in PK15 cells, respectively. mAbs to rdCap protein also revealed the neutralizing ability to PCV2 particles. These results demonstrated that rGST-dCap protein expressed in E. coli was folded correctly or at least partly, and mAbs to rdCap protein possessed the binding epitopes of PCV2 particles whereas mAbs 4C4 and 3F6 to rdCap protein remained the neutralization epitope of PCV2 particle, showing a possibility of neutralizing mAb to rdCap protein as an immnuotherapeutic agent and a potential of rGST-dCap protein as a vaccine antigen or serodiagnostic reagent.
Assuntos
Proteínas do Capsídeo/biossíntese , Circovirus/genética , Sinais de Localização Nuclear/genética , Proteínas Recombinantes de Fusão/biossíntese , Animais , Anticorpos Monoclonais/imunologia , Anticorpos Antivirais/imunologia , Antígenos Virais/imunologia , Proteínas do Capsídeo/genética , Proteínas do Capsídeo/imunologia , Linhagem Celular , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/imunologia , Suínos , Vacinas Virais/genética , Vacinas Virais/imunologiaRESUMO
To seek a new delivery system of vaccine for infectious bronchitis virus (IBV), transgenic potato expressing full-length spike (S) protein of IBV was produced and its immunogenicity in chickens was investigated. One to three copies of S gene of IBV were randomly and stably inserted into potato (Solanum tuberrosum cv. Dongnong 303) genome by Agrobacterium tumefaciens-mediated transformation. Transcription and translation of S gene for IBV were confirmed by Northern blot and Western blot analyses in transgenic plantlets. Chickens immunized orally and intramuscularly with transgenic potato tubers expressing S protein generated the detectable levels of serum neutralizing antibodies and were protected against the challenge with the virulent IBV. In vitro secretion of interleukin 2 and T lymphocyte proliferation of spleen cells from the immunized chickens varied with the dose and the manner of vaccination with S protein derived from transgenic plants. The results indicated that S protein expressed in transgenic plants might be a new source for the production of Coronaviridae IBV vaccine.
Assuntos
Vírus da Bronquite Infecciosa/genética , Vírus da Bronquite Infecciosa/metabolismo , Glicoproteínas de Membrana/biossíntese , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/imunologia , Solanum tuberosum/genética , Solanum tuberosum/metabolismo , Proteínas do Envelope Viral/biossíntese , Proteínas do Envelope Viral/genética , Proteínas do Envelope Viral/imunologia , Animais , Galinhas , Clonagem Molecular , Glicoproteínas de Membrana/química , Plantas Geneticamente Modificadas/metabolismo , Engenharia de Proteínas/métodos , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/imunologia , Glicoproteína da Espícula de Coronavírus , Proteínas do Envelope Viral/químicaRESUMO
The expression of infectious bronchitis virus (IBV) S1 glycoprotein in potatoes and its immunogenicity in mice and chickens were investigated. Potato plants were genetically transformed with a cDNA construct encoding the IBV S1 glycoprotein with the Agrobacterium system. Genomic DNA and mRNA analyses of the transformed plantlets confirmed the integration of the foreign cDNA into the potato genome, as well as its transcription. Mice and chickens vaccinated with the expressed IBV S1 glycoprotein produced antibodies that neutralized IBV infectivity. After three immunizations, vaccinated chickens were completely protected from virulent IBV infection. These results demonstrate that transgenic potatoes expressing IBV S1 glycoprotein can be used as a source of recombinant antigen for vaccine production.
