RESUMEN
Pseudorabies virus (PRV) is an alphaherpesvirus that causes pseudorabies (PR), an economically important viral disease of pigs. Marker vaccines were widely used in PR prevention and eradication programs. The purpose of this study was to construct a novel recombinant virus with deletions at defined regions in the glycoprotein E (gE) and thymine kinase (TK) genes by homologous recombination. This study also evaluated the safety and efficacy of the virus for a live attenuated marker vaccine. No significant difference was observed in virus replication between gE gene-deleted (gE(-)), gE/TK double gene-deleted (gE(-)TK(-)), and wild-type PRV by growth curve analysis. However, gE(-)TK(-) PRV was completely attenuated in mice. To evaluate the immunogenicity of gE(-)TK(-) PRV, four 12-week-old specific-pathogen-free pigs per group were immunized intramuscularly with viral titers of 1×10(4), 1×10(5), or 1×10(6) TCID50, followed by intranasal challenge infection with virulent PRV (1×10(8) TCID50) at 3 weeks post vaccination. The gE(-)TK(-) PRV-vaccinated pigs displayed no general adverse effects after immunization and had protective immune responses after PRV challenge. Thus, gE(-)TK(-) PRV was safe and efficacious and might be a potential candidate for a live attenuated marker vaccine against PRV.
Asunto(s)
Herpesvirus Suido 1 , Seudorrabia/virología , Timidina Quinasa/inmunología , Vacunas de ADN , Proteínas del Envoltorio Viral/inmunología , Animales , Anticuerpos Antivirales/sangre , ADN Recombinante/genética , Femenino , Herpesvirus Suido 1/genética , Herpesvirus Suido 1/inmunología , Ratones , Ratones Endogámicos BALB C , Seudorrabia/prevención & control , Porcinos , Timidina Quinasa/genética , Timidina Quinasa/metabolismo , Vacunas Atenuadas/genética , Vacunas Atenuadas/inmunología , Vacunas de ADN/genética , Vacunas de ADN/inmunología , Proteínas del Envoltorio Viral/genética , Proteínas del Envoltorio Viral/metabolismoRESUMEN
Porcine circovirus type 2 (PCV2) capsid (Cap) protein has been successfully used as a vaccine to control porcine circovirus associated disease (PCVAD). Most PCV2 subunit vaccines are recombinant Cap protein expressed in baculovirus/insect cell expression system, but using this eukaryotic system is laborious and expensive. In our previous study, full-length of PCV2Cap protein expressed in Escherichia coli formed virus-like particles (VLPs). This expression system has the advantages of being relatively simple and inexpensive. In this study, we constructed a recombinant plasmid containing the full-length codon-optimized cap (ORF2) gene to improve high-level expression of recombinant Cap protein (rCap) with no changed amino acids. The highly water-soluble rCap protein was purified by a single-column, high-throughput fractionation procedure based on size exclusion chromatography. Yield was 10mg per 200ml bacterial culture. The rCap protein self-assembled into VLPs of diameter 25-30nm that contained exogenous nucleic acids. The immunogenicity of PCV2 VLPs was analyzed by immunizing mice. VLP-immunized mice mounted specific immune responses to PCV2. Thus, expression of rCap in E. coli was feasible for large-scale production of PCV2 VLPs, which could potentially be used for a VLP-based PCV2 vaccine.
Asunto(s)
Infecciones por Circoviridae/virología , Circovirus/genética , Escherichia coli/química , Escherichia coli/metabolismo , Vacunas de Partículas Similares a Virus/biosíntesis , Vacunas de Partículas Similares a Virus/genética , Animales , Anticuerpos Antivirales/sangre , Anticuerpos Antivirales/inmunología , Formación de Anticuerpos , Western Blotting/métodos , Proteínas de la Cápside/biosíntesis , Proteínas de la Cápside/genética , Proteínas de la Cápside/inmunología , Proteínas de la Cápside/aislamiento & purificación , Cromatografía en Gel/métodos , Infecciones por Circoviridae/genética , Infecciones por Circoviridae/inmunología , Circovirus/inmunología , Circovirus/metabolismo , Clonación Molecular , Electroforesis en Gel de Poliacrilamida/métodos , Ensayo de Inmunoadsorción Enzimática , Ensayo de Immunospot Ligado a Enzimas/métodos , Escherichia coli/genética , Femenino , Ratones , Ratones Endogámicos BALB C , Microscopía Electrónica de Transmisión , Datos de Secuencia Molecular , Pruebas de Neutralización , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/genética , Proteínas Recombinantes/inmunología , Proteínas Recombinantes/aislamiento & purificación , Alineación de Secuencia , Porcinos , Enfermedades de los Porcinos/inmunología , Enfermedades de los Porcinos/prevención & control , Enfermedades de los Porcinos/virología , Vacunas de Subunidad , Vacunas de Partículas Similares a Virus/inmunología , Virión/genéticaRESUMEN
Porcine circovirus type 2 (PCV2) is the primary causative agent of porcine circovirus-associated diseases in pigs. The sole structural capsid protein of PCV2, Cap, consists of major antigenic domains, but little is known about the assembly of capsid particles. The purpose of this study is to produce a large amount of Cap protein using Escherichia coli expression system for further studying the essential sequences contributing to formation of particles. By using codon optimization of rare arginine codons near the 5'-end of the cap gene for E. coli, a full-length Cap without any fusion tag recombinant protein (Cap1-233) was expressed and proceeded to form virus-like particles (VLPs) in normal Cap appearance that resembled the authentic PCV2 capsid. The N-terminal deletion mutant (Cap51-233) deleted the nuclear localization signal (NLS) domain, while the internal deletion mutant (CapΔ51-103) deleted a likely dimerization domain that failed to form VLPs. The unique Cys108 substitution mutant (CapC/S) exhibited most irregular aggregates, and only few VLPs were formed. These results suggest that the N-terminal region within the residues 1 to 103 possessing the NLS and dimerization domains are essential for self-assembly of stable Cap VLPs, and the unique Cys108 plays an important role in the integrity of VLPs. The immunogenicity of PCV2 VLPs was further evaluated by immunization of pigs followed by challenge infection. The Cap1-233-immunized pigs demonstrated specific antibody immune responses and are prevented from PCV2 challenge, thus implying its potential use for a VLP-based PCV2 vaccine.
