RESUMO
Virus-like particles composed of the core antigen of hepatitis B virus (HBcAg) have been shown to be an effective platform for the display of foreign epitopes in vaccine development. Heterologous sequences have been successfully inserted at both amino and carboxy termini as well as internally at the major immunodominant epitope. We used cryogenic electron microscopy (CryoEM) and three-dimensional image reconstruction to investigate the structure of VLPs assembled from an N-terminal extended HBcAg that contained a polyhistidine tag. The insert was seen to form a trimeric spike on the capsid surface that was poorly resolved, most likely owing to it being flexible. We hypothesise that the capacity of N-terminal inserts to form trimers may have application in the development of multivalent vaccines to trimeric antigens. Our analysis also highlights the value of tools for local resolution assessment in studies of partially disordered macromolecular assemblies by cryoEM.
Assuntos
Vírus da Hepatite B/ultraestrutura , Proteínas do Core Viral/ultraestrutura , Vírion/ultraestrutura , Microscopia Crioeletrônica , Modelos Moleculares , Estrutura Quaternária de Proteína , Estrutura Secundária de ProteínaRESUMO
The core antigen of the hepatitis B virus (HBcAg) has been used widely as a diagnostic reagent for the identification of the viral infection. However, purification using the conventional sucrose density gradient ultracentrifugation is time consuming and costly. To overcome this, HBcAg particles displaying His-tag on their surface were constructed and produced in Escherichia coli. The recombinant His-tagged HBcAgs were purified using immobilized metal affinity chromatography. Transmission electron microscopy and enzyme-linked immunosorbent assay (ELISA) revealed that the displayed His-tag did not impair the formation of the core particles and the antigenicity of HBcAg.
Assuntos
Antígenos do Núcleo do Vírus da Hepatite B/imunologia , Antígenos do Núcleo do Vírus da Hepatite B/isolamento & purificação , Cromatografia de Afinidade , Escherichia coli/genética , Expressão Gênica , Antígenos do Núcleo do Vírus da Hepatite B/genética , Microscopia Eletrônica de Transmissão , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/imunologia , Proteínas Recombinantes de Fusão/isolamento & purificação , Virossomos/ultraestruturaRESUMO
The nucleocapsid protein of Nipah virus produced in Escherichia coli assembled into herringbone-like particles. The amino- and carboxy-termini of the N protein were shortened progressively to define the minimum contiguous sequence involved in capsid assembly. The first 29 aa residues of the N protein are dispensable for capsid formation. The 128 carboxy-terminal residues do not play a role in the assembly of the herringbone-like particles. A region with amino acid residues 30-32 plays a crucial role in the formation of the capsid particle. Deletion of any of the four conserved hydrophobic regions in the N protein impaired capsid formation. Replacement of the central conserved regions with the respective sequences from the Newcastle disease virus restored capsid formation.
Assuntos
Capsídeo/fisiologia , Vírus Nipah/genética , Proteínas do Nucleocapsídeo/genética , Sequência de Aminoácidos , Proteínas do Capsídeo/genética , Primers do DNA , Escherichia coli/virologia , Dados de Sequência Molecular , Mutagênese , Vírus Nipah/ultraestrutura , Proteínas do Nucleocapsídeo/ultraestrutura , Deleção de SequênciaRESUMO
The nucleocapsid (N) protein of Nipah virus (NiV) is a major constituent of the viral proteins which play a role in encapsidation, regulating the transcription and replication of the viral genome. To investigate the use of a fusion system to aid the purification of the recombinant N protein for structural studies and potential use as a diagnostic reagent, the NiV N gene was cloned into the pFastBacHT vector and the His-tagged fusion protein was expressed in Sf9 insect cells by recombinant baculovirus. Western blot analysis of the recombinant fusion protein with anti-NiV antibodies produced a band of approximately 62 kDa. A time course study showed that the highest level of expression was achieved after 3 days of incubation. Electron microscopic analysis of the NiV recombinant N fusion protein purified on a nickel-nitrilotriacetic acid resin column revealed different types of structures, including spherical, ring-like, and herringbone-like particles. The light-scattering measurements of the recombinant N protein also confirmed the polydispersity of the sample with hyrdrodynamic radii of small and large types. The optical density spectra of the purified recombinant fusion protein revealed a high A(260)/A(280) ratio, indicating the presence of nucleic acids. Western blotting and enzyme-linked immunosorbent assay results showed that the recombinant N protein exhibited the antigenic sites and conformation necessary for specific antigen-antibody recognition.
Assuntos
Vírus Nipah/metabolismo , Proteínas do Nucleocapsídeo , Proteínas Recombinantes de Fusão , Animais , Anticorpos Antivirais/imunologia , Baculoviridae/genética , Baculoviridae/metabolismo , Células Cultivadas , Eletroforese em Gel de Poliacrilamida , Ensaio de Imunoadsorção Enzimática , Infecções por Henipavirus/diagnóstico , Infecções por Henipavirus/veterinária , Infecções por Henipavirus/virologia , Microscopia Eletrônica , Vírus Nipah/genética , Vírus Nipah/imunologia , Proteínas do Nucleocapsídeo/química , Proteínas do Nucleocapsídeo/imunologia , Proteínas do Nucleocapsídeo/isolamento & purificação , Proteínas do Nucleocapsídeo/metabolismo , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/imunologia , Proteínas Recombinantes de Fusão/isolamento & purificação , Proteínas Recombinantes de Fusão/metabolismo , Spodoptera/virologia , Doenças dos Suínos/diagnóstico , Doenças dos Suínos/virologiaRESUMO
The nucleocapsid (N) protein of Nipah virus (NiV) can be produced in three Escherichia coli strains [TOP10, BL21(DE3) and SG935] under the control of trc promoter. However, most of the product existed in the form of insoluble inclusion bodies. There was no improvement in the solubility of the product when this protein was placed under the control of T7 promoter. However, the solubility of the N protein was significantly improved by lowering the growth temperature of E. coli BL21(DE3) cell cultures. Solubility analysis of N- and C-terminally deleted mutants revealed that the full-length N protein has the highest solubility. The soluble N protein could be purified efficiently by sucrose gradient centrifugation and nickel affinity chromatography. Electron microscopic analysis of the purified product revealed that the N protein assembled into herringbone-like particles of different lengths. The C-terminal end of the N protein contains the major antigenic region when probed with antisera from humans and pigs infected naturally.
Assuntos
Vírus Nipah/química , Vírus Nipah/imunologia , Proteínas do Nucleocapsídeo/química , Proteínas do Nucleocapsídeo/imunologia , Animais , Antígenos Virais , Sequência de Bases , DNA Viral/genética , Escherichia coli/genética , Infecções por Henipavirus/imunologia , Infecções por Henipavirus/virologia , Humanos , Microscopia Eletrônica , Vírus Nipah/genética , Proteínas do Nucleocapsídeo/genética , Coelhos , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Deleção de Sequência , SolubilidadeRESUMO
The coding region of the nucleocapsid (N) gene was amplified from the viral RNA and inserted into the bacterial expression vector, pTrcHis2, for intracellular expression in three Escherichia coli strains: TOP 10, BL 21 and SG 935. The N protein was expressed as a fusion protein containing the myc epitope and His-tag at its C-terminal end. The amount of the fusion protein expressed in strain SG 935 was significantly higher than the other two strains, and was detected by the anti-myc antibody, anti-His and swine anti-NiV serum. Hence, the N(fus) protein produced in E. coli could serve as an alternative antigen for the detection of anti-NiV in swine.
