Structural characterization by transmission electron microscopy and immunoreactivity of recombinant Hendra virus nucleocapsid protein expressed and purified from Escherichia coli

Autor: William J. McKinstry, Lynne J. Waddington, Jennifer A. Barr, Judith A. Scoble, Meng Yu, Gary Crameri, Lesley A. Pearce
Rok vydání: 2015
Předmět:
Swine
viruses
Gene Expression
SDS–PAGE
sodium dodecyl sulfate polyacrylamide gel electrophoresis
medicine.disease_cause
Antibodies
Viral
law.invention
IMAC
immobilized metal affinity chromatography
law
TBS
TRIS-buffered saline
Cloning
Molecular
Purification
chemistry.chemical_classification
Henipavirus Infections
biology
nsNSRV
non-segmented negative-sense single-stranded RNA virus
virus diseases
MERS-CoV
Middle East respiratory syndrome coronavirus
Nucleocapsid Proteins
NiV N
Nipah virus nucleocapsid protein
Recombinant Proteins
HeV NFL
Hendra virus nucleocapsid protein (full length
amino acid residues 1–532)
Recombinant DNA
Antibody
N-RNA
Hendra virus nucleocapsid protein complexed with viral RNA
Biotechnology
Plasmids
RNP
ribonucleocapsid protein complex
EIDs
emerging infectious diseases
ESI-TOF-MS
electro-spray ionization time-of-flight mass spectrometry
HeV
Hendra virus
Viral protein
Molecular Sequence Data
Luminex assay
HeV N
Hendra virus nucleocapsid protein
Recombinant protein expression
IPTG
isopropyl-β-d-thiogalactopyranoside
P
phosphoprotein
Article
SARS-CoV
Severe acute respiratory syndrome coronavirus
Hendra Virus
sG
soluble G glycoprotein
medicine
Escherichia coli
L
Hendra virus RNA polymerase
Electron microscopy
Animals
Humans
Amino Acid Sequence
Horses
TEM
transmission electron microscopy
Nucleocapsid
MeV
Measles virus
RNA virus
biology.organism_classification
Virology
digestive system diseases
HeV NCORE
Hendra virus nucleocapsid protein CORE region (amino acid residues 1–402)
IDR
intrinsically disordered region
chemistry
CedPV
Cedar virus
NiV
Nipah virus
biology.protein
SEC
size exclusion chromatography
Heterologous expression
Glycoprotein
N
nucleocapsid
Zdroj: Protein Expression and Purification
ISSN: 1096-0279
Popis: Highlights • Recombinant HeV N was expressed in a soluble from in E. coli. • HeV N purified by IMAC and SEC formed higher order oligomers. • Negative-stain EM images of recombinant HeV N indicated self-assembly to form helical chains of nucleocapsids. • Recombinant forms of HeV N were immuno-reactive with sera from infected animals and humans.
Hendra virus (family Paramyxoviridae) is a negative sense single-stranded RNA virus (NSRV) which has been found to cause disease in humans, horses, and experimentally in other animals, e.g. pigs and cats. Pteropid bats commonly known as flying foxes have been identified as the natural host reservoir. The Hendra virus nucleocapsid protein (HeV N) represents the most abundant viral protein produced by the host cell, and is highly immunogenic with naturally infected humans and horses producing specific antibodies towards this protein. The purpose of this study was to express and purify soluble, functionally active recombinant HeV N, suitable for use as an immunodiagnostic reagent to detect antibodies against HeV. We expressed both full-length HeV N, (HeV NFL), and a C-terminal truncated form, (HeV NCORE), using a bacterial heterologous expression system. Both HeV N constructs were engineered with an N-terminal Hisx6 tag, and purified using a combination of immobilized metal affinity chromatography (IMAC) and size exclusion chromatography (SEC). Purified recombinant HeV N proteins self-assembled into soluble higher order oligomers as determined by SEC and negative-stain transmission electron microscopy. Both HeV N proteins were highly immuno-reactive with sera from animals and humans infected with either HeV or the closely related Nipah virus (NiV), but displayed no immuno-reactivity towards sera from animals infected with a non-pathogenic paramyxovirus (CedPV), or animals receiving Equivac® (HeV G glycoprotein subunit vaccine), using a Luminex-based multiplexed microsphere assay.
Databáze: OpenAIRE
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