EV71 virus-like particles and preparation method and application thereof
Inventors
GU, Meirong • WEI, Wenjin • Liu, Jiankai • SONG, Linlin • Xu, Shanshan • LI, Guoshun • Guo, Lin
Assignees
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Publication Number
US-9782471-B2
Publication Date
2017-10-10
Expiration Date
Abstract
The present invention provides EV71 virus-like particles and a preparation method and application thereof. The method comprises: connecting a P1 protein gene and a 3CD protease gene of an EV71 virus with a PMV plasmid to construct a PMV-P1-3CD recombinant expression plasmid; then transforming a Hansenula polymorpha AU-0501 expression strain with the PMV-P1-3CD recombinant expression plasmid to obtain an AU-PMV-P1-3CD recombinant expression strain; fermenting and culturing the recombinant expression strain, and inducing the recombinant expression strain to express the EV71 virus-like particle protein with methanol; centrifuging and collecting mycelia for homogeneous breakage at a high pressure; and purifying the supernatant through ion-exchange chromatography, hydrophobic chromatography, and molecular sieve chromatography, so as to obtain EV71 virus-like particles.
Core Innovation
The invention relates to EV71 virus-like particles based on a PMV recombinant expression plasmid carrying EV71 P1 and 3CD genes. The PMV-P1-3CD recombinant expression vector comprises a P1 gene with a nucleotide sequence shown in SEQ ID No. 1 and/or a 3CD gene with a nucleotide sequence shown in SEQ ID No. 3.
EV71 VLP proteins are expressed using the Hansenula polymorpha AU-0501 recombinant expression system. The platform includes methanol induction for VLP protein expression and a workflow for isolating and purifying the EV71 VLPs to obtain particles.
The disclosed processing yields EV71 VLPs characterized as approximately icosahedral particles, including visualization by transmission electron microscopy. The invention further provides an aluminum-phosphate-adjuvanted HFMD/EV71 vaccine formed using the EV71 VLPs together with an aluminum phosphate adjuvant under defined formulation specifications including VLP concentration and vaccine pH.
Claims Coverage
The provided claims include two independent claim sets directed to a recombinant expression vector and EV71 virus-like particles prepared by a defined expression-and-purification workflow. Across these, the inventive features are centered on the PMV-P1-3CD recombinant expression vector containing EV71 P1 and/or 3CD genes with specific sequence identifiers, and on preparing EV71 VLPs using a Hansenula polymorpha AU-0501 expression strain with subsequent purification steps.
PMV-P1-3CD recombinant expression vector with SEQ ID gene sequences
The expression vector is PMV-P1-3CD comprising an EV71 virus P1 gene having a nucleotide sequence as shown in SEQ ID No. 1 or an EV71 virus 3CD gene having a nucleotide sequence as shown in SEQ ID No. 3.
EV71 virus-like particles prepared using AU-0501 and PMV-P1-3CD with SEQ ID gene sequences
EV71 virus-like particles prepared by transforming a Hansenula polymorpha AU-0501 expression strain with the PMV-P1-3CD recombinant expression vector, fermenting and culturing the recombinant expression strain, and isolating and purifying the EV71 virus-like particles, wherein the PMV-P1-3CD recombinant expression vector comprises a P1 gene with a nucleotide sequence as shown in SEQ ID No. 1 or a 3CD gene with a nucleotide sequence as shown in SEQ ID No. 3.
Overall, the claim coverage is focused on the PMV-P1-3CD recombinant expression vector using EV71 P1 and/or 3CD nucleotide sequences specified by SEQ ID No. 1 and SEQ ID No. 3, and on producing EV71 virus-like particles in Hansenula polymorpha AU-0501 followed by isolation and purification.
Stated Advantages
Industrial scalability.
Safety and immunogenicity of the VLP-based vaccine.
Documented Applications
A hand, foot and mouth disease (HFMD) vaccine comprising EV71 virus-like particles, including an aluminum-phosphate adjuvanted formulation.
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