Improved production and in vivo assembly of soluble recombinant icosahedral virus-like particles

A recombinant virus, soluble technology, applied in the direction of virus/phage, virus, virus peptide, etc., can solve the problem of icosahedral virus CP-peptide fusion particle assembly and other problems

Inactive Publication Date: 2010-07-21
菲尼克斯股份有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The reason why the observed icosahedral viral CP-peptide fusion particles fail to assemble in Escherichia coli as VLPs in vivo is not fully understood

Method used

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  • Improved production and in vivo assembly of soluble recombinant icosahedral virus-like particles
  • Improved production and in vivo assembly of soluble recombinant icosahedral virus-like particles
  • Improved production and in vivo assembly of soluble recombinant icosahedral virus-like particles

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0318] Example 1: Cloning of expression plasmids for expression of codon- and hydrophilicity-optimized CCMV capsid proteins in Pseudomonas fluorescens

[0319] clone:

[0320]A codon and hydrophobicity optimized CCMV CP nucleotide sequence (SEQ ID NO: 3) was designed. A shuttle plasmid (DNA 2.0, MenloPark, CA). The insert was gel purified on a 1% agarose gel and ligated into vector pDow1169 (a medium copy plasmid with RSF1010 origin, pyyF, tac promoter, and rrnBT1T2 terminator from pKK223-3 (PL-Pharmacia)) to create an expression plasmid (SEQ ID NO: 23) for expression of CCMV CP in Pseudomonas fluorescens. After purification with a Micro Bio-spin 6 column, the ligated product was transformed into Pseudomonas fluorescens strain DC454 (ΔpyrFRXF01414(lsc)::lacIq1) by electroporation. After shaking in LB medium at 30°C for 2 hours, the transformants were plated on M9 glucose plates. The presence of the insert was confirmed by restriction digest and sequencing of plasmid DNA i...

Embodiment 2

[0511] Example 2: Introduction of restriction sites into loops of codon and hydrophilicity optimized CCMV capsid proteins

[0512] Site-directed mutagenesis reactions were performed using Quikchange II-XL (Stratagene, TX) according to the manufacturer's protocol. The Pseudomonas fluorescens expression plasmid (SEQ ID NO: 23) containing the codon-optimized CCMV-CP served as template. After purification with Micro Bio-spin 6, the resulting plasmid with the introduced restriction sites was transformed into Pseudomonas fluorescens strain DC454 (ΔpyrF RXF01414(lsc)::lacIq1) by electroporation. Protein expression was performed as described in Example 1.

[0513] Primers used to introduce the blunt-end cleavage restriction site AfeII into loop 63:

[0514] CCMV-AfeI-63-F (SEQ ID NO: 24): 5'-TGCGCGGCTGCCGAGAGCGCTGCCAAGGTCACCAGT-3'

[0515] CCMV-AfeI-63-R (SEQ ID NO: 25): 5'-ACTGGTGACCTTGGCAGCGCTCTCGGCAGCCGCGCA-3'

[0516] Primers used to introduce the 3' overhang cleavage restrict...

Embodiment 3

[0528] Example 3: Restriction digest based cloning and expression of influenza vaccine M2e peptide fused to the codon and hydrophilicity optimized 129 surface loop of the CCMV capsid protein

[0529] Peptide Synthesis:

[0530] Inserts were synthesized by overlapping DNA oligonucleotides described below and the thermal cycling procedure detailed below:

[0531]

[0532]

[0533] *(from Invitrogen Corp, Carlsbad, CA)

[0534] use PCR purification kit (Qiagen) purified PCR products, digested with XbaI (NEB), and The kit was purified again and ligated with T4 DNA ligase (NEB) into the XbaI-restricted CCMV CP Pseudomonas fluorescens expression vector (from Example 2) containing the XbaI restriction site in loop 129 point. After purification with a Micro Bio-spin 6 column (Biorad), the ligated product was transformed into Pseudomonas fluorescens strain DC454 (ΔpyrFRXF01414(lsc)::lacIq1) by electroporation. After shaking in LB medium at 30°C for 2 hours, the transforman...

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Abstract

The present invention provides an improved method for the in vivo production of soluble assembled virus-like particles (''VLPs'') in bacterial cells of Pseudomonad origin. The Pseudomonad cells support assembly of VLPs from icosahedral viral capsid proteins (''CPs'') in vivo, and allow the inclusion of larger recombinant peptides as monomers or concatamers in the VLP. The invention specifically provides an improved method for the in vivo production of soluble assembled Cowpea Chlorotic Mottle Virus (''CCMV'') VLPs by introducing modifications into the CCMV CP that result in high yield production of soluble CP fusions in a Pseudomonas fluorescens bacterial system. These soluble VLPs can subsequently be purified and used as vaccines.

Description

[0001] priority claim [0002] This application claims the filing date benefit of US Provisional Patent Application Serial No. 60 / 914,677, filed April 27,2007. [0003] Statement of Government Interest [0004] This invention is made under U.S. Government contract with the National Institutes of Health, National Institute of Allergy and Infectious Disease (NIAID), Collaborative Agreement No. 1-U01-AI054641-01 generated with government support. The government has certain rights in this invention. field of invention [0005] The present invention provides an improved method for producing soluble, assembled virus-like particles ("VLPs") in bacterial host cells. Background of the invention [0006] Bacterial, yeast, Dictyostelium discoideum, insect, and mammalian cell expression systems are currently used with varying degrees of success to produce recombinant peptides for use as human and animal therapeutics. One goal in creating expression systems for the production of hete...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N7/04C07K14/08C12P21/02
CPCC12N2770/14023A61K2039/5256C07K2319/00C12N7/00A61K2039/5258C12N2770/14022C12N2760/16022C07K14/005C12N2795/10243
Inventor 拉达·罗索乔瓦杰米·P·费尔普斯
Owner 菲尼克斯股份有限公司
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