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Recombinant icosahedral virus like particle production in pseudomonads

A technology of icosahedron and pseudomonas, which is applied to viruses, antiviral agents, single-celled algae, etc., can solve problems such as the destruction of protein geometric structure and the failure of chimeric virus assembly

Inactive Publication Date: 2008-03-26
DOW GLOBAL TECH LLC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Chapman reasoned that the presence of larger peptides at the intrinsic insertion site in the icosahedral viral capsid might lead to disruption of the protein's geometry and / or ability to successfully interact with other capsids, leading to failure of chimeric virus assembly

Method used

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  • Recombinant icosahedral virus like particle production in pseudomonads
  • Recombinant icosahedral virus like particle production in pseudomonads
  • Recombinant icosahedral virus like particle production in pseudomonads

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0346] Example 1: Production of Peptide PD1 in CCMV VLPs in Pseudomonas

[0347] 1.A. Construction of chimeric CCMV-PD1 gene

[0348] 20 amino acid antigenic peptides were selected as inserts in CCMV capsid virus to express. Antigenic peptides were not related to CCMV and Pseudomonas fluorescens. Oligonucleotides encoding peptides were amplified from plasmid pCP7Pavol DNA using primers Parvo-BamHI-F (nucleotide sequence: 5'-cgggatcctg gacccggatg-3' (SEQ ID NO: 16)) and Parvo-BamHI-R (nucleotide sequence : 5'-cgggatcccc gggtctcttt c-3' (SEQ ID NO: 17)). (These primers were obtained from Integrated DNA Technologies, Inc., Coralville, IA, USA, hereinafter referred to as "IdtDNA"). These primers amplified from the canine parvovirus peptide coding sequence with the addition of BamHI restriction sites at both ends for insertion into the CCMV 129 coding sequence at their BamHI restriction sites.

[0349] Use PTC225 thermal cycler (MJ Research, South San Francisco, CA, USA) to c...

Embodiment 2

[0374] Example 2: Production of D2A21 AMP trimers in CCMV VLPs in Pseudomonas and collection of AMP therefrom

[0375] 2.A. Synthesis of D2A21 insert

[0376] The nucleic acid sequence encoding the antimicrobial peptide ("AMP") trimer ("D2A21 trimer", i.e. three D2A21 monochimeric AMPs) was amplified from plasmid pET-(D2A21)3 using primer D2A21-BamHI- F (nucleic acid sequence: 5'-cgggatcctg ggacagcaaa tgggtcgcga tccg-3' (SEQ ID NO: 5)) and D2A21-BamHI-R (nucleic acid sequence: 5'-cgggatcccg tcgacggagc tcgaattcgg atcacc-3' (SEQ ID NO: 6)) . PCR reactions were performed according to the same protocol as described in Example 1.A. above.

[0377] The resulting amplified inserts contained added BamHI restriction sites at each end for insertion of the D2A21 trimer CDS into the CCMV129 CDS at the engineered BamHI sites. The nucleic acid coding sequence and amino acid sequence of the D2A21 trimer are shown in SEQ ID NO: 19 and 20, respectively.

[0378] Nucleotide sequence enc...

Embodiment 3

[0409] Example 3: Production of anthrax antigen in CCMV VLPs in Pseudomonas

[0410] 3.A. Synthesis of PA peptide insert

[0411] Four different B. anthracis protective antigen ("PA") peptides (PA1-PA4) were independently expressed in CCMV VLPs. Nucleic acids encoding PA1-PA4 were synthesized by SOE (Splice Overlap Extension) of synthetic oligonucleotides. The resulting nucleic acid contains the BamHI recognition site ends. The nucleic acid coding sequence and amino acid sequence of these PA peptides are respectively as follows: 1) for PA1, SEQ ID NO: 8 and 9; 2) for PA2, SEQ ID NO: 10 and 11; 3) for PA3, SEQ ID NO: 12 and 13; and 4) for PA4, SEQ ID NO: 14 and 15. The resulting nucleic acid was digested with BamHI to create cohesive ends for cloning into the shuttle vector. Each resulting PA insert was cloned into the pSEC-CCMV129BamHI shuttle plasmid at the BamHI site of the CCMV129 CDS. Each of the resulting shuttle plasmids was digested with SpeI and XhoI restriction...

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Abstract

The present invention provides an improved process for the production of recombinant peptides by fusion of recombinant peptides with icosahedral viral capsids and expression of the fusion in bacterial cells of Pseudomonad origin. The Pseudomonad cells support formation of virus like particles from icosahedral viral capsids in vivo, and allow the inclusion of larger recombinant peptides as monomers or concatamers in the virus like particle. The invention specifically provides cells expressing viral capsid fusions, nucleic acids encoding fusions of toxic proteins with icosahedral viral capsids and processes for manufacture of recombinant proteins.

Description

[0001] Cross References to Related Applications [0002] This application claims priority to U.S. Provisional Patent Application No. 60 / 525,982, filed December 1, 2003, entitled "Efficient Peptide Production in Pseudomonas." technical field [0003] The present invention provides improved methods for producing recombinant peptides. In particular, the present invention provides improved methods for the production or presentation of recombinant peptides in bacterial cells utilizing virus-like particles of icosahedral viruses. Background of the invention [0004] The genetic engineering revolution has been extended to develop recombinant peptides for use as human and animal therapeutics. Currently, there are more than 100 biotechnology-derived therapeutics and vaccines approved by the U.S. FDA for medical use, and more than 1,000 others are in various stages of clinical trials. (See, M. Rai & H. Padh, (2001) "Expression systems for production of heterologous proteins", Cur. S...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07H21/02C07H21/00C07H21/04C12P21/06C12P21/04A01N63/00A01N65/00C12N1/12C12N1/20C12N15/00C12N15/09C12N15/63C12N15/70C12N15/74C12N5/04C12N5/10A61K39/108A61K39/02A61K39/295A61K48/00A61K38/00C07K14/00C07K14/005C07K14/015C07K14/08C07K14/18C12N7/04C12N15/62C12P21/02G01N33/53
CPCC12N2770/14023A61K2039/5256C07K2319/40C12N2750/14322C12N2770/36122A61K2039/5258C07K14/00C07K14/005A61K38/00C12N7/00C12N15/62C12P21/02A61P31/04A61P31/12C12N15/03C12N15/11
Inventor L·拉索霍娃P·P·达奥
Owner DOW GLOBAL TECH LLC
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