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Soluble fragments of the SARS-cov spike glycoprotein

A technology of fragments and coupled proteins, which can be used in the fields of peptide/protein components, resistance to vector-borne diseases, medical preparations containing active ingredients, etc., and can solve problems such as alveolar wall damage

Inactive Publication Date: 2006-10-25
THE GOVERNMENT OF THE U S A REPRESENTED BY THE SEC DEPT OF HEALTH & HUMAN SERVICES NAT INST OF HEALTH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0009] The pathophysiology of the disease has yet to be determined; however, the mechanism of acute lung injury may involve direct damage to the alveolar walls by the virus attacking endothelial or epithelial cells

Method used

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  • Soluble fragments of the SARS-cov spike glycoprotein
  • Soluble fragments of the SARS-cov spike glycoprotein
  • Soluble fragments of the SARS-cov spike glycoprotein

Examples

Experimental program
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preparation example Construction

[0145] The preparation of monoclonal antibodies is also routine (see Kohler & Milstein, Nature, 256:495 (1975); Coligan et al., sections 2.5.1-2.6.7; and Harlow et al., Antibodies: A Laboratory Manual, page 726 ( Cold Spring Harbor Pub. 1988)), incorporated herein by reference. Simply put, monoclonal antibodies can be injected into mice with a composition containing an antigen, and after the presence of antibodies is confirmed from the collected serum samples, the spleen is taken to obtain B lymphocytes, and the B lymphocytes are fused with myeloma cells to form hybridomas cells, hybridoma cells are cloned, positive clones producing antibodies against the antigen are screened, and antibodies are isolated from the culture medium of the hybridoma cells. Antibodies can be isolated and purified from the culture medium of hybridoma cells by various known methods. These separation techniques include affinity chromatography with Protein A Sepharose, size exclusion chromatography, an...

Embodiment 1

[0282] Cloning of the spike protein

[0283] The nucleic acid sequence encoding the full-length Spike protein was obtained by overlapping polymerase chain reaction (PCR). Overlapping clones containing the spike protein were obtained from the British Columbia Cancer Agency (Vancouver, British Columbia). The following primers were used in a PCR reaction to amplify the nucleic acid sequence encoding the full-length SARS-CoV Spike protein: Clone 1: Forward primer: 5′-AGTC GGA TCC GGT AGG CTT ATC ATT AGA G-3' (SEQ ID NO: 3); reverse primer: 5'-CCA TCA GGG GAG AAA GGC AC-3 (SEQ ID NO: 4). Clone 2: forward primer: 5'-GTG CCT TTC TCC CCT GAT GG-3' (SEQ ID NO: 5); reverse primer: 5'-GAA GAG CAG CGC CAG CAC C-3' (SEQ ID NO: 6). Clone 3: Forward primer: 5'-GGT GCT GGC GCT GCT CTT C-3' (SEQ ID NO: 7); Reverse primer: 5'-A CTG TCT AGA GTT CGT TTA TGT GTAATG-3 (SEQ ID NO: 8).

[0284] The nucleic acid fragment generated by overlapping PCR amplification of the nucleic acid fragment b...

Embodiment 2

[0287] Generation of amino-terminal (S1) and carboxy-terminal (S2) fragments of the full-length Spike protein

[0288] Computer analysis was used to determine the potential functional segmentation points of the amino-terminal (S1) and carboxy-terminal (S2) of the spike protein, and the segmentation points between S1 and S2 were at 758 and 761 of SEQ ID NO: 1 ( 758 RNTR 761 )between. The method of PCR was used to prepare the amino-terminal fragment (S1) and the carboxy-terminal (S2) fragment encoding the spike protein.

[0289] The following primer pair, S1 forward primer: 5′-AGTC GGA TCC GAC CGG TGCACC ACT TTT G-3' (SEQ ID NO: 9), and S1 reverse primer: 5'-AGTC GGG CCC CTG TTC AGC AGC AAT ACC-3' (SEQ ID NO: 10) was used to prepare a nucleic acid fragment encoding amino acid residues 17 to 757 of the Spike protein. Two restriction sites, BamHI and ApaI (underlined parts of the two primer sequences), were used to clone the nucleic acid fragment (S1) encoding the N-termi...

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Abstract

The invention relates to the spike protein from the virus (SARS-CoV) that is etiologically linked to severe acute respiratory syndrome (SARS); polypeptides and peptide fragments of the spike protein; nucleic acid segments and constructs that encode the spike protein, polypeptides and peptide fragments of the spike protein, and coupled proteins that include the spike protein or a portion thereof; peptidomimetics; vaccines; methods for vaccination and treatment of severe acute respiratory syndrome; antibodies; aptamers; and kits containing immunological compositions, or antibodies (or aptamers) that bind to the spike protein.

Description

[0001] This application claims priority to US Application Serial No. 60 / 489,166, filed July 21, 2003, and US Application Serial No. 60 / 524,642, filed November 25, 2003, which are hereby incorporated by reference in their entirety. [0002] government fund [0003] Development of the invention described herein was supported by funding from the Department of Health and Human Services. The US Government has certain rights in this invention. field of invention [0004] The present invention generally relates to spike glycoproteins encoded by coronaviruses associated with the etiology of severe acute respiratory syndrome (SARS), here SARS-CoV. The present invention further relates to polypeptides, nucleic acids and their conservative variants having an amino acid sequence consistent with the spike glycoprotein fragment of SARS-CoV. The present invention also relates to using these nucleic acids, polypeptides, variants and fragments to pro...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/165A61K38/16A61K39/215A61K39/42
CPCY02A50/30
Inventor D·S·迪米特罗夫X·肖
Owner THE GOVERNMENT OF THE U S A REPRESENTED BY THE SEC DEPT OF HEALTH & HUMAN SERVICES NAT INST OF HEALTH
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