High throughput proteomics

A protein and nucleotide sequence technology, applied in the field of high-throughput proteomics, can solve the problem of not being able to provide high-throughput methods for characteristic antigens

Active Publication Date: 2012-10-10
RGT UNIV OF CALIFORNIA
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, since they require the isolation of single clones for each protein, they do not provide a high-throughput method for identifying characteristic antigens of infectious agents that represent the full range of possible antigenic protein or peptide moieties

Method used

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  • High throughput proteomics
  • High throughput proteomics
  • High throughput proteomics

Examples

Experimental program
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Embodiment 1

[0109] Prepare vector and insert

[0110] A linear T7 vector encoding an N-terminal histidine tag and a C-terminal HA tag was prepared by extensive restriction digestion followed by PCR; this procedure allows residual loops to be transformed when transformed without a complementary insert into chemically competent E. coli The amount of vector-like vectors and background colonies was reduced to close.

[0111] The plasmid used to make the linear recombinant vector pXT7 is shown in figure 1 . This vector contains a T7 promoter followed by an ATG start codon, a 10×histidine sequence, a spacer sequence before the first codon of the open reading frame to be cloned, a BamH1 site and a T7 terminator. The vector was double digested at the BamH1 site to eliminate residual circular vector, as incompletely digested vectors produced background colonies lacking the insert. This linearized vector is amplified by PCR to generate a stock of linear recombinant vectors. Each batch of lin...

Embodiment 1A

[0117] Example 1A : Applying these methods to vaccinia virus required preparation of primers for 213 genes, of which 211 PCR products were isolated (>99%). All 211 were cloned and 181 of the products were sequenced; 93% (169 of 181) provided predicted sequences.

Embodiment 1B

[0118] Example 1B : Similarly, applying this method to Plasmodium falciparum requires the preparation of primers for 720 genes. 462 PCR products (64%) were thus obtained, resulting in 266 clones (58%). A group (63) of these were sequenced and 97% yielded the expected sequence.

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Abstract

Methods to obtain expression systems and proteins in a high-throughput protocol by utilizing mixtures of cells cultured from those transformed with a desired nucleotide sequence permit rapide production of protein for use in arrays to assess activity. In one embodiment, the proteins (or peptides) in the array are assessed for their immunological activity with regard to an infectious agent.

Description

[0001] Cross-references to related applications [0002] This application claims the benefit of US Provisional Application 60 / 585,351, filed July 1, 2004, and US Provisional Application 60 / 638,624, filed December 23, 2004. The contents of these applications are incorporated herein by reference. [0003] Claims of Rights to Inventions Made Under Federally Sponsored Research [0004] This work was supported in part by the National Department of Health / National Society of Allergy and Infectious Diseases. The US Government has certain rights in this invention. technical field [0005] The present invention relates to methods for preparing proteins or peptides from coding open reading frames (ORFs), and methods for identifying immunologically active proteins. The invention also relates to methods of making protein / peptide arrays from multiple coding ORFs, and the use of these arrays in assaying immunologically active proteins. The invention also relates to these immunologica...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/68
CPCY02A50/30
Inventor P·L·费尔格纳H·达维斯X·凌
Owner RGT UNIV OF CALIFORNIA
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