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Rearranged squamous cell carcinoma antigen genes II

a squamous cell carcinoma and antigen gene technology, applied in the field of fusion protein transcripts, can solve problems such as false positives

Inactive Publication Date: 2007-09-20
CANAG DIAGNOSTICS
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

"The present invention provides a fusion protein consisting of parts of SCCA1 and SCCA2, which are genes found in different cell lines. The invention also provides methods for detecting these fusion proteins using specific immunological reagents. The fusion protein is defined by a specific amino acid sequence and is found in different cell lines of cervix, lung, and pharynx. The invention also provides the DNA sequence that encodes the fusion protein. The technical effect of the invention is the detection and validation of a new fusion protein that can be used as a target for diagnosis and treatment of cancer."

Problems solved by technology

It has been reported that false positive results may often be caused by contamination with saliva or sweat during assay procedure (1).

Method used

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  • Rearranged squamous cell carcinoma antigen genes II
  • Rearranged squamous cell carcinoma antigen genes II
  • Rearranged squamous cell carcinoma antigen genes II

Examples

Experimental program
Comparison scheme
Effect test

example 1

Cloning of SCCA

1.1. PCR Amplification

[0021] mRNA from the cell-lines Caski (cervix), C4-I (cervix), A549 (lung), CaLu3 (ung), SkMes (lung), and RPMI2650 (pharynx) was prepared using QuickPrep Micro mRNA Purification kit (Pharmacia) and cDNA was prepared using First-Strand cDNA Synthesis kit (Pharmacia). A 1218 bp DNA fragment covering the coding sequence of SCCA was amplified by PCR in a 100 μl reaction containing 10 mM Tris-HCl pH 8.85, 25 mM KCl, 5 mM (NH4)2SO4, 2 mM MgSO4 (Boehringer), 0.2 mM DNTP (Pharmacia), 10 μM SCCA 1-7F (DNA sequences for all primers are shown in Table 1), 10 μM SCCA 391-397B, 2 μl cDNA and 2.5 U Pwo-polymerase (Boehringer). After denaturing samples for 5 min at 96° C. a total of 30 cycles were performed, each consisting of denaturation for 15 sec at 96° C., annealing for 15 sec at 60° C., and extension for 30 sec at 72° C. The PCR reaction was completed by a final extension for 10 min at 72° C.

TABLE 1PCR-primersPrimer nameSequence1. SCCA 1-7F5′-CGGGA...

example2

Protein Expression and Purification

2.1. Protein Expression

[0027] Expression conditions were determined by small-scale preparations. For large scale expression 500 ml cultures of 2×YT and 100 μg / ml ampicillin were inoculated with 5 ml over-night culture and grown at 37° C. Protein expression was induced at OD600=0.5-1.3 by adding IPTG to a final concentration of 0.1 mM. Cultures producing SCC1 were grown for 4-16 h, SCCA1 / A2 for 16-18 h. Cultures producing the SCCA2 protein were induced at OD600=1.2-1.4 and were grown for 2-3 h.

2.2. Protein Purification

[0028] Cells were harvested by centrifugation for 10 min at 2000 g, washed with 50 ml TE pH 8.0, and dissolved in 3 ml TE / g bacterial pellet. Lysozyme was added to a final concentration of 800 μg / g pellet and the mixtures were incubated on ice for 30-60 min and then frozen over night at −70° C. Magnesium chloride and DNase were added to a final concentration of 12 mM and 20 μg / g pellet, respectively. After incubation on ice for ...

example 3

DNA Analysis

3.1. Southern Blot Analysis

[0030] Approximately 10 μg of DNA prepared from SCC cell-lines as well as from blood samples from normal healthy volunteers, were digested with restriction endonucleases PstI or BamHI. Digested DNA were separated on 0.8% agarose and transferred to membranes (Hybond N+, Pharmacia). Filters were prehybridized for 1 h and hybridized over night at 60° C. in 20 ml of a solution containing 5×SSC, 0.1% SDS, 5% Dextrane sulfate, Liquid block (Pharmacia) diluted 1:20 and salmon sperm DNA 100 μg / ml. Probe concentration during hybridization was 10 ng / ml. After hybridization filters were stringency washed for 15 min in 1×SSC / 0.1% SDS and for 15 min in 0.2×SSC / 1% SDS, both at 60° C. Probe hybridization was detected using Gene Images CDP-Star detection module (Pharmacia) with minor modifications. Filters were blocked for 1 hour at room temperature in a solution containing liquid block diluted 1:7.5. Then they were incubated in buffer A (0.1M Tris, 0.3M N...

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Abstract

The present invention relates to a SCCA1 / SCCA2 fusion protein; plasmid containing the same; antibodies of said fusion protein; methods for detecting said protein; methods for diagnosing the presence or absence of SCC by determining the presence of SCCA1 / SCCA2 fusion protein.

Description

FIELD OF THE INVENTION [0001] The present invention relates to a fusion protein transcript for the production of fusion protein, a fusion gene found in squamous cell carcinomas, detection of the rearrangement and monoclonal antibodies specific for SCCA1; SCCA1 / A2, SCCA2 / A1 and SCCA2. BACKGROUND OF THE INVENTION [0002] Squamous cell carcinoma antigen (SCCA) is a serological marker for squamous cell carcinomas (SCC) of the uterine cervix, lung, head and neck, vulva, and esophagus (1, 2). It was originally purified from the TA-4 complex from human cervical squamous cell carcinoma, with a molecular weight of 42-48 kDa (1, 3). The antigen consists of more than 10 proteins and isoelectric focusing of the antigen reveals two subfractions, an acidic (pI<6.25) and a neutral (pI∃36.25) isoform (4). The difference in molecular weight is probably due to modification (5). [0003] Cloning of the cDNA of SCCA shows that it belongs to the family of serine protease inhibitors (serpins) (6). Furthe...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): C12Q1/68C07H21/04C12N1/21G01N33/574C12N15/12G01N33/53C07K14/47C07K14/81C07K14/82C07K16/18C07K16/32C07K19/00C12N15/09C12N15/15C12N15/62C12P21/02C12P21/08
CPCC07K14/4731C07K14/811C12N15/62C07K2319/40C07K2319/00A61P35/00
Inventor ROJER, EVA
Owner CANAG DIAGNOSTICS
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