ICBP90 polypeptide and its fragments and polynucleotides coding for said polypeptides and applications for diagnosing and treating cancer
a polypeptide and cancer technology, applied in the field of icbp90 polypeptides and its fragments, can solve problems such as emergence of resistance to drugs
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example 1
Evidence of a New Binding Protein for the ICB Sequence
[0143] 1.1 Reporter Construction for the Screening of the Library
[0144] The simple hybrid system is a powerful technique for detecting, in vivo, in yeast the interaction of proteins with specific DNA sequences when screening cDNA libraries. This technique allows you to evaluate directly cDNA corresponding to the protein to be linked. Several studies using this technique resulted in the identification of novel proteins. The protocols are well described by Inouye et al. (1994) and Wang and Reed (1993).
[0145] Briefly, the following oligonucleotides have been synthesized:
(SEQ ID NO:15)5′-AATTCGGGGCGGGGCCGGGGCGGGCCCGGGGCGGGGCT-3′(SEQ ID NO:16)5′-CTAGAGCCCCGCCCCGGCCCCGCCCCGGCCCCGCCCCGG-3′
[0146] These nucleotides were then hybridised. According to the documentation of the manufacturer (Clontech, Palo Alto, Calif.), the reporter construct targeted possesses three copies in tandem of the ICB2 sequence (ICB2X3). As mentioned above, on...
example 2
Characterisation of the ICBP90 Protein
[0157] 2.1. Synthesis of Antibodies
[0158] Mouse monoclonal antibodies were synthesized in our laboratory by injection of ICBP-59 protein using traditional methods (Brou et al., 1993); the protein was purified beforehand by a fusion GST system. Two monoclonal antibodies from 1RC1C-10 and 1RC1H-12 were selected for their ability to detect the ICBP-59 endogenous protein; their specificity was demonstrated in both Western blotting and immunocytochemistry experiments. Before use, the antibodies were purified on a DEAE-cellulose column (DE52, Whatmann) from ascites fluid.
[0159] 2.2 Detection of the Endogenous Protein by Western Blotting
[0160] To detect endogenous ICBP-59 protein, we first used 1RC1C-10 in a Western blot (0.4 μg / ml 1RC1C-10 monoclonal antibodies) of nuclear extracts from confluent and proliferating HeLa cells (FIG. 1). COS-1 and HeLa cells were cultivated as previously described (Brou et al., 1993; Gaub et al., 1998; Rochette-Egly ...
example 3
Isolation and Characterization of Gene ICBP90
[0185] 3.1. Material and Methods
[0186] 3.1.1. Construction and Screening of a Human Placental Genomic Library
[0187] After partial digestion with MboI enzyme, the placental genomic DNA was split up according to size on a 10 to 40% sucrose gradient. Fifteen kb DNA Fragments were ligated in a λGEM 12 vector previously digested with BamHI (Promega, Madison Wis., USA). After packaging, phage λ particles were assayed on TAP 90 cells. The genomic library contains 3.106 plaque-forming units (pfu). 106 clones were spread out for analysis. A 620 bp probe corresponding to a 5′ terminal extremity of the ICBP90 cDNA used for the screening was labelled with α32P-dCTP by a random priming method (Sambrook et al., 1989). The labelled probe is used according to a classic on plaque hybridisation protocol to screen the genomic library (Sambrook et al., 1989). Hybridisation was achieved at 68° C. in 5× SSC (15 mM NaCl, 1.5 mM sodium citrate pH 7.0), 5 × De...
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