Methods for identifying fragile histidine triad (fhit) interaction and uses thereof
一种fhit-his6、fhit-his6cdna的技术，应用在鉴定脆性组氨酸三联体(Fhit)相互作用及其用途领域，能够解决Fhit知之甚少等问题
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Embodiment I
[0112] Materials and methods
[0113] Cells, Vectors and Antisera - A549, H1299, MKN74-E4 and A116 and HCT116 cells were maintained in RPMI1640 medium plus 10% fetal bovine serum and penicillin / streptomycin (Sigma). HEK293 cells (Microbix) used to prepare recombinant adenoviruses were cultured in Dulbecco's modified Eagle's medium plus 10% fetal bovine serum and penicillin / streptomycin. AdFHIT-His6 virus was prepared as described in Example II herein. [His6-SEQ ID NO:32][penta-His-SEQ ID NO:33].
[0114] Full-length FDXR was cloned from human brain cDNA (Clontech), subcloned into the pcDNA3.1 / V5-HisTOPO TA vector (Invitrogen) and sequenced; details are described in the accompanying methods below. Using Lipofectamine TM (Invitrogen) cells were transfected according to the manufacturer's instructions.
[0115] Western blot analysis - Western blot analysis was performed as described in (13) using: monoclonal anti-pentaHis (Qiagen); rabbit polyclonal anti-Fhit (Zymed Laborator...
Embodiment III
[0125] Production of Recombinant Adenovirus - Recombinant adenovirus carrying wild-type FHIT cDNA (AdFHIT) was prepared as previously described (Ishii et al., 2001 Cancer Res 61: 1578-1584). His-tagged FHIT cDNA was generated by PCR using the following oligonucleotides: 5'-ACgTggATCCCTgTgAggACATgTCgTTCAgATTTggC-3' (forward) [SEQ ID NO:26] and 5'-TTgTggATCCTTATCAgTgATggTgATggTgATgCgATCCTCCTGAAAgTAgCCCgCAg-3' [SEQ ID NO:27]. These primers were designed with a BamHI restriction site for subcloning into the transfer vector pAdenoVator-CMV5-IRES-GFP. Ad-His6 is using AdenoVator TM Kits (Qbiogene, Carlsbad, CA) were produced according to the manufacturer's protocol. Ad GFP used as a control was purchased from Qbiogene (Carlsbad, CA).
[0126] Generation of recombinant expression vector carrying FDXR cDNA - full-length wild-type ferredoxin reductase was amplified from human brain cDNA (Clontech, Palo Alto, CA) using the following primers: 5'-CTgTTCCCAgCCATggCTTCgCgCTg-3' (forward) ...
Embodiment approach
[0151] 1. An adenovirus capable of isolating Fhit-His6, the adenovirus comprising an adenovirus carrying FHIT-His6 cDNA.
[0152] 2. A method for isolating exogenously overexpressed Fhit-His6, the method comprising using an adenovirus carrying FHIT-His6 cDNA, wherein said Fhit-His6 is isolated by means of a His tag.
[0153] 3. A recombinant adenovirus carrying a fragile histidine triplet (Fhit) FHIT cDNA (AdFHIT-His6) modified at its 3' with a sequence encoding a hexahistidine epitope tag.
[0154] 4. A method of mediating an apoptotic process in at least one cell, the method comprising exposing said cell to a fragile histidine triad (Fhit triad) in an amount sufficient to mediate said apoptotic process in said cell ) gene product.
[0155] 5. A method of inducing an apoptotic process in a cell, the method comprising exposing said cell to a fragile histidine triad (Fhit) gene in an amount sufficient to cause generation of reactive oxygen species (ROS) in said cell product.
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