Oculospanin as a tumor specific antigen and methods and compositions utilizing same
a tumor specific and antigen technology, applied in the field of oculospanin as a tumor specific antigen and methods and compositions utilizing same, can solve the problems of limited range of monoclonal antibodies useful for tumor treatment, and the relationship between human oculospanin and tumors is still unknown, and achieve the effect of inhibiting oculospanin activity
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example 1
Screening of a Gene Specifically Expressed in a Cancer Cell
[0388] Expression profile analysis was performed, using an EST probe (Affymetrix GeneChip HG-133 probe 223795_at: manufactured by Affymetrix) having a nucleotide sequence partially overlapping with the sequence represented by Sequence ID No. 1 of the sequence listing, by use of the data base (GeneExpress Software System Release 1.4.2) provided by Genelogic company.
[0389] Expression of the human oculospanin gene in various cells was quantitatively compared by considering its transcription. As a result, the expression levels in 8 melanocyte samples were to found to be significantly high, compared to the levels in other cells samples, including 12 blood-cell samples, 6 glia cell samples, 62 epithelial cell samples (P values thereof were <0.0001, =0.0007, and <0.0001 sequentially in order, FIG. 1, upper panel).
[0390] Next, the expression levels of the human oculospanin gene were compared in samples derived from tissue. More s...
example 2
Acquisition of the Human Oculospanin Gene and Construction of Expression Plasmid
[0393] a) PCR Reaction
[0394] As a primer for amplifying human oculospanin cDNA by PCR, oligonucleotides having the following sequences were synthesized in accordance with a customary method.
5′-CACCATGGAGGAGGGGGAGAGGAGCC(Primer 1,C-3′Sequence ID No. 5of the sequencelisting)5′-GCCCCGGGCGGGTTTGGCAGCGG-3′(Primer 2,Sequence ID No. 6of the sequencelisting)
[0395] Note that Primer 1 is an oligonucleotide constructed by adding 4 bases, CACC, as a KOZAK sequence, upstream of the initiation codon of the human oculospanin gene, in other words, an oligonucleotide constructed by adding the 4 bases, the CACC sequence, to the 5′ side of the nucleotide sequence consisting of nucleotides No. 1 to 23 of the Sequence ID No. 1 of the sequence listing. The CACC sequence, since it forms a chain complementary to the 3′ terminus of the vector when it is integrated into the cloning vector pENTR / D-TOPO, makes it possible to in...
example 3
Introduction of the Human Oculospanin Gene into Cells, Confirmation that the Human Oculospanin Gene Product is Expressed, and Preparation of a Membrane Fraction from Human Oculospanin Expressing Cells for Use as an Immunogen
[0400] a) Transfection of NIH3T3 cells with the plasmid pcDNA3.1-DEST40-NM—031945
[0401] NIH3T3 cells were transfected with plasmid pcDNA3.1-DEST40-NM—031945 obtained in Example 2 as follows. The transfection of the NIH3T3 cells was performed by lipofection using the Lipofectamine 2000 Reagent manufactured by Invitrogen. To explain more specifically, first, NIH3T3 cells were grown in a 6 well plate up to a semi-confluent state. Next, the cells were washed once with antibiotic-free DMEM containing 10% fetal calf serum, then 200 μl of antibiotic-free DMEM containing 10% fetal calf serum was added to the cells. Then, to a 1.5 ml Eppendorf tube, 100 μl of serum-free medium (DMEM) and 2 μg of plasmid DNA (pcDNA3.1-DEST40-NM—031945) recovered in the aforementioned man...
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