2010 Fiscal Year Final Research Report
Genome Analysis through Reverse Translation of Human Bone Metastasis-Specific Protein
| Project/Area Number |
19591741
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| Research Category |
Grant-in-Aid for Scientific Research (C)
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
Orthopaedic surgery
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| Research Institution | Kurume University |
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Principal Investigator |
TSURU Michiyo Kurume University, 医学部, 助教 (90368887)
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| Project Period (FY) |
2007 – 2010
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| Keywords | 骨・軟部腫瘍学 |
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| Research Abstract |
Primary cancer control has become almost feasible, and controlling the spread of metastases to remote organs is the main focus of future cancer treatment. Even if a patient receives the best primary cancer treatment, metastasis of cancer occurs mostly five or ten years after the treatment when the cancer is considered to be completely cured. For the clinical test, a time-series follow-up study was conducted on blood samples obtained from each patient after the primary cancer was completely cured. Clinically, the target protein is present in the blood before the spread of definitive bone metastases (screening for bone metastases can be achieved by bone scintigraphy) and shows a decreasing trend after the initiation of chemotherapy and radiotherapy. A time-series follow-up was conducted on an individual basis. Regarding the purification method, the target peak was detected for the unattached fraction and the elution fraction (pH 7.0) was obtained after fractionating 200 μL of the blood s
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erum in an anion exchange column. Using this fraction as a partial purification fraction, the unattached and elution fractions (pH 7.0) were added to the cation exchange column and eluted by increasing the level of NaCl concentration. Consequently, the target peak was confirmed for 0.2 M and 0.3 M NaCl fractions.These fractions were then applied onto reverse-phase HPLC (2 mm) and eluted by the concentration gradient of acetonitrile. However, because the peak immediately adjacent to the target peak could not be separated and there was a possibility that high molecular protein, which could not be confirmed by SELDI may have existed, the fraction was applied onto the normal-phase HPLC system, where the target peak was successfully obtained. A tertiary structure analysis was performed on the amino acid sequence of the target protein using a super computer. First, an identified amino acid was synthesised. An antibody was generated by immunizing a rabbit with this amino acid, and the antibody was then purified. Furthermore, the result of tertiary structural analysis of this target protein has suggested that its synthesis for antibody pharmaceuticals can be expected. The target antigenic peptide and cancer cells were transplanted into the artery of a nude mouse, and systemic metastasis was confirmed by CT scan ten days after transplantation. Less
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