| Project/Area Number |
03454175
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| Research Category |
Grant-in-Aid for General Scientific Research (B)
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| Allocation Type | Single-year Grants |
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| Research Field |
Experimental pathology
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| Research Institution | Tokai University School of Medicine |
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Principal Investigator |
MORIUCHI Tetsuya Associate Professor Department of Molecular Life Science, 医学部, 助教授 (20174394)
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| Co-Investigator(Kenkyū-buntansha) |
TANIGUCHI Yasushi Instructor Department of Molecular Life Science, 医学部, 助手 (30207188)
YOSHIMURA Shinichi Instructor Department of Molecular Life Science, 医学部, 助手 (30230808)
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| Project Period (FY) |
1991 – 1992
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| Project Status |
Completed (Fiscal Year 1992)
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| Budget Amount *help |
¥6,600,000 (Direct Cost: ¥6,600,000)
Fiscal Year 1992: ¥1,300,000 (Direct Cost: ¥1,300,000)
Fiscal Year 1991: ¥5,300,000 (Direct Cost: ¥5,300,000)
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| Keywords | Human / Mouse / Homeobox Gene / Antibody / Recombinant Protein / Immunohistochemistry / ホメオボックス蛋白 / 大腸菌発現ベクタ- / マルト-ス結合蛋白 / 蛋白精製 |
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| Research Abstract |
A whole cDNA fragment of homeobox 4B was inserted into the expression vector pIH888 and the recombinant plasmid was transfected to E.coli JM109. In this experiment, transformants were not obtained. The expression of homeobox sequence was suspected to be toxic to E.coli. Therefore, the second exon of Hox4B gene, which contains the homeobox sequence, was removed and the first exon was inserted into pIH888 (designated pIH 4Bb). Transformants were obtained by transfection of the pIH4Bb to JM109. Thereafter, translation termination codonTAG was inserted at the end of the first exon of Hox4B(pIH 4Bc). The nucleotide sequence analysis revealed that a linker, the first exon of Hox4B, termination codon and a linker were ligated in-frame for the translation to the fusion protein. Transformants were obtained by transfection of the pIH 4Bc plasmid to JM109. Proteins from bacterial periplasms were analyzed by SDS-PAGE. A dense band of 57 kDa was observed in the stained gel. The protein of 57 kDa was purified by preparative acrylamide gel electrophoresis. The antibody against Hox4B was obtained by immunization of the fusion protein to rabbits. The antibody was used after purification with an affinity column. The specificity of the antibody was demonstrated by immunoblot analysis using mouse embryo homogenate. The immunoblot revealed a single band of 64 kDa. This antibody was used for the immunostaining of paraffin sections of mouse embryos. Immature chondroblasts, spinal neurons, and medulla of adrenal glands were stained using this antibody.
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