| Co-Investigator(Kenkyū-buntansha) |
YOSHIMURA Kentaro Akita University, School of Medicine, Emeritus Professor (2001 only), 医学部(平成13年度のみ), 名誉教授(平成13年度のみ) (90053058)
ISHIDA Kazuto Akita University, School of Medicine, Research Associate, 医学部, 助手 (60006731)
MATSUDA Shinji Akita University, School of Medicine, Associate Professor, 医学部, 助教授 (70199800)
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| Budget Amount *help |
¥2,200,000 (Direct Cost : ¥2,200,000)
Fiscal Year 2002 : ¥800,000 (Direct Cost : ¥800,000)
Fiscal Year 2001 : ¥1,400,000 (Direct Cost : ¥1,400,000)
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| Research Abstract |
Our previous data show that eosinophils increased in cerebrospinal fluid are involved in the killing of intracranial worms in mice infected with Angiostrongylus cantonensis. However, It is still unclear what effector molecules are released from eosinophils to kill the worms. In this study, therefore, we attempted to produce recombinant major basic protein-1 (MBP-1), one of basic proteins containing eosinophil granules. Total RNA was extracted from bone marrow of infected mice or IL-5 transgenic mice and reverse-transcribed to cDNA. Mouse mature MBP-1 cDNA coding from C-terminal to N-terminal (GENBANK L46768, Larson et al., 1995) was amplified by PCR. The MBP-1 cDNA was ligated into the plasmid vectors, pIVEX2.3 fusing the gene with a C-terminal his-tag and pIVEX2.4a, b, c with a N-terminal his-tag gene. Sequencing analysis showed that all sequences, except to one base (silent after translation), were identical with those of GENBANK L46768. Three plasmids ligated with MBP-1 cDNA were ta
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nsformed with E. coli and amplified by large scale plasmid preparation method. Purified pIVEX2.3 and pIVEX2.4c were applied on in vitro protein expression system (Rapid Translation System RTS 500, Roche Molecular Biochemicals). However, It is sill unsuccessful to get mature MBP-1 protein, suggesting that MBP-1 may be highly cytotoxic. Next, we examined a deposition of MBP on the surface of intracranial worms or on the brain tissure. Rabbit anti-mouse MBP polyclonal antibody was kindly provided by Dr. James Lee in Mayo Clinic Scottsdale Research in USA. Male C57BL/6 mice were infected with 24 third stage larvae and killed from days 8 to 22 p.i. At necropsy, brains were fixed in formalin and paraffin sections were prepared. According to the protocol in Dr. Lee's lab, MBP was immunohistochemically stained. Prominent eosinophil infiltration was noted in pia-arachnoid and MBP particles were distributed around eosinophils. Interestingly, degenerating intracranial worms were found surrounded by numerous eosinophils and MBP was deposited on the cuticular surface of the worms. These results indicate that MBP is one of possible effector molecules from eosinophils for worm killing. Less
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