| Project/Area Number |
13660330
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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 |
Applied veterinary science
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| Research Institution | Nippon Institute for Biological Science |
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Principal Investigator |
IWATA Akira Nippon Institute for Biological Science, Research Dept. Senior Scientific Stuff, 研究部, 主任研究員 (70193745)
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| Co-Investigator(Kenkyū-buntansha) |
KANAI Tomoko Nippon Institute for Biological Science, Research Dept. Researcher, 研究部, 研究員 (10300790)
YAMAMOTO Akira Nippon Institute for Biological Science, Research Dept. Researcher, 研究部, 研究員 (40290986)
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| Project Period (FY) |
2001 – 2002
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| Project Status |
Completed (Fiscal Year 2002)
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| Budget Amount *help |
¥3,300,000 (Direct Cost: ¥3,300,000)
Fiscal Year 2002: ¥1,600,000 (Direct Cost: ¥1,600,000)
Fiscal Year 2001: ¥1,700,000 (Direct Cost: ¥1,700,000)
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| Keywords | dog / parvovirus / treatment / CDR grafting / canonized / neutralization / monoclonal antibody / Contact determination / パルボウィルス / モノクローナル抗体 |
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| Research Abstract |
(1) Monoclonal antibody (mAb) against a canine parvovirus (CPV) and its cDNA from the hybridoma cells.
CPV was propagated in CRFK cells and purified through density gradient ultracentrifugations. Hybridomas were obtained by immunization of mice with the purified CPV and subsequent fusion of spleen cells with P3U1 myeloma cells. After screening by ELISA, IFA and virus neutralization, six clones (CP1a-6a) were established. RNA was isolated from CP3a, which produced immunoglobulin (Ig) of G1 subtype and showed the strongest binding activity, then the cDNA of Ig light (L) and heavy (H) chain were cloned by PCR, and their nucleotide sequences were determined.
(2) Cloning of cDNA of canine Ig
Spleen cells were isolated a beagle dog, and RNA was extracted, cDNA of Ig was isolated using primer designed from the nucleotide sequences reported previously. Four clones of H chain were consisted of about 470 amino acid residues (aa) and the constant regions have a homology of 60-70% to human or mouse Ig gamma H chains. Two clones of L chain (one kappa (242 aa) and one lambda (231 aa)) were obtained, which have a homology of 58% and 85% in constant region with human Ig, respectively.
(3) Construction of a caninized antibody by complementary determining region (CDR) grafting.
CDR was determined in H and L chain of the CP3a mAb cDNA by the Contact definition. The CDR of canine H and L chains was assumed by the same definition. The caninized Ig gene was designed by replacing the canine CDR with CP3a's. The DNA fragments of H and L variable domains were synthesized by PCR, and conjugated to the DNA encoding corresponding constant region of Ig, respectively. Both DNA fragments were inserted into a transfer vector and the recombinant baculoviruses were obtained. The expression of caninized Ig was demonstrated by anti-canine Ig antisem in the culture supernatant of the Sf21 cells infected with the recombinant baculovirus.
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