| Project/Area Number |
03454109
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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 |
基礎獣医学
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| Research Institution | National Institute of Health (N.I.H.) |
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Principal Investigator |
TATSUMI Masashi N.I.H. Dept. Veterinary Science, Senior Researcher, 獣医科学部, 主任研究官 (00133629)
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| Co-Investigator(Kenkyū-buntansha) |
AMI Yasushi N.I.H. Div. Experimental Animal Research, Researcher, 動物管理室, 研究員 (10202699)
MORIKAWA Shigeru N.I.H. Dept. Virology I, Senior Researcher, ウイルスI部, 主任研究官 (00167686)
YAMADA Yasuko N.I.H. Div. Experimental Animal Research, Senior Researcher, 動物管理室, 主任研究官 (20158223)
MATSUURA Yoshiharu N.I.H. Dept. Virology II, Chief, ウイルスII部, 室長 (50157252)
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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 |
¥6,500,000 (Direct Cost: ¥6,500,000)
Fiscal Year 1992: ¥2,100,000 (Direct Cost: ¥2,100,000)
Fiscal Year 1991: ¥4,400,000 (Direct Cost: ¥4,400,000)
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| Keywords | Cynomolgus monkey / CD4 / CD8 / Molecular cloning / Chimeric CD4 / in vitro expression / SIV / HIV / 形質転換細胞 / CD4遺伝子、 / CDRー1領域、 / PCR, / アミノ酸置換 / cDNA / 塩基配列決定 |
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| Research Abstract |
To elucidate the interaction between SIV and monkey CD4 and CD8 molecules, we attempted molecular cloning and to compare those with human counterparts. Cynomolgus monkey thymocyte cDNA library was constructed in a lambdaZAP phage vector system by inserting cDNA synthesized from thymocyte mRNA with Gubler-Hoffmann method, and screened by plaque hybridization with PCR-generated putative monkey CD4 and CD8 gene fragments. The coding region of monkey CD4 gene was revealed to consist of 1377 nucleoside acids and show high homology (95%) to human CD4 gene. Alignments of deduced amino acid sequences revealed that only one amino acid was substituted in both signal peptide and transmembrane domains, and that there was no difference in its cytoplasmic domain. The substitution of amino acids was concentrated on CDR-1 region of extracytoplasmic Ig-like V1 domain, suggesting the possible conformational change in this region reported to be important for HIV binding. We constructed mammalian expression vectors with monkey, human and their chimeric CD4 genes and established Hela transformants expressing respective CD molecules on the cell surface. The susceptibility of these cells to HIV and SIV was examined by synthcium formation and by detection of provirus with PCR method. HIV could infect not only transformant expressing human V1 domain but also monkey V1 domain suggesting that monkey CD4 molecule might serve as the cellular receptor to both SIV and HIV.
The coding region of monkey CD8 gene was revealed to consist of 708 residues and also show high homology (95%) to human CD8. Alignments of deduced amino acid sequences displayed that the substitution was distributed evenly in the entire coding region, differing from monkey CD4. Expected 26KDa molecule was produced by in vitro translation method with monkey CD8 gene.
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