| Project/Area Number |
14572064
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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 |
Biological pharmacy
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| Research Institution | Kitasato University |
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Principal Investigator |
INOKOSHI Junji Kitasato University, School of Pharmaceutical Sciences, Assistant Professor, 薬学部, 講師 (30151640)
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| Co-Investigator(Kenkyū-buntansha) |
CHIBA Harumi Kitasato University, School of Pharmaceutical Sciences, Research associate, 薬学部, 助手 (90276163)
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| Project Period (FY) |
2002 – 2003
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| Project Status |
Completed (Fiscal Year 2003)
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| Budget Amount *help |
¥2,200,000 (Direct Cost: ¥2,200,000)
Fiscal Year 2003: ¥700,000 (Direct Cost: ¥700,000)
Fiscal Year 2002: ¥1,500,000 (Direct Cost: ¥1,500,000)
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| Keywords | Actinohivin / anti-HIV / recombinant protein / syncytium formation / production / actinomycete / Eschelichia coli / entry inhibitor / アクチノヒビン / 組換えたん白質 / 糖鎖結合モジュール / 高マンノース型糖鎖 |
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| Research Abstract |
Actinohivin (AH) which is a novel anti-HIV protein isolated from the culture broth of Longispora albida gen. novo., sp. novo., consists of 114 amino acid residues. The proposes of this project were to clarify the structure-activity relationship of AH and to obtain the basic evidence for the creation of new low moleculer weight anti-HIV compounds.
AH gene coloned from an AH producing strain was introduced into E. coli expression vector pET30 Xa/LIC to construct the AH expression plasmid. The truncated AHs were amplified by PCR using full length AH encoding gene cloned in pBluescript II SK+ as a template. Site-directed mutagenesis on pBluescript II SK+::2K3A was performed using appropriate primers. The mutant constructs were expressed in E. coli and the recombinant proteins obtained were subjected to syncytium formation assay using env-expressing HeLa cells and CD4/CXCR4expressing HeLa cells.
AH consists of highly conserved triple tandem segments. Six kinds of the truncate variations which consist of one or two segments of AH were constructed and tested for the syncytium formation inhibitory activity. It was considered that three segments of AH are essential for the syncytium formation inhibitory activity. Since the mutant AH in which two cysteine residues in a segment 2 were replaced by serine also had a similar inhibitory activity to AH, which indicates that the cysteine residues do not affect the activity of AH. Then, we identified that six amino acid residues, ^<15>D, ^<23>Y, ^<25>L, ^<28>N, ^<32>Y and ^<33>Q between LD and QXW consensus sequence of segment 1 were essential for binding to the sugar chain. Furthermore, the mutant AHs in which the LD-QXW regions of three segments were replaced with that of segment 1(Seg 1 trimer) had increased activities. These results suggest that the region between LD and QXW is important for binding to the sugar chain and the other regions are needed for suitable arrangement of each segment.
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