| Co-Investigator(Kenkyū-buntansha) |
OWHASHI Makoto Tokushima Univ. Faculty of Integrated Arts and Sciences Associate Professor, 総合科学部, 助教授 (40128369)
HORII Yoichiro Miyazaki Med. Coll. Faculty of Medicine Research Associate, 医学部, 助手 (80173623)
IMAI Junichi Miyazaki Med. Coll. Faculty of Medicine Associate Professor, 医学部, 助教授 (00039918)
丸山 治彦 宮崎医科大学, 医学部, 助手 (90229625)
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| Budget Amount *help |
¥4,400,000 (Direct Cost: ¥4,400,000)
Fiscal Year 1991: ¥2,100,000 (Direct Cost: ¥2,100,000)
Fiscal Year 1990: ¥2,300,000 (Direct Cost: ¥2,300,000)
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| Research Abstract |
Because of limitted availability of Strongyloides stercoralis and because of antigenic complexity, initially our aim of gene cloning was focused on the neutrophil chemotactic factor (NCF) of S. ratti (Sr). By using combination of DE52 ion exchange colum chromatography and SW3000 HPLC or Procion Red affinity chromatograophy, we obtained NCF as a single protein peak. However, this peak was still heterogenous by SDS-PAGE. Furthermore, an attempt to make cDNA library was failed because only about 5000 clones were established from mRNA of Sr-L_3. As an alternative approach, therefore, Dirofilaria immitis (Di) NCF was used as the stating material because it has been already purified by our hand, and Di-NCF epitopes were expressed in several antigenic components of Sr including Sr-NCF by Western-blot analysis. Poly-A-RNA was isolated from Di adult worms and the cDNAs were inserted into EcoRI site of lambdagT11. About 2 x 10^5 clones produced as the cDNA library were further selected by using
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anti-Di-NCF. The phage DNAs of positive clones were purified, fragmented by EcoRI, and clones having 0.7-2.0 Kb inserts were subcloned in to a plasmid vector Bluescript SK(-). Their DNA sequences were determined by dideoxy method using M13 primer. In parallel, N-terminal 45 amino acid sequence of purified Di-NCF was determined by antomatic sequencer. When the expected amino acid sequences from the DNA sequences of four subclones were compared to the actual N-terminal amino acid sequences, subclone named pD4 showed complete homology at the DNA sequence of 268-402 from 5' terminal. Open rcading frame was 166-606 coding the leader sequence of 34 amino acid residues and the Di-NCF sequence of 112 amino acid recidues. By comoputer -aided homology search, 99-101 amino acid sequence (Met-Phe-Lys) had a similarity to known NCF peptide so that this sequence was thougt as the epitope of active site of the Di-NCF. In fact, synthesised Met-Phe-Lys and also fMet-Phe-Lys showed NCF activity at the concentrations lower than of known NCF peptides. The clone pD4 was subcloned into plasmid vector pGEM EX and fusion protein with gene 10 was produced. This fusion protein showed NCF activity and antigenicity similar to those of purified Di-NCF. When mice were immunized with this fusion protein, their susceptibility against Brugia pahagi rather increased, suggesting the induction of blocking antibody or suppressor system. Further study is required as to the methods of immunization. Less
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