Chemistry: natural resins or derivatives; peptides or proteins; – Peptides of 3 to 100 amino acid residues – 8 to 10 amino acid residues in defined sequence
Reexamination Certificate
2000-02-22
2002-11-05
Borin, Michael (Department: 1631)
Chemistry: natural resins or derivatives; peptides or proteins;
Peptides of 3 to 100 amino acid residues
8 to 10 amino acid residues in defined sequence
C530S330000, C514S015800
Reexamination Certificate
active
06476189
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates to antibacterial peptides and antibacterial agents containing such peptides as an effective ingredient.
2. Description of the Related Art
An intention of keeping our living environmnent dean and comfortable has been emphasized by sensational prevalence of pathogenic
Escherichia coli O-
157, etc. On the other hand, drug resistant microbes such as MRSA are developed as a result of excessive use of antibiotics, which would throw a shadow of feature uneasiness.
Various kinds of antibacterial agents including antibiotics have been provided and used not only for medical purposes but as a variety of so-called antibacterial goods.
Antibacterial peptides have been getting popular in the course of developing antibacterial agents.
The above mentioned peptides are present in semen or blood serum of mamals and is considered as one of the most noteworthy antibacterial ingredients because of a wider antibacterial spectrum and high safety, as well as infrequent appearance of drug resistant microbe by use thereof.
Existence of an antibacterial peptide derived from body fluid of insects has been known as such peptides. Further, it has been known that various kinds of antibacterial peptides are derived in body fluid of insects when bacteria or different blood corpuscles are inoculated to insects or stimulation is given to a body surface thereof by wounding, etc.
For example, an antibacterial active peptide has been identified, which is derived from body fluid of a larval
Zophobas atratus
, an insect of Tenebriodae (see, J. Biol. Chem., vol. 266, pp. 24,524 to 24,525, 1991).
Further, physical and chemical properties of an antibacterial peptide named as Coleoptericin have been investigated.
On the other hand, an antibacterial peptide of cecropin group which is derived from body liquid of larval
Hyalophora cecropia
, an insect of Lepidoptera, itself and physical and chemical properties thereof have been found and investigated.
Antibacterial peptides derived from these insects have a wide antibacterial spectrum and accordingly are considered to bear important relation to biological defense of such insects which are lack of antibody forming ability.
One of problems associated with these antibacterial peptides includes antigenicity. Each of antibacterial peptides has not so low molecular weight and can be antigen in the body when they are vascularly administered as an antibacterial agent.
Because of the wide but somewhat biased antibacterial spectrum, there are not necessarily provided antibacterial peptides which are sufficiently effective to both Gram-positive and Gram-negative bacteria.
Further, there is no gainsaying possible development of some cytotoxicity when these insect-derived antibacterial peptides are given to mammals.
SUMMARY OF THE INVENTION
Accordingly, it is an object of the present invention to provide antibacterial measure by use of antibacterial peptides which overcome conventional problems as described above.
In order to solve conventional problems, structure and functions of an antibacterial peptide derived from insects of Lepidoptera such as Scarabaeidae, Tenebriodae, etc., especially
Oryctes rhinoceros
which is an insect of Eophileurus chinensis of Scarabaeidae have been investigated as an insect-derived peptide by the present inventor.
It is known that an antibacterial peptide is induced in body liquid of larva
Oryctes rhinoceros
when stimulated by, for example, wounding. Using cDNA of larva
Oryctes rhinoceros
, its nucleotide sequence encoding an antibacterial peptide (hereinafter referred to as natural antibacterial peptide gene) was identified.
Then, amino acid sequence of an antibacterial peptide derived from
Oryctes rhinoceros
(hereinafter referred to as natural antibacterial peptide unless otherwise noted) was estimated from the nucleotide sequence thus identified. Development of novel antibacterial peptides has been done on the basis of information of such amino acid sequence.
[1] Nucleotide sequence identification of natural antibacterial peptide cDNA
The nucleotide sequence of natural antibacterial peptide cDNA was identified by a conventionally known method as in the following.
(1) The natural antibacterial peptide was extracted from larva
Oryctes rhinoceros
and purified to identify N-terminal amino acid sequence thereof Based on the amino acid sequence, a degenerate primer was synthesized. In addition, another degenerate primer was also synthesized based on the conserved amino acid sequence of the C-terminal region of the insect antibacterial peptide family designated defensive.
(2) Using these oligonucleotides as primers, a portion which would code the antibacterial peptide cDNA, was subjected to manipulation of gene amplification (PCR method).
(3) The nucleotide sequence of PCR product was determined.
In order to identify N-terminus of the natural antibacterial peptide in the above mentioned process (1), separation and purification of the natural antibacterial peptide from larva
Oryctes rhinoceros
were carried out as in the following.
Ten of the third instar larvae of
Oryctes rhinoceros
were kept on ice for several minutes to slow down their movement and wounded by piercing a hypodermic needle. The thus wounded larvae were kept in a breeding box with leaf mold for 24 to 48 hours at a temperature of 25° C.
After cutting legs of wounded larva
Oryctes rhinoceros
, drops of their body liquid was squeezed and collected in a tube on ice by pressing abdomen thereof. As a result, about 1.5 ml of body liquid was obtained from each larva.
After the above mentioned body liquid was subjected to centrifugal separation (39,000× g) for 50 minutes, a blood corpuscle component was removed and the supernatant thus obtained was kept at a temperature of−20° C. Then, 15 ml of the supernatant was applied to Sep-Pak C18-cartridge (available from Waters Associates Co., Ltd.) which had been equilibrated with a solvent A containing 20% of acetonitrile, followed by washing the column with the solvent A, to elute an antibacterially active substance (active fraction) with a 20%-acetonitrile/0.05%-trifluoroacetic acid solution.
The active fraction was freeze-dried to remove acetonitrile and form a dried powder, which was dissolved in a 0.05%-trifluoroacetic acid solution to apply to Resource RPC Column (1 ml; available from Pharmacia Co., Ltd.) which had been equilibrated with a 0.05%-heptafluorobutytir acid (HFBA) solution.
The column was washed with the 5%-HFBA solution, followed by gradient elution under a condition of 0% to 20%-acetonitrile/0.05%-trifluoroacetic acid solution for 1.25 minutes; similarly a 20% to 40%-solution for 10 minutes and a 40% to 100%-solution for 1.25 minutes. It was observed that an 38 to 40%-acetonitrile fraction is antibacterially active.
The antibacterial activity was observed as in the following.
To 0.99 ml of a bouillon medium prepared by dissolving 1 g of beef extract (available from Difco Co., Ltd), 2 g of Bacto-peptone (available from Difco Co., Ltd.) and 1 g of NaCl in 200 ml of water, 10 &mgr;l of bacteria suspension prepared by culturing
Staphylococcus aureus
in bouillon medium overnight was added and cultured up to period of logarithmic multiplication.
To bouillon-agar medium prepared by adding 1.5% by weight of agar to the bouillon medium, 200 &mgr;l of the thus obtained culture solution was added and poured to a laboratory dish to gelate it. Then, a well of 2 mm in diameter was formed on the gelled bouillon-agar medium by means of a gel puncher (available from Parmacia Co., Ltd.). Five &mgr;l of 38 to 40%-acetonitrile fraction (sample) dissolved in water was poured into the well and cultured overnight at a temperature of 37° C. to observe a hollow formed thereon.
A procedure described above was repeated similarly.
The active fraction was further freeze-dried to remove acetonitrile and form a dried powder, which was dissolved in the solvent A to apply to a column (3.2×30 mm, &mgr; RPC C2/C18 PC3.2/3 column, available from
Ishibashi Jun
Sakanaka Hisako
Yamakawa Minoru
Borin Michael
Flynn ,Thiel, Boutell & Tanis, P.C.
National Institute of Agrobiological Sciences
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