JPS6022000B2 - Novel bioactive peptide - Google Patents

Description

[Detailed description of the invention]

The present invention has antiviral effects, cell proliferation suppressive effects, and N
The present invention relates to a novel physiologically active peptide with excellent physiological activity such as K cell activity enhancement, a method for producing the same, and its uses. Conventionally, metabolic inhibitors, alkylating agents, antibiotics, etc. have been used as antiviral agents or cell growth inhibitors, but these are highly toxic and are not preferred. Therefore, the development of antiviral agents or cell proliferation inhibitors with low toxicity is strongly desired. Gichin, interferon-Q
(hereinafter referred to as "IFN-Q") and interferon-8 (hereinafter referred to as "IFN-3") are effective as low-toxicity antiviral agents, and research on these has progressed. However, the cell proliferation inhibitory effects of IFN-Q and IFN-8 are not satisfactory. On the other hand, interferon-
The research on y (hereinafter referred to as "PoN-y") was carried out by IFN-Q.
Research on IFN-B and IFN-B has not progressed much. However, the second annual meeting of the International Conference on Interferon Research (The SecoM Annual Meeting) held in San Francisco, United States in October 2013,
tema0onaI Con scale ess for
In rferonResearch), mN
There was no report that Y was singled out, but David
The sequence of the messenger RNA (hereinafter abbreviated as "mRNA") deoxynucleotide of IFN-y was developed by David V. (Genentech, IM., California, USA). Pro-IFN-y estimated from ``
The amino acid sequences of Ny and Ny-like bebutides have been published. The amide/acid sequence is represented by the following formula (1). Formula (1): JC Scale T's C$ GinAspProT's VaI
LysG1u AIaGIu Asn LeuLys
Lys Tyr Phe AsnNa GIy His
Ser ASp VaI Na Asp Asn G
IyThr LeuPhe LeuGIy ne Le
uL$ AsnTrpLys GIu GIu Ser
Asp Arg L$ lie Met GinSe
r Gin lie VaI Ser Phe Tyr
Phe Lys LeuPhe L scale Asn Ph
e L侭 Asp ASP Gin Ser lieG
in Lys Ser VaI GIu Thr 11
e Lys GIu AspMetAsn VaI L
ysPhe Phe Asn Ser ASn Lys
Lys Lys ArgASp ASpPheGIuL
ys LCuThrASn Tyr Ser VaI
Thr ASp LeuAsn VaI GinA

[g
Lys AIa lie His GIu Leu
lie Gin VaIMet AIa GIu Le
u Ser Pro AIa AIa Lys Thr
o1y L Pine ArgLyS Arg Ser Gin
Met Leu PheAJgGIy ArgFurthermore, at the aforementioned meeting, it was reported that a culture solution of cells containing a gene containing mN-y-like peptide information has antiviral activity, but a culture solution of cells containing a gene containing pro-IFN-y information has antiviral activity. It was also announced that there was no viral activity. On the other hand, unlike the Misaki Ny-like peptide, IFN-y does not have an artificial methionine trailing group added to its N-terminus, so it is possible to use a culture solution of cells containing the gene that has more desirable information on the IFN-y. Manufacturing has not yet been successful. In view of the current situation as described above, the present inventors have conducted intensive research to find a bioactive agent that not only has high antiviral activity but also has excellent cell proliferation inhibiting action and NK cell enhancing action. Therefore, an object of the present invention is to provide a physiologically active bebutide which not only has excellent antiviral activity but also has excellent cell proliferation inhibiting action and NK cell enhancing action. Other objects and advantages of the invention will become apparent from the detailed description below, along with the claims. According to the present invention, a novel physiologically active peptide represented by formula (1) (wherein J represents a hydrogen atom in formula (1)) is provided. The novel bioactive peptide of the present invention can be prepared using the conventional three-seal method, namely, "Synthetic Chemistry Series - Bebutide Synthesis" by Nobuo Izumiya et al., 1975, published by Ohmaruzen Co., Ltd.; Japan Biochemical Association Line, "Biochemical Experiment Course" / Chemistry of protein costs 1-W” 1977
It can be produced by using the method described in the publication published by Tokyo Kagaku Dojin. As one of the embodiments, the solid phase method will be specifically explained below. After the carboxylic acid group of the amino acid having a Q-amino protecting group is bonded to a support such as a polystyrene resin crosslinked with divinylbenzene, the Q-amino protecting group is selectively removed. The free ami/group is reacted with a second amino acid having a Q-amino protecting group. The desired peptide chain is obtained by repeating the removal of the Q-amino protecting group and the introduction of the amino acid having the Q-amino protecting group. Next, the covalent bond between the carboxylic acid group of the C-terminal amino acid of the peptide and the resin is cleaved. At this time, depending on the reagent used to cleave the covalent bond, the Q-amino protecting group or side chain protecting group can be removed simultaneously with the cleavage of the covalent bond.
In the solid-phase method described above, all reactions can be carried out in one container, so the production of peptides is easy in terms of operation, and the obtained product is soluble in the solvent used for the reaction. Since appropriate amounts of reagents and byproducts used in the reaction can be removed by filtration, the desired peptide can be produced in a short time and in high yield, making this method more advantageous than the so-called liquid phase method. The support used in the first method is generally a 2% styrene polymer sealed with divinyl besozene and preferably has a size of 200 to 400 mesh. Before reacting and bonding the resin with amino acids, functional groups are introduced into the resin to impart reactivity. Generally, in order to introduce a functional group into a resin, the aromatic ring of styrene is reacted with chloromethyl methyl ether by a Friedel-Krach reaction using SNC12 as a catalyst, and the aromatic ring of styrene is chloromethylated. method is taken. The Q-amino protecting group used in the first method includes the support and the Q-amino protecting group.
-It is preferable to use one that is stable upon coupling with an amino acid having an amino protecting group and that can be easily removed without adversely affecting the side chain protecting group or peptide chain. Preferred Q-amino protecting groups as mentioned above include tertiary-butoxycarbonyl (hereinafter abbreviated as "B ratio") group. This BM group can be mixed with INHCI/acetic acid mixture, YamaHCI/dioxane mixture or 50% (W/V) T
It can be easily removed from the peptide chain using fOH (tetrafluoroacetic acid)/CH2CI2 mixture. The side chain protecting group is preferably a group that is stable during coupling and removal of the Q-amino protecting group and that can be easily removed at the stage of peptide synthesis. Such preferable side chain protecting groups include N02 or tosyl group for protecting the side chain of arginine, penzyl group (hereinafter abbreviated as "&1") group for protecting the side chain of aspartic acid, cysteine and For protecting the side chain of glutamic acid, a Bzl group or -methylbenzyl (hereinafter abbreviated as "OBz") group is used, and for protecting a side chain of histidine, a Bzl group or -benzyloxycarponyl (hereinafter abbreviated as "Z") group is used. or BM group, Z group, tosyl group or diisopropylmethyloxycarbonyl (hereinafter abbreviated as "DipmM") group for side chain protection of lysine, sulfoxide group for side chain protection of methionine, and serine, Bzl groups are included for side chain protection of threonine and tyrosine. The method for determining the amino acid sequence of the novel bioactive peptide of the present invention is as follows. First, the methionyl bond of the novel bioactive peptide of the present invention is cleaved with bromo cyanide. The peptide fragment obtained by cutting was treated with Sephadex G-100 (Pharmacia).
The amino acid sequence of each fragment is sequentially determined from the N-terminus of each fragment using a well-known high-precision amino acid sequence analysis. On the other hand, the novel bioactive peptide of the present invention was partially degraded with tribucin, and the obtained peptide fragment was
Separate using 100 columns. The amino acid sequence of each separated fragment is sequentially determined from the N-terminus of each fragment in the same manner as described above. The sequence of each peptide fragment is determined by comparing the amino acid sequence of each peptide fragment obtained by cleavage with promosyan and the amino acid sequence of each peptide fragment obtained by cleavage with trypsin. In this way, the amino acid sequence of the novel bioavailable peptide of the present invention is determined. When the purpose is to inhibit cell proliferation, for example, to inhibit the proliferation of malignant tumor cells, the peptide of the present invention can generally be administered by injection intravenously, intramuscularly, or subcutaneously. The daily dosage of the peptide of the present invention will, of course, vary depending on the age, medical condition and weight of the patient, but is usually 1 dose per adult.
It is administered by injection to children of x1 to 1 x 1 units per day. To treat viral diseases, an ointment containing lxl to lxl units of the peptide of the present invention can be applied to the skin 10 times a day. Ointments containing the peptides of the present invention can be prepared using conventionally known pharmaceutically acceptable ointment bases.
The daily dosage of the ointment containing the peptide of the present invention will of course vary depending on the age and medical condition of the patient, but it is usually an amount of 1 x 1 to 1 x 1 unit based on the peptide of the present invention. Can be divided and applied. EXAMPLES Hereinafter, the present invention will be explained in more detail with reference to examples, but the present invention is not limited to the following examples. Explanation of abbreviations used in the examples - Z-lysine: Penzyloxycarbonyl group (Z-) is bonded to the -amino group of lysine G-N02-Arginine: Nitrogen is bonded to the guanidino group (G-) of arginine. imBzl-histidine: a compound with a pendyl group (im) attached to the imidazolyl group (im) of histidine
Bzl-) is bonded to ○-Bzl-serine: A pendyl group (Bzl-) is bonded to the hydroxyl group (0-) of serine BX-valine: A tert-butoxycarbonyl group (8X-) is bonded to the Q-amino group of valine. are combined, OBzl
: Orthomethylbenzyl group V, which is one of the side chain protecting groups
SV: Vesicular stomatitis virus (Vesic arSPm)
atitisVims) AMV: Avian Myeloblastosis Virus (sisVir vessel of AvjanMyelobias)
BSA: Bovine serum albumin SEA: Staphylococcal enterotoxin A, a type of mitogen Example 1 Step 1 (Washing of resin) Resin [Bio-Bead S-1 x 2, 200-400 mesh (2% divinylbenzene cross-linked) Product name of styrene polymer, manufactured by Bio-Rad, USA)] 150
Add the liquid to the distilled benzene while stirring. After 30 minutes, the resin is filtered through a glass filter and dried. Next, the dried resin was added to the methanol while stirring, and after 30 minutes, it was removed in an oven using a glass filter and dried. The resin obtained by drying is washed successively with tatanol, methanol/water and water. The washed resin is placed in IZ INNa.
Add to OH aqueous solution. After heating the resulting mixture in a boiling water bath for 1 hour, the resin is removed from the oven and washed with water. The resulting resin is then added to an IN aqueous solution of hydrochloric acid and simmered in a boiling water bath for 1 hour. Next, the resin is taken out of the oven and washed with water.
The resin obtained by washing with water is suspended in distilled water, and then left to stand. After the Shizuma 3 piece, the floating fine resin is removed by decantation. In this way, the resin is obtained as a precipitate. The precipitated resin is filtered onto a glass filter and washed with methanol. The washed resin was added to a small amount of dimethylformamide (hereinafter abbreviated as "DMF"), heated at 8,000 ℃ for 30 minutes, and then removed from the furnace and D
Wash with MF and methanol. Further, the resin was boiled in methanol for 1 hour and then taken out. After the resulting resin is air-dried, it is further vacuum-dried at 100 oo for 2 hours. Step 2 (manufacture of chloromethyl resin) Put 25 g of the resin obtained in step 1 and 100 g of distilled chloromethyl methyl ether into a round-bottom three-necked flask, and stir gently at 25 g for 1 hour. After the resin is swollen, it is cooled to 0°C. 1. To this swollen resin. 5 containing 14 in S of machine'
Add methyl methyl ether to the mixture over a period of 30 minutes while keeping the temperature at 0°C, and then stir for another 30 minutes to obtain a reaction product. The product thus obtained is filtered through a glass filter and gently washed with 500 parts of dioxane-water (1:1) and then with 500 parts of dioxane-water and hydrochloric acid (3:1). Furthermore, the product is mixed with water,
After sequentially washing with dioxane-water and methanol, it was dried under reduced pressure at 100 pm. Step 3A (Go-Z-lysine)
L-lysine steel salt prepared on November 11th, NaHC03 and 1 prepared on October 10th.
2. Add a quarter of the Z-CI on the table every 10 minutes while keeping it cool on ice, and stir for another 3.5 hours. The precipitate thus obtained is filtered off and washed successively with water, ethanol and acetone/ether. The washed precipitate was suspended in 250' of water and then this suspension was
The above precipitate is dissolved by adding 12' aqueous carp hydrochloric acid solution. Then, the resulting solution is added with a specific S gas of 1~
Vent for 2 hours. The resulting C-taka is removed using a Hyflo Super Cell (Wako Pure Chemical Industries, Japan),
Wash the uS with IN aqueous hydrochloric acid solution. The furnace liquor and wash liquor obtained in the above operation are collected and passed through air to remove the ratio S.
Then, this solution was adjusted to pH by adding concentrated ammonia water under ice-cooling.
6.5 and leave it in the refrigerator for 2 hours. The resulting precipitate is filtered off and washed successively with water, ethanol and ether. In this way, melting point 25400-Z-
10 ml of lysine is obtained. The yield of Do-Z-lysine produced is 65%. The obtained Go-Z-lysine is recrystallized by dissolving it in dilute hydrochloric acid and then neutralizing it with aqueous ammonia.
B (G-N02-Arginine) A mixture of oleum nitric acid 120 and fuming sulfuric acid 75 is cooled on ice, and 11M L-arginine hydrochloride is gradually added to the mixture while stirring. Furthermore, 45' of concentrated sulfuric acid is added to the above mixture. After the resulting mixture was allowed to stand for 1 hour, it was poured into ice cubes and the pH was adjusted to 8 with concentrated aqueous ammonia. Next, the mixture was adjusted to pH with acetic acid.
After adjusting the temperature to 6, leave it in the refrigerator for about 4 hours. The resulting precipitate is collected in an oven and recrystallized from hot water. The obtained crystals are sequentially washed with ethanol and diethyl ether and dried. In this way, G-N02-arginine with a melting point of 251° C. is obtained. The yield of G-N02-arginine is 50%. C (
jmBzl-histidine) 20 evening L-histidine hydrochloride is dissolved in 200' liquid ammonia cooled to -30 qo in a dry ice-acetone chamber, and a small piece of metallic sodium is added to this solution until the blue color of the solution disappears. Add the mixture while stirring until it remains. A small amount of histidine is then added until the blue color of the solution disappears, and then penzyl chloride is added dropwise to the solution. After this mixed solution is rope-pressed for 30 minutes, the N ratio in this mixed solution is evaporated at room temperature under atmospheric pressure, and then the NH3 is removed under reduced pressure using a water jet pump. Pour the liquid after NH3 removal into 100 ml of ice water. After extracting the resulting mixed solution with diethyl ether, the remaining residue is removed in an oven. Dilute sulfuric acid is added to the resulting solution to adjust the pH to 8, and the solution is left in the refrigerator for 3 hours. The resulting precipitate is filtered and recrystallized from 7% by weight ethanol. ImBzl-histidine 13 is obtained with a melting point of 248°C. The yield of imBzl-histidine is 50%. D (01 Bzl-serine) Dissolve the acetyl-L-serine of 237 in the aqueous NaOH solution of 235 and adjust the pH to 7.3. Add Takajustase (manufactured by Sankyo Co., Ltd.) to this solution for 4 days.
25' of 0.1M citrate buffer (Book 6.7, Ca
After adding a solution obtained by dissolving in C120.029M (containing 0.029M) and adding several drops of toluene, the resulting mixture was
Leave in CO for 10 days. After removing the resulting precipitate and concentrating the solution, acetone is added to the concentrated solution to obtain crystals. In this way, 100 grains of 1-serine are obtained. According to a similar method, 100 ml of acetyl-L-threonine is obtained from 240 ml of acetyl-L-threonine. E (0 one loss, 1-tyrosine) 1.8 evening L-tyrosine and 1.2 evening CuS04.8LO, 5 units are NaOH
Suspend it in a solution of 10 parts water and pray for 2 hours. Add 60 methanol to this suspension and then add 1.
Slowly add 2' of pendyl bromide and 0.7' of NaOH aqueous solution. After 15 minutes, additional 0.3' pendyl bromide and 0.8' NaOH aqueous solutions are added. After stirring the resulting mixture for 1 hour, the resulting precipitate is filtered and washed with methanol-water (1:3).
In this way, two days of copper salt of Bzl-tyrosine are obtained. This ○1 Bzl-chi. Copper salt of Shin and 20 [I of
The solid content is collected from the mixture obtained by kneading the NEDTA aqueous solution in a mortar, and the solid content is washed with water. Recrystallization of the washed product from hot water containing a small amount of EDTA yields ○-Bzl-tyrosine with a melting point of 22 years. The yield of 0-Bzl-tyrosine is 30%
It is. F (S-Bzl-cysteine)
Dissolve in an aqueous solution, add 256 grams of pendyl bromide to this solution by vigorously stirring under ice-cooling, and keep in a cold room for 5 hours. The solution is then adjusted to pH 5 with acetic acid to form a precipitate. The generated precipitate is taken out from the furnace and washed with water. In this way, S-Bzl-cysteine with a melting point of 2120°C is obtained. The yield of S-Bzl cysteine is 70%. In the same manner as described above, 150 days of ○-Bzl-aspartic acid is obtained from 160 days of aspartic acid, and 150 days of ○-Bzl-glutamic acid is obtained from 160 days of glutamic acid. G (corrosion-amino acid) Water is added to a solution obtained by dissolving 29% L-valine in 250% INNaOH aqueous solution to make a 400% solution. After adding 150 tons of tetrahydrofuran to this solution, 1
Add B above 100 to the solution while stirring vigorously at 000.
Add one-fifth of M-CI every 10 minutes. On the other hand, state N
Add aOH to the solution each time the Boc-CI is added to maintain the pH of the solution at 8-9. After 2 hours, the mixture is extracted with diethyl ether, the resulting aqueous layer is made acidic with a 0.8M aqueous citric acid solution, and the precipitated oil is extracted with ethyl acetate. The resulting extract is washed with a small amount of water, dried over Na2SO4, and concentrated under reduced pressure. Add petroleum ether to the concentrated extract and leave it in the refrigerator to form crystals. The resulting crystals are taken out and dried. In this way, BM with a melting point of 78 qo
- Obtain 30 evenings of balin. The yield of B-valine is 55%. (Other BX-amino acids) By performing the same method as above, BX-glycine 1
5 evenings, B ratio - alanine 16 evenings, B ratio - o isin 15 evenings,
BX-isoleucine 15 times, BM-serine (Bzl) 1
4 evenings, B-threonine (Bzl) 14 evenings, regular c-cysteine (BZI) 15 evenings, European c-methionine 15 evenings, B
M-proline 12m, Boc-aspartic acid (OBz
l) 15 evenings, Boc-glutamic acid (OBzl) 15 evenings, European c-glutamine 15 evenings, BoC-histidine (Bz
l) 12 evenings, BX-leucine (Z) 13 evenings, BM-arginine (N02) 15 evenings, BX-phenylalanine 15 evenings
evening, BX-tyrosine (Bzl) 15 evenings and BX-tryptophan 10 evenings, glycine 30 evenings, alanine 30 evenings, leucine 30 evenings, isoleucine 30 evenings, serine (Bzl) 30 evenings, stereonin (Bzl) 30 evenings, cysteine ( &1) 30 evenings, methionine 30 evenings, proline 30 evenings, aspartic acid (0&1) 30 evenings, glutamic acid (OBzl) 30 evenings, glutamine 30 evenings, histidine (Bzl) 30 evenings, lysine (Z) 30 evenings, arginine (
N02) 30 evenings, phenylalanine 30 evenings, tyrosine (
Bzl) 30 days and tryptophan 30 days. Step 4 (Coupling of glutamine and resin in 8) 2 chloromethyl resin (4 mmol of total CI), 2 mmol of B-L-glutamine, 1.8 mmol of triethylamine and 12' of dimethyl The formamide mixture is shaken at room temperature for two periods. Next, the solid content is removed from the mixture using a glass filter, and the resulting solid product is sequentially washed with 700 g of dimethylformamide, ethanol, acetic acid/ethanol, and methylene chloride. The product is then dried at room temperature under reduced pressure. To measure the amino acid content of the product, 50M of the product was weighed and hydrolyzed in a 1:1 volume ratio mixture of aqueous hydrochloric acid and dioxane for 2 hours, and then measured using an amino acid analyzer. do. The amino acid content of the product is 0.2 mmol/day. Step 5 (BX-Se
r-Ginn resin) The 2-day-old c-glutamine (hereinafter abbreviated as "B-Gin") resin obtained in step 4 was placed in 160% acetic acid and left at room temperature for 6 hours. This mixture is then filtered through a glass filter. The resulting solid was placed in 160 ml of acetic acid, shaken, and filtered. Repeat this operation three times. Next, the above solid was placed in an IN aqueous solution of hydrochloric acid/acetic acid with 160 kg at room temperature.
Shake for 0 minutes to remove BM groups from the resin. The product thus obtained is taken out of the oven and sequentially washed three times each with acetic acid, ethanol and dimethylformamide. A neutralization reaction is carried out by placing the washed product in 1:160% by weight triethylamine nodimethylformamide and shaking for 10 minutes. The resulting product is then taken out and washed with dimethylformamide and then with methylene chloride. The washed product is placed in a solution of 2 mmol of BM-serine (Bzl) in 12 methylene chloride and shaken. After 1 minute, a solution of 20 mmol of dicyclohexylcarbodiimide in 60 volumes of methylene chloride is added to this mixture, and the coupling is effected by shaking at room temperature for 2 hours. The resulting product is taken out of the oven and washed successively with methylene chloride and ethanol. Step 6 (B-beptidyl resin) Repeat the same operation as step 5 to elongate the amino acids to obtain BX-beptidyl resin. Step 7 (cleavage of peptide from the resin) The 1.4-day knot c-beptidyl-resin obtained in step 6 and the anisole of 1 were covered with polytetrafluoroethylene in a rope sieve. Put into a reaction vessel, introduce 20% of hydrogen fluoride into the reaction vessel while cooling with dry ice-acetone, and after heating at 0°C for 1 hour, remove the hydrogen fluoride in the reaction vessel under reduced pressure at 0°C. do. A 1% by weight aqueous acetic acid solution is added to the resulting residue to extract the peptides, and the extract is placed in a separating funnel, washed with ether, and freeze-dried. In this way, peptides without protecting groups are separated. Step 8 (Physical properties of synthetic peptides) The molecular weight, amino acid composition, and amino acid sequence of each peptide obtained in Step 7 are measured. The molecular weight of each peptide is determined from the moving temperature by SDS gel flea spectroscopy. The amino acid composition (mol %) is determined by hydrolyzing a certain amount of each peptide in an aqueous carp hydrochloric acid solution at 110° C. for 24 hours, and subjecting the resulting mixture to two-dimensional paper chromatography. Tryptophan, cystine and cysteine are not detected by this method. Therefore, by decomposing another fixed amount of each peptide with a protease, the composition of tryptophan and cystine or cysteine is determined. Note that glutamic acid and glutamine, and aspartic acid and asparagine cannot be distinguished. The amino acid sequence of each peptide is
Analyze using an automatic amino acid analyzer manufactured by Hitachi. Step 9 (Measurement of antiviral activity) The antiviral activity of each peptide bound in step 7 was measured by P.C. Merrigan, a monolayer of human neonatal dermal fibroblasts and a monolayer of bovine vesicular stomatitis virus. Evaluation of inhibition of plaque formation using human interferon - "In vitro method for cell-mediated immunity", published by DP R Bloom and P R Grade Academic Press, New York 1971, p. 489 [P
．． C. Merjgan, PIaq Fertilizer Inhibiti
on ASsay for Human Inteher
on Employing Human NeoMte
SkinFibroblaStMonolayerS
& BoVj ship VestiC mountain arS matit
is Vims,''ln-vitroMethod
nCell-Mediated Immunity”,
edi d by E. D. B. R. Bloom &
P. R. Grade, A upper academic pres
s, N. Y. 1971, pp. 489] and the same method. Specifically, each bebutide obtained in step 7 is diluted variously and placed in a growth medium containing 1% by volume fetal calf serum. In this medium, FS-4 cells (human neonatal dermal fibroblasts) are cultured in a single cell. After 1 plaque hour, marine stomatitis virus (V
SV) and cultured for an additional 1 hour at 370°C. Next, the cells are washed twice with the above growth medium, and then cultured again in the growth medium 370 for 2 hours. Whether or not a virus has been generated is examined by microscopically observing the cells after culturing. The resulting cell damage is due to the virus. The peptide exhibits antiviral activity at a concentration of 1 muta/ml. Step 10 (Measurement of cell proliferation inhibitory effect) 2×1 FS-4 cells were transplanted into the growth medium described in Step 9 in a 6-diameter cellar. After 5 hours, the growth medium was removed and, instead, fresh medium containing the peptides obtained in step 7 at a concentration of 0.1 overnight/secretion was poured into the above-mentioned petri dish and cultured at 370 °C for 1 hour. continue. After culturing, the medium is removed, and a medium containing 3-day-thymidine at a concentration of 5 rCi/c is added, followed by further culturing at 370° C. for 2 hours. After culturing, the cells were washed with IJ acid buffered saline, and then
Add 5% by weight trichloroacetic acid to the washed cells. A certain amount of the resulting precipitate was dried and placed in a liquid scintillation counter (Packard, USA).
Measure the radioactivity of the dried precipitate after 3 days. For comparison, David V.
Pro-IFN-y, IF elucidated by G℃eddel)
Peptides having the same amino acid sequences as the N-y and IFN-y uniform peptides are prepared by the solid-phase synthesis method described above, and the cell proliferation inhibitory effect is measured by the method described above. The results are shown below. Professionals obtained in step 9
The results of the antiviral activity of -y and Ny-like peptides are shown below. A case in which antiviral activity is observed without dilution and not observed in a twofold dilution is defined as 1 unit, and a case in which antiviral activity is observed in an n-fold dilution but not observed in a mechanical dilution is defined as an n unit. From the above, BromN-y has no ability to suppress cell growth, and IF
The cytostatic ability of N-y and IFN-y-like peptides is 2.
You can see that it is 0%. BX-bebutidyl resin obtained in step 6: I BMC (BZI) Tyr (BZI) C (BZI) Gin mother p
(OBZI) ProTyr (BZI) VaI L$ (Z
)GIu(OBzl)NaGIu(OBzl)AsnL
euLys(Z)L$(Z)TM(Bzl)PheAs
nAIaGIyHis(BZI)Ser(BZI)mother p
(OBZI) VaI Ala*p (OBZI) $nGl
ymr(BZI)LeuPheUuGlyneLeuL
侭(Z)AsnTrpLys(Z)GIu(OBzl)
GIu (OBzl) Ser (Bzl) Matsup (OBZI)
A[g(N02)Lys(Z)lieMetGinSe
r(Bzl)GinlieVaISer(Bzl)Ph
eTyr (Bzl) Phe Lys (Z) u Phe
Lys(Z)AsnPheL Momme(Z)Asp(0 losses 1
)Asp(OBzl)GinSer(Bzl)lieG
inLys(Z)Ser(&1)VaIGIu(OBz
l)Thr(Bzl)lieLys(Z)GIu(OB
zl)Asp(OBzl)MetAsnVaILys(
Z) PhePheAsnSer (BZI) false nLys (
Z)Lys(Z)L$(Z)Arg(N02)Asp(
OBzl) $p (OBzl) PheGIu (OBzl)
Lys(Z)JuThr(Bzl)AsnTyr(Bz
l) Ser(BZI) VaIThr(BZI) mother p(O
BZI) AsnVaIGinArg (N02)L
侭(Z)AIalieHis(&1)GIu(OBzl
) Buddha uliieGinVaIMetAIaG1u (0 & 1
) Buddha u Ser (Bzl) Pro AIa AIaLy
s(Z)Thr(BZI)GIy L$(Z)Arg(
N02) L (Z) A (NQ) Ser (BZI) Gi
nMetLeuPheArg(N02)GIyArg(
N02)~g(N02)AIaSer(Bzl)Gin
-Resin, 2 BMMetC$(Bzl)TM(Bzl)
Cys(Bzl)GinAsp(OBzl)ProTy
r(Bzl)VaILys(Z)GIu(OBzl)A
IaGIu (OBzl) Asn Buddha uLys (Z) L I (
Z)T(BZI)PheAsnNaGIyHis(&
1) Ser(Bzl)Asp(0&1)VaIAIaA
sp(OBzl) $nGIyThy(Bzl) Buddha uPh
e LeuGIy ne LeuLys(Z)AsnT
rpLys(Z)GIu(OBzl)GIu(OBzl
)Ser(&1)Asp(OBzl)~g(N02)L
ys(Z)lieMetGinSer(Bzl)Gin
neVaISer (Bzl) PheTyr (BZI)
PheL Ore(Z)LeuPheLys(Z)AsnPh
eL$(Z)Asp(OBzl)Asp(OBzl)G
inSer(Bzl)lieGinLys(Z)Ser
(Bzl) VaIGIu (OBzl) Thr (Bzl)
lieLys(Z)GIu(OBzl)Asp(OBz
l) MetAsnVaILys(Z)PhePheAs
nSer(BZI) False nLys(Z)Lys(Z)Lpine(Z)Arg(N02)Asp(OBZI)Fup(OB
ZI) PheGlu (OBZI) Lys (Z) Buddha uTh
r(BZI)Asn Tyr(BZI)Ser(Bzl
) VaIThr (Bzl) $p (OBzl) ratio uAsn
VaIGinArg(N02)Lys(Z)AIali
eHis(&1)GIu(OBzl)julieGin
VaIMetAIaGlu(0&l)Ju Ser(B
ZI)Pro AlaAlaLys(Z)Thr(B
ZI) GIy L 侭 (Z) Arg (N02) L pine (Z)
Arg(N02)Ser(BZI)GinMetLeu
PheArg(N02)GIy Arg(NQ)~g(
N02) AIaSer (Bzl) Gin-resin, 3 8
MetLys(Z)T〆(Bzl)Thr(Bzl)
Ser(BZI)T×(BZI)lie Buddha u NaPh
eGinUuC Matsu(&1)lieValuiuGIySe
r (Ship 1) Uu GIyC 侭 (BZI) T's (BZI
)C$(BZI)Gin fake p(OBZI)ProTyr
(BZI) VaILys (Z) Giu (OBzl) AI
aGIu(OBzl) Asn ratiouLys(Z)L侭(Z
)TM(BZI)PheAsnNaGIyHis(Bz
l)Ser(Bzl)Asp(OBzl)VaIAIa
Asp (OBzl) AsnGIyThr (Bzl) Buddhau Phe LeuGIy ne LeuLys (Z)
AsnTrpLys(Z)Glu(OBZI)GIu(
0board 1) Ser(Bzl)Asp(OBzl)ng(N
02) Lys(Z)lieMetGinSer(Bzl
)Gin neVaISer(Bzl)PheTyr(
BZI)PheLys(Z)LeuPheLys(Z)A
snPheL侭(Z)Asp(OBZI)Asp(OB
zl) GinSer(Bzl)lieGinLys(Z
)Ser(Bzl)VaI Gin(OBzl)Thr
(Bzl)lieLys(Z)GIu(OBzl)As
p(OBzl)MetAsnVaILys(Z)Phe
PheAsnSer(BZI) GoldnLys(Z)Lys
(Z)L is (Z)Arg(N02)Asp(OBZI)
pseudop(OBZI)PheGlu(OBZI)Lys(Z
)juThr(BZI)AsnTyr(BZI)Se[
(BZI) VaIThr (B21) false p (OBZI) Buddha uAsnVaIGinArg (N02) Lys (Z) A
IalieHis (BZI) Glu (OBZI) ul
ieGinVaIMetAlaG1u (OBzl) comparison u
Ser(Bzl)Pro AIa AIaLys(Z
)Th(BZI)GIy L$(Z)Arg(N02
)L$(Z)Arg(N02)Ser(Bzl)Gin
MetLeuPheArg(N02)GIyArg(
N02)~g(N02)AIaSer(Bzl)Gin
one resin (however, the symbols are the same as defined above). Physical properties obtained in step 8: Peptide molecular weight of 1 mN-y: approximately 20,000 Tanomolar amino acid composition (mol%): Histidine (1.4) Tryptophan (0.3) Lysine (13.6) Arginine (5.5 ) Noasparagine aspartate (13.6) Serine (7.6) Noglutamine glutamate (11.3) Threonine (3.
4) Glycine (3.4) Proline (1.4) Alanine (5.5) Valine (5.5) 1/2 Cystine (1
．． 4) Methionine (2.7) Isoleucine (4.8
) Leucine (6.8) Phenylalanine (6.8)
Tyroshiso (3.4) amino acid sequence: Cys Tys
Cys Gin Asp Pro TM VaI Ly
sGIu Na GIu ASn Leu Lys L
Tyr PheASn AIa GIy His
Ser Asp VaI AIa AspASn GI
y ThrLeuPhe LeuGIy lie Le
uLysA6n Trp L佼 GIu GIu Se
r Asp A [g Lysne Met Gin S
er Gin lie VaI Ser Phe TM
Phe Lys Leu Phe Lys Asn P
he Lys AspASp Gin Ser ne
Gin Lys Ser VaI GIuThrne
LysGIuAspMetASn VaI L侭Ph
e PheASn Ser ASn LyS LyS
L$ A Stopg ASP ASPPhe GIu Lys
Leu Thr ASn T Ser VaITh
rAspLeuAsnVaI GinArgLys A
Ia lieHis GIuLeu ne Gin V
a]MetAIa GIuLeuSer Pro AI
a Na L$ Thr GIy La ArgLys
Arg Ser Gin Met Leu Phe
AJg GIyAJgArgAIaSerGin2 m
N-y-like peptide molecular weight: approx. 20,000 tanomol Amino acid composition (mol%): Histidine (1.4) Tryptophan (0.3) Lysine (136) Arginine (5.4) Aspartic acid/asparagine (13.6) Serine (7.5) Glutamic acid/Glutamine (12.2) Threonine (3.4
) Glycine (3.4) Proline (1.4) Alanine (5.4) Valine (5.4) 1/2 Cystine (1.
4) Methionine (3.4) Isoleucine (4.8)
Leucine (6.8) Phenylalanine (6.8)
Tyrosine (3.4) amino acid sequence: Met Cys T
X Cys Gin Asp Pro TM VaIL
ys GIu AIa GIu Asn Leu L侭 Lys TMPhe Asn AIa GIy Hi
s Ser Asp VaI AIaASp Asn
GIy Thr Leu Phe Leu GIy l
ieLeu Lys Asn Trp GIu
GIu Ser AspA Stopg LySI1e Met
Gin Ser Gin11e VaISerPhe
Tyr Phe Lys Leu Phe Lys
ASn PheLys AspAspGinSer l
ie Gin Lys Ser VaIG1uThr
lie Lys GIu ASp Met AsnVa
I LysPhe Phe ASn Ser Asn
Lys Lys Lys ArgASP ASp Ph
e GIu L is Leu Thr Asn TMSe
r VaI Thr Asp Leu ASn VaI
Gin ArgLys AIa lie His G
Iu Leu lie Gin VaI MetNa
GIu Leu Ser Pro AIa AIa L
ys Thr GIyLyS A岱 LyS AJg
Ser Gin Met Leu PheAJgGIy
Arg AJgAIa Ser Gin3 Pro ``N
-Y peptide molecular weight: approximately 20,000 units/mol Amino acid composition (mol%): Histidine (1.2) Tributophane (0.3) Lysine (12.7) Arginine (4.8) Asbaragic acid/Asparagine (12. 0) Serine (7.8) Glutamic acid/Glutamine (9.4) Threonine (3.6
) Glycine (4.2) Proline (1.2) Alanine (5.4) Valine (5.4) 1/2 Cystine (1.
8) Methionine (3.0) Isoleucine (5.4)
Leucine (8.4) Phenylalanine (6.6)
Tyrosine (4.5) amino acid sequence: Met LyST
yr Thr Ser Tyte 11e Leu AI
aFhe Gin LeuCys lieVaI Le
uGIy Ser LeuG1y C$ Tyr Cy
s Gin Asp Pro Tyr VaILys
GIu AIa GIu Asn Leu L's Ly
s TMPhe Asn AIa GIy His S
er Asp VaI AIaASp Asn GIy
Thr Leu Phe Leu GIy lieL
eu Lys Asn Trp L$ Gin GIu
Ser AspA【g Lys lie Met G
in Ser Gin lie VaI SerPhe
T's Phe Lys Leu Phe Lys A
Sn PheLys Asp AspGinSer l
ie Gin Lys Ser VaIG1uThrl
ie Lys GIu ASpMet AsnVaI
LysPhe Phe ASn Ser Asn Ly
s Lys Lys ArgASp ASp Phe
GIu Lys Leu Thr Asn T Hit Ser
VaI Thr Asp Leu ASn VaI
Gin ArgLys AIa lie His GI
u Leu lie Gin VaI MetNa G
Iu Leu Ser ProAIaAIaLys T
hr GIyLys Arg Lys Arg Ser
Gin Met Leu PheA [gGIy Ar
gA[gAlaSer Gin Furthermore, the interferon y of the present invention has the following properties. '1- Isorat point PH8.6 '2' Ultraviolet absorption spectrum shows a maximum peak at 278 Kite Buddha (Figure 1). t31 SDS electrophoresis plate SDS-PAGE Laemm
li, U. K. (1970) Natme
227, spots 0 to 5, and then dyed with Coomassie Brilliant Blue (R). The marker is Pospho hilase b (94k), B
SA (67k), Ovalb mountain min (4 fall)
, Carbnica skin hydrase (30k), Soy
PanTripsinl skin ibitor (20.1k)
, Q-Lactalbumin (14.4k) (
Figure 2). In addition, examples of acute denaturation and preparation of interferon, a product of the present invention, are shown below. ○ - Acute Toxicity When we conducted an acute toxicity test on mice regarding the product of the present invention, the following results were obtained, and even at the limit of physical administration, the animals did not die and the LD5o value remained low. is impossible to calculate. {2} Preparation Example For intravenous injection, 5% glucose solution of product 1 of the present invention, 1/100, is used. Brief Description of the Drawings Figure 1 shows the ultraviolet absorption spectrum of the bebutide of IFN-y obtained in Example 1, and Figure 2 shows its SDS gel fin aerophoresis. Markers include ovalbumin (molecular weight 4), carbonic anhydrase (30k), soybean trypsin inhibitor (20.1k) and Q-lactalbumin (14k).
4k) was used. Figure 1 Figure 2

Claims (1)

[Claims] 1 Formula (I): J CysTyrCysGlnAspProTyrVa
lLysGluAlaGluAsnLeuLysLys
TyrPheAsnAlaGlyHisSerAspV
alAlaAspAsnGlyThrLeuPheLe
uGlyIleLeuLysAsnTrpLysGlu
GluSerAspArgLysIleMetGlnS
erGlnIleValSerPheTyrPheLy
sLeuPheLysAsnPheLysAspAsp
GlnSerIleGlnLysSerValGluT
hrIleLysGluAspMetAsnValLy
sPhePheAsnSerAsnLysLysLys
ArgAspAspPheGluLysLeuThrA
snTyrSerValThrAspLeuAsnVa
lGlnArgLysAlaIleHisGluLeu
IleGlnValMetAlaGluLeuSerP
roAlaAlaLysThrGlyLysArgLy
sArgSerGlnMetLeuPheArgGly
ArgArgAlaSerGln (However, Cys, Ty
r, Gln, Asp, Pro, Val, Lys, Glu
,Ala,Asn,Leu,Phe,Gly,His,
Ser, Thr, He, Trp, Arg and Met
is cysteine, tyrosine, glutamine, aspartic acid, proline, valine, lysine, glutamic acid, alanine, aspartic acid, leucine, phenylalanine, glycine, histidine, serine, threonine, isoleucine, tryptophan, arginine and methionine residues; J represents a hydrogen atom) is a human interferon γ-peptide. 2. A cell proliferation inhibitor consisting of a peptide of human interferon-γ. 3. Antiviral agent consisting of human interferon-γ peptide.