JPS62223191A - Production of novel physiologically active peptide - Google Patents

Abstract

PURPOSE:To obtain the titled compound useful as interferon gamma, etc., by successively reacting a resin containing no functional group except a functional group participating in peptide bond as a substrate with an amino acid under a strongly acidic condition and simply carrying out washing even if chains are extended and molecules are agglomerated. CONSTITUTION:In synthesizing peptide by solid phase method, a resin (e.g. divinylbenzene crosslinked chloromethylated polystyrene resin, etc.) containing no functional group except a functional group participating in peptide bond is used as a substrate is reacted with an alpha-amino group-protecting amino acid under a strongly acidic condition according to amino acid sequence of peptide, the alpha-amino-protecting group is removed, these operations are repeated to successively react the amino acid. By this method, washing with methylene chloride is simply carried out even if molecules are agglomerated with extension of peptide chain and the aimed peptide is obtained.

Description

[Detailed description of the invention]

The present invention relates to a novel method for producing bioactive R-butide, and more specifically, during the synthesis of R-buty 12 by a solid phase method, a resin having no functional groups other than the functional groups involved in R-butide bonding is used as a support. The present invention relates to a method for producing R peptide, which is characterized in that the reaction is carried out under strong acid conditions, and washing with methylene chloride is simplified even in areas where molecules aggregate as the peptide chain becomes longer. The present invention also relates to iptides obtained by the above-mentioned production method, and these peptides include novel physiologically active iptides that have excellent physiological activity such as antiviral activity, cell proliferation inhibiting effect, and NK cell activity enhancing effect. be done. Conventionally, as antiviral agents or IIB cell proliferation inhibitors,
Antimetabolites, alkylating agents, antibiotics, etc. have been used, but these are highly toxic and undesirable. Therefore, the development of antiviral agents or cell proliferation inhibitors with low toxicity is strongly desired. Recently, interferon-α (hereinafter referred to as [pN-aJ)] and interferon-β (hereinafter referred to as 1'-rpN-β)
These studies have progressed because of the effectiveness of antiviral agents (referred to as ``viruloids'') as low-toxicity antiviral agents. However, if
The cell proliferation inhibitory effects of N-α and IFN-β are not satisfactory. On the other hand, interferon-r (r
Research on IFN-α and I
Compared to research on FN-β, there has been little progress. However, in October 1981, the United States
7 International Center for Interferon Research held in Cisco City
The 5th annual meeting (The 5th annual meeting)
International Congress for
Although there was no report that IFN-γ was isolated in the Irr + Erferon Research, David V. Gödel (David V, G.
oeddel) C Genen Tesoku Co., Ltd. (Genen +
Metsenger RN of IFN-γ by Eek*Inc, ), California, USA]
Complementary DNA of A (hereinafter abbreviated as 1'-mRNJ)
(hereinafter referred to as “cDN person”) pro-IFN-r, IPN-γ, and I deduced from the deoxynucleotide sequences of
The amino acid sequence of FN-γ-like pepti r was published. Its amino acid sequence is represented by the following formula (I). Formula (1): %Formula % and MOt are /stine, tyrosine, glutamine, aspartic acid, proline, valine, lysine, glutamic acid, alanine, asparagine, leucine, phenylalanine, glycine, histidine, serine, threonine, inroinone, trizotopha/, arginine and methionine residues; and in the case of proeFN-γ, Me
t L78Tyr 'rhr Ser Tyr Ile
Leu Ala Phe Gln Lou Cys]
: lo Van l, eu Gl, y Ser Leu
Guy (however, Mo2゜Lys, Tyr, Thr,
Ser, Eng1e, Leu, Ala, Phe, Gln, C
ys and Van are the same residues as defined above),
In the case of N-r, it represents a hydrogen atom, and in the case of an IFN-γ-like peptide, it represents a methionyl group] Furthermore, at the aforementioned conference, rFN-r-like peptide 1-
It was also announced that a culture solution of cells containing the gene containing the information of PloeFN-r has antiviral activity, but a culture solution of cells containing the gene containing the information of PloeFN-r does not have antiviral activity. On the other hand, unlike IFN-γ-like peptides, XFN-γ does not have an artificial methionine residue added to its N-terminus. has not yet been successful. In view of the current situation as described above, the present inventors have developed a drug that not only has high antiviral activity but also has cell proliferation inhibitory effect and NK
We conducted intensive research to develop a bioactive agent that also has excellent cell-enhancing effects. Therefore, the object of the present invention is to provide a method for producing Seikou-activated Bobuti 12 and a sterilized product obtained by the method, which not only has excellent antiviral activity but also has cell proliferation inhibiting action and NK cell enhancing action. It is an object of the present invention to provide an excellent physiologically active butide. Other aspects and advantages of the present invention will be readily apparent from the detailed description provided below, along with the claims. According to the present invention, physiologically active compounds are provided, and these molecules include a novel useful compound represented by the formula (1):
1: J! l-active bezotide (in formula (1), J represents a hydrogen atom) is included. The novel physiologically speaking waveputide of the present invention can be produced by a conventionally known method, namely, by Nobuo Izumiya et al. [Synthetic Chemistry Series - Waveputide]
! Synthesis J Published by Maruzen Co., Ltd. in 1975; Edited by Japan Tooth Chemical Society "Biochemistry Experimental Course/Chemistry of Tan and Acid 1-IVJ"
By using the publication published by Tokyo Kagaku Dojin University in 1977, it is possible to manufacture DIJ, combine Dψ, T,, +7. That fruit/It! As one of the P4 methods, the solid phase method will be specifically explained below. In addition, when synthesizing the sterptide by the solid-phase method, as described in Example 1 of the explanation, a resin having no other functional groups other than the functional groups involved in the Rzotide bond is used. Good results can be obtained if the reaction is carried out under strongly acidic conditions using as a support, and if the molecules are aggregated as the isotide chain becomes longer, washing with methylene chloride is simplified. In the solid-phase method, the carboxylic acid group of an amino acid having an α-amine-protecting 11φ group is bonded to a support such as a polystyrene resin cross-linked with divinylbenzene, and then the α-amine protecting group is selectively removed. Remove. The free amino group is reacted with a second amino acid having an α-amine protecting group. Removal of this α-amine protecting group and α
-The introduction of an amino acid having an amine protecting group is repeated to obtain the desired butylene r chain. The covalent bond between the carbo/acid group of the C-terminal amino acid of the peptide and the resin is then cleaved. At this time, depending on the reagent used to cleave the covalent bond, the α-amine 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 4-butyl-R is easy in terms of operation, and the obtained product is soluble in the solvent used for the reaction. This method is more advantageous than the so-called liquid phase method because the appropriate amount of reagents and byproducts used in the reaction can be removed by filtration, and the desired peptide can be produced in a short time and in high yield. The support used in the first method is generally a styrene polymer crosslinked with divinylbenzene with a degree of crosslinking of 2, and preferably has a size of 200 to 400 meshes. Before the resin is reacted with the amino acid and bonded, 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 5nC12 as a catalyst, and the aromatic ring of styrene is chloromethylated. Be taken. The α-amine protecting group used in the first method is stable during camplation between the support and the amino acid having an α-amine protecting group, 17 and does not adversely affect the side chain protecting group or Vepti P chain. Those that can be easily removed are preferred. Preferred α-amine protecting groups as mentioned above include tert-butoxy-7carzenyl (hereinafter abbreviated as “Boc”) group. This Boc group is 1NHCl! /acetic acid mixture, aNHcz/dioxane mixture or so% (w/
v) It can be easily removed from the Vepti P chain using a TroH (tetrafluoroacetic acid)/CH2Cl2 mixture. The side chain protecting group is preferably a group that is stable during coupling and removal of the α-amine protecting group and that can be easily removed at the stage of peptide synthesis. As such preferable side chain protecting groups, NO2 or tosyl group is used to protect the side chain of arginine, and benzyl group (hereinafter abbreviated as 1'-B, J) is used to protect the side chain of as-ξlagic acid. ) group is B11 group or 0-methylbenzyl (hereinafter "OBz
) group is used to protect the side chain of histidine.
l group, benzyl, oxycarcenyl (hereinafter abbreviated as rZJ) group, or Boc group, two groups for side chain protection of lysine, tosyl group or diisopropylmethyloxycarzenyl (hereinafter abbreviated as "DlpmOC") group, Sulfoxy r groups are included for side chain protection of methionine, and Bzl groups are included for serine, threonine, and tyrosine/glue I chain protection. The method for determining the amino acid sequence of the novel physiologically active peptide of the present invention is as follows. First, the methionyl bond of the novel bioactive peptide of the present invention was
Cut with. The peptide fragments obtained by the cleavage were separated using Sephadex a-1OO (Pharmacia Fine Chemicals, Sweden), and the amino acid sequence of each fragment was determined by known high-precision amino acid sequence analysis.
-Determine in order starting from the end. On the other hand, the novel physiologically active peptide of the present invention is partially digested with trypsin, and the obtained peptide fragments are separated using a Sephadex G-Zoo column. The amine sequence of each separated fragment is determined in the same manner as described above starting from the N-terminus of each fragment. The sequence of each fragment of PeptiP is determined by comparing the amino acid sequence of each fragment of the peptide obtained by cleaving with bromo cyanide and the amino acid sequence of each fragment of the peptide obtained by cleaving with trypsin. In this way,
The amino acid sequence of the novel physiologically active peptide of the present invention is determined. When the purpose is to suppress cell proliferation, for example, suppress the proliferation of malignant tumor cells, the bezotir of the present invention can generally be administered by injection intravenously, intramuscularly, or subcutaneously. The daily dosage of the uninvented peptide will vary (depending, of course, on the age, medical condition and weight of the patient), but is usually between 1X10' and 10' per adult.
It is administered by injection in the amount of X 10' units/mole. To treat viral diseases, ointments containing 4 peptide I X 10' to I X 10' units of the present invention per Log ointment can be applied to the skin. Ointments containing Vepti F of the present invention can be prepared using conventionally known pharmaceutically acceptable ointment bases. The Ir14Q value of the soft-11 agent containing the peptide of the present invention varies depending on the patient's age and medical condition, but it is usually 1 x 104 ~) for the peptide group M of the present invention.
1 [+' can be divided and applied in small units of fl. 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. 013Zl: Ortho-methylbenzyl group which is one of the side chain protecting groups 85 people: Bovine serum albumin Example 1 Step 1 (Resin washing) Resin [Bio-Bead 5-X2. Trade name of cross-linked styrene polymer, -z Iorad Company, USA)) 1
Add 50 g to 11 distilled benzene with stirring. 3
After 0 minutes, the resin is filtered through a glass filter and dried. Next, the dried resin was added to 11 methanol with stirring, and after 30 minutes, it was dried using a glass filter. The resin obtained by drying is washed successively with methanol, methanol/water and water. The washed resin was diluted with 11 lNNa.
Add to OH aqueous solution. After stirring the resulting mixture in a boiling water bath for 1 hour, the resin is removed and washed with water. Next, the resulting resin is added to a 11 IN aqueous hydrochloric acid solution and stirred for 1 hour in a boiling water bath. Next, the resin is washed with water. 41 resins obtained by washing with water! After suspending in distilled water,
Leave it still. After 30 minutes of standing, fine resin t-deca/
Removed by station/. In this way, the resin is obtained as a precipitate. The precipitated resin is placed on a glass filter.
Remove F and wash with methanol. The washed resin was added to a small amount of dimethylformamide (hereinafter abbreviated as "DMF") and stirred at 80°C for 30 minutes.
Wash with MF and methanol. , further add 2g of resin
After stirring for 1 hour in methanol, strain. The resulting resin is air-dried and then vacuum-dried at 100° C. for 2 hours. Step 2 (Manufacture of chloromethyl resin) Put 2'5 g of the resin obtained in Step 1 and 100 d of distilled chloromethyl methyl ether into a 1e round bottom two-headed flask, and stir gently at 25°C for 1 hour to prepare the resin. After swelling, cool to 0°C. 1.88-
50ml containing 5nC14! of chloromethyl methyl ether was added over 30 minutes while stirring while maintaining the temperature at 0°C, and the mixture was further stirred for 30 minutes to obtain a reaction product. The product thus obtained is filtered through a glass filter and gently washed with 500° dioxane-water (1:1) and then with 500° dioxane-3N hydrochloric acid (3:1). Furthermore, the product is washed successively with water, dioxane-water and methanol, and then dried at iooo<0>C under reduced pressure. Step 3: (ε-Z-lysine) Add 10 g of Na HC03 to 11 g of L-lysine copper salt.
and 1/64 of 12-z-cz were added every 10 minutes while stirring under water cooling, and the mixture was further stirred for 15 hours. The precipitate thus obtained is separated and washed successively with water, ethanol and acetone/ether. The washed precipitate was suspended in 2501 nt water, and then 412 rnI! was added to this suspension. The above precipitate is dissolved by adding 0.06N aqueous hydrochloric acid solution. Then, the resulting solution was added with H2S
Bubble the gas for 1-2 hours. The resulting CuS is removed using a Hyfloss-.ξ-cell (manufactured by Wako Junta, Japan), and the OuS is washed with an IN aqueous solution of hydrochloric acid. Collect the lachrymal fluid and lavage fluid obtained in the above procedure and pass through the air
Remove 2S. After that, this solution was cooled with water and concentrated aqueous ammonia was added to adjust the pH.
6.5 and leave it in the refrigerator for 2 hours. The resulting precipitate is separated and washed successively with water, ethanol and ether. In this way, ε-2-lysine Log with a melting point of 254°C is obtained. The yield of 1-2-lysine produced is 65%. The resulting ε-z+7 gin! The crystal is
After dissolving in dilute hydrochloric acid and neutralizing with aqueous ammonia, a mixture of 120 tnt of fuming nitric acid and 751 nt of oleum was cooled on ice, and 110 g of L-arginine hydrochloride was added to the mixture. Gradually add while stirring. Furthermore, concentrated sulfuric acid 45- is added to the above mixture. After stirring the resulting mixture for 1 hour, it is poured into ice cubes and the pH is adjusted to 8 with concentrated aqueous ammonia. Next, the pH of the mixture was adjusted to 61° C. with acetic acid, and then left in the refrigerator for about 4 hours. The resulting precipitate is separated and recrystallized in hot water. The obtained crystals are washed successively with ethanol and diethyl ether and dried. In this way, 60 g of G-NO2-arginine having a melting point of 251°C is obtained. The yield of G-NO2-arginine is 5
I am in charge of 0. C-(imBzl-histidine) 20 g of histidine hydrochloride H20 are dissolved in 200+d of liquid ammonia cooled to -30°C in a bath of Plyice-acetone and a small piece of sodium metal is added to this solution until the blue color of the solution Add while stirring until it remains. A small amount of histidine is then added until the blue color of the solution disappears, and then benzyl chloride is added dropwise to the solution. After stirring this mixed solution for 30 minutes, NH, in this mixed solution is evaporated at room temperature under atmospheric pressure, and then NH3 is removed under reduced pressure using a water jet pump. Pour the residue after removing NH into 100-g ice water. After the resulting mixture is extracted with diethyl ether, the insoluble matter is removed. 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. Count the resulting sediment,
Recrystallize from 70% by weight ethanol. 13 g of imBzl-histidine with a melting point of 248° C. are obtained. The yield of imBzl-histidine is 50%. D, (OBzl-serine) 237g of acetyl-L-serine was mixed with 235rnt of 5N
Dissolve in aqueous NaOH and adjust pH to 7.3. 4 g of Takajustase (manufactured by Sankyo Co., Ltd.) was added to this solution in 25-0.1 M citrate buffer (pH 6,7,
After adding a solution obtained by dissolving in CaC/20.025M (containing 20.025M) and adding several drops of toluene, the resulting mixture was left at 36° C. for 10 days. After removing the resulting precipitate and concentrating the tear fluid, acetone is added to the concentrated tear fluid to obtain crystals. 100 g of 0--Bzl-serine are thus obtained. Following a similar method, 240 g of acetyl-
Get 100g of L-threonine or Q-Bzl-threonine. B, (0-Bzl-tyrosine)゛1.8g L-tyrosine and 1.2g CuS04
- 5H20 is suspended in a solution of 5-2N NaOH aqueous solution and 10-water and stirred for 2 hours. This suspension contains 60-
of methanol is added, followed by the gradual addition of 1.2-benzyl bromide and 0.7-2N NaOH aqueous solution. 15
After min 0.3-benzyl bromide and 0.8-2N NaO
Add more H aqueous solution. After stirring the resulting mixture for 1 hour, the resulting precipitate is separated and washed with methanol-water (1:3). In this way, 2g of 0-B
A copper salt of zl-tyrosine is obtained. This copper salt of 0-Bzl-tyrosine was dissolved in 20 rnl of INEDTA. The solid content is removed from the mixture obtained by kneading the liquid and the liquid in a mortar, and the solid content is washed with water. Add a small amount of E to the washed product.
Recrystallization from hot water containing DTA gives 1 g of 0-Bzl-tyrosine with a melting point of 223°C. The yield of OBzl-tyrosine is 30%. F, (S-Bzl-cystine) 157g of L-cystine hydrochloride 21! 2NNaO
256 g of benzyl bromide was added to this solution with vigorous stirring under water cooling, and the mixture was stirred in a cold room for 5 hours. Thereafter, the pH of the solution is adjusted with acetic acid to form a precipitate. Collect and wash the formed precipitate with water. In this way, 160 g of 5-Bzl-cysteine with a melting point of 212 DEG C. are obtained. The yield of 5-Bzl-cysteine was 70%. 15 from 160 g of aspartic acid in the same manner as above.
0 g of 0-Bzl-aspartic acid is obtained, and 150 g of OBzl-glutamic acid is obtained from 160 g of glutamic acid. G, (Boc-amino acid) Dissolve 29g of L-valine in 250+nl of 1N NaOH aqueous solution and add water to the resulting solution to give 400rnt.
Let it be a solution of After adding 150% of tetrahydrofuran to this solution, 150% of tetrahydrofuran was added to the solution while stirring vigorously at 10°C.
Add one-fifth of 00-Boc C1 every 10 minutes. Meanwhile, 2N NaOH is added to the solution each time the Boa-C2 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 0.5M aqueous citric acid solution, and the precipitated oil is extracted with 9 ethyl acetate. The resulting extract is washed with a small amount of water, dried over Na2S04 and concentrated under reduced pressure. Add petroleum ether to the condensed extract and leave it in a refrigerated layer to form crystals. The resulting crystals are dried several times.In this way, Bo with a melting point of 78°C is
Obtain 30 g of a-valine. The yield of Boc-valine is 55
It is Chi. H, (Other Boe-amino acids) By performing the same method as above, Boc-glycine 15g1 Boc-alanine 16g5 Boc-leucine lsg, Boc-isoleucine 15g, BoC-serine (Bzl) 14g, Boc-threonine ( B
, I) 14g, Boc-cystine (Bzl) 15
g, BoC-methionine 15g% Boc-proline 12g, Boc-asnoξlagic acid (OBzl) 15
g, BoC-glutamic acid (OBzl) 15
g, Boc-glutamine 15 g1 Boa-histidine (Bzl) 12 g, Boc-leucine (Z)
1'3 gSBoa-Arginine (NO2) 15
g, 15 g of Boa-phenylalanine, 15 g of Boa-tyrosine (B, I) and Boa)! J Butofan L
og is 30 g of glycine and 30 g of arania, respectively.
Leucine 30g1 Isoleucine 30g1 Serine (B
z+) 30 g1 threonine (szl), 30 g
, cysteine (nz+) 30 g. Methionine 30 g 5 Zoroline 30 g 1 Aspartic acid (OBz I) 30 g 1 Glutamic acid (OB
zl) 30 g. Glutamine 30g, Histidine (Bzl) 30g
, Lysine (Z) 30g, Arginine (NO2) 30g
g, phenylalanine 30g1 tyrosine (az+)3
0 g and tryptophan 30 g. Step 4 (Coupling of Boa-glutamine with resin) A mixture of 2 g of chloromethyl resin (total C/' content 4 mmol), 2 mmol of Boa L-glutamine, 1.8 mmol of triethyl°amine and 12 ml of dimethylformamide. Incubate at room temperature for 24 hours. Next, the solid content is removed from the mixture using a glass filter, and the resulting solid products are washed sequentially with 700-dimethylformamide, ethanol, acetic acid/ethanol, and methylene chloride, respectively. The product is then dried at room temperature under reduced pressure. To determine the amino acid content of the product, weigh 50 ml of the product,
The mixture was placed in a mixture of 12N hydrochloric acid and dioxane in a volume ratio of 1:1 and hydrolyzed for 24 hours, and then measured using an amino acid analyzer. The amino acid content of the product is 0.2 mmol/g
It is. Step 5 (Boc-Ser-Gln-resin) 2 g of Boc-glutamine obtained in step 4 (rBoc-Gln-resin)
The resin (abbreviated as InJ) is 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 substance was shaken in 160-molecular acetic acid and weighed. Repeat this operation three times. Next, add 160% of the above solid matter.
- in an aqueous IN hydrochloric acid solution/acetic acid and shaken at room temperature for 30 minutes to remove the Boa group from the resin. The product thus obtained is separated and sequentially washed three times each with acetic acid, ethanol and dimethylformamyl. washing product (160-10ffit%)
C in ethylamine/dimethylformamide 1
Shake for 0 minutes to perform a neutralization reaction. The resulting product is washed several times with dimethylformamide and then with methylene chloride. The washed product is placed in a solution of 2 mmol of Boa-serine (B, I) in 12-methylene chloride and shaken. After 10 minutes, this mixture was added to a solution of 20 mmol of dicyclohexylcardashiimide in 60-methylene chloride, and the mixture was shaken at room temperature for 2 hours to effect coupling. The resulting product is filtered and washed sequentially with methylene chloride and ethanol. Step 6 (Boa-4 butidyl resin) The same operation as Step 5 is repeated to elongate the amino acid to obtain Boc-peptidyl resin. Step 7 (cleavage of peptide from resin) Boa-peptidyl-resin and 1-anisole obtained in step 6 were placed in a reaction vessel containing a stirring bar covered with polytetrafluoroethylene. Add hydrogen fluoride 2 while cooling with dry ice-acetone.
0- was introduced into the reaction vessel, stirred at 0°C for 1 hour, and then heated to 0°C.
Hydrogen fluoride in the reaction vessel is removed under reduced pressure. A 1-weight aqueous acetic acid solution is added to the resulting residue, stirred, and then the peptides are extracted.The extract is placed in a separating funnel, washed with ether, and freeze-dried. In this way, peptides without protecting groups are separated. Step 88 (Physical properties of synthetic peptides) Measure the molecular weight, amino acid composition, and amino acid sequence of each peptide obtained in Step 7. The molecular weight of each peptide is determined from the mobility measured by SDS gel electrophoresis. Amino acid composition (molti) is 6N
It is hydrolyzed in an aqueous hydrochloric acid solution at 110°C for 24 hours, and the resulting mixture is subjected to two-dimensional penoξ-chromatography for determination. 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 PeptiP is analyzed using an automatic amino acid analyzer manufactured by Hitachi. Step 9 (Measurement of antiviral activity and sex) The antiviral activity of each peptide R obtained in step 7 was measured by using a monolayer of human neonatal skin fibroblasts and bovine varicella stomatitis virus (written by B.C. Merrigan). Evaluation of inhibition of plaque formation using human interferon - "In vitro methods in cell-mediated immunity" Edited by E.D.B.R. Bloom and B.R. Gray 1, Akademi Tsuku Press, New York 1971, 489 page(
p, c, λ (erigan + Plaque In
hibition As5ay for Huma
n Interferon Employing
Hum≠■ Neonale 5kir+ Fibroblast
Monolayers & Bovine Ves+i
eular Stomatitis Virus +”
In-vi+ro Method in Ce1
l −Mediatearmrr+uni +y”, e
dited-'by E, D, B, ordinary, 'BIoom
l P, R, Grade + Academic Pr
ess r N, Y, 1971 + I) I) 489
') Measured using the same method as described. Specifically, each peptide obtained in step 7 is diluted into various dilutions into a growth medium containing 10 volumes of fetal microsera. FS-4 cells (human neonatal skin fibroblasts) are cultured in a monolayer in this medium. After 18 hours, the cells are infected with varicella stomatitis virus (VSV) capable of forming 920 cells per cell and incubated for an additional hour at 37°C. The cells were then grown in the above growth medium for 2 hours.
After washing twice, the cells are cultured again in the growth medium at 37° C. for 24 hours. Whether or not a virus has been generated is determined by observing the cells after culturing using a lacrimal microscope. The resulting cell damage observed is due to the virus. 1 peptide
It exhibits antiviral activity at a concentration of μg/-. Step 10 (Measurement of cell proliferation inhibitory effect) 2×105 FS-4 cells are transplanted into the growth medium described in Step 9 in a Petri dish with a diameter of 60 III. After 5 hours, the growth medium is removed and, instead, a fresh medium containing VeptiP obtained in step 7 at a concentration of 0.1 μg/pod is poured into the above petri dish, and culture is continued at 37° C. for 18 hours. After culturing, the medium is removed, a medium containing 3H-thymidine at a concentration of 5 μOj/lnl is added, and the cells are further cultured at 37° C. for 2 hours. After culturing, the cells are washed with phosphate buffered saline, and then 5% by weight of trichloroacetic acid is added to the washed cells. A certain amount of the resulting precipitate is dried, and the 3H radioactivity of the dried precipitate is measured using a liquid scintillation counter (manufactured by Paracard, USA). For comparison, David P.
V. Goeddel) elucidated pro-IFN-γ, I
Peptides having the same amino 3 sequences as FN-γ and IFN-r, respectively, are prepared by the solid-phase synthesis method described above, and their cell proliferation inhibitory effects are measured by the method described above. The results are shown below. OIFN-r obtained in step 9, IFN-r and IF
The results of the antiviral activity of N-r-like peptide are shown below. A case where antiviral activity is observed without dilution but not observed at 1:2 dilution is defined as 1 unit, and a case where antiviral activity is observed at 0 times dilution but not observed at 21 times dilution is defined as n units. From the above, Boo61FN-γ has no ability to suppress cell proliferation;
It can be seen that IFN-γ and IFN-r-like peptides have a 20% cell growth suppressive ability. Boa-peptidyl resin obtained in step 6: 1, Boa
Cys (BzQ Tyr (Bzl)C!
ys (Bzl)Gln Asp(oBz+) Pr
o Tyr (Bzl) Val Lys (Z),
Glu (OBzl)Ala Glu (oBz+
) person sn Leu Lys (Z)Lys
(Z)Tyr(Bzl) Phe Asn人1a G
ly His (Bzl)Ser (Bzl)人s
p (0,Bzl)Vat Ala'Asp
(OBzl) 人SN Gly Thr(Bzl)
Leu Phe Leu Gly Ile
Leu Lys (Z) 人＄nTrpLYs
(Z) Glu (OBzl) Glu (OBzl)
Ser (Bzl)人sp (OBzl)Arg
(NO2) Lys (Z)Ile Met
G1n5er (Bzl) Gin Ile Val
Ser (Bzl) Phe Tyr(Bzl)
Phe Lys (Z) Leu Phe
Lys (Z) Asn PheLys (Z)
Person sp (OBil)Asp (OBzl)Gl
n Ser (Bzl)11e Gln Ly
s (Z) Ser (Bzl) Vat
Glu (OBzl)Tbr (Bzl)Ile
Lys (Z)Glu (oBz+) Human sp
(OBzl)Met Asn Vat Lys
(Z) Phe Phe Asn Ser
(Bzl) person sn l, ys (z) Lys (
Z) Lys (Z) Person rg (NO2) Person 5p (OBzl) Asp (OBzl) Phe Gl
u (OBzl)Lys (Z)Leu Thr
(Bz"l') 人SN Tyr (Bzl)
Ser (Bzl)MalThr (Bzl)As
p (Of3zl)'Leu Asn Val
Gin Arg(NO2)Lys(Z)AIa
Ile His (Bzl) Glu (OBz
l) Leu Ile Gln VaI Met
Person 1a Glu (OBzl)LeuSet
(Bzl)Pro Person 1a Ala Lys
(Z) Thr (Bzl) Gly Lys (Z
) Arg (NO2) Lys (Z) person rg
(NO2) Ser (Bzl) Gln Met L
eu Phe Arg (NO2) Gly person rg
(NO2) Human rg (No2) AIa Ser
(Bzl)Gin-resin, Z BOCMet Cy
s (Bzl) Tyr (Bzl) Oys (
Bzl) GIIIAsp, (OBzl) Pro T
yr (Bzl) Vat Lys (Z) Glu
(OBzl)Ala Glu (OBzl) person sn Leu Lys ('Z)Lys(Z)T
yr (Bz'1)Phe, Asn AIa G
Iy'His (Bzl)Ser (Bzl)As
p (OBzl)Vat Ala As, p
(OBzl) Asn Gly Thr (Bzl) L
eu Phe Leu Gly Ile LeuLys
(Z) Asn Trp Lys (Z) Glu
(OBzl) Glu(OBzl)Ser (Bz
l) Person sp (OBzl) Person rg (NO2
) Lys(Z) Ile Met Gln S
er (Bzl) Gla Ile Mal 5
er (Bzl) Phe Tyr (Bzl) Phe
Lys (Z) Leu PheLys (Z)A
sn Phe Lys (Z) λ$1)(Q
Bzl) Asp(OBzl) Gln Ser
(Bzl) Ile Gln Lys (Z) 5er
(Bzl) Val Glu (OBzl) Thr
(Bzl) Ile Lys (Z) Glu (O
Bzl) Person sp (OBzl) λ(et As
n Val Lys (Z) Phe Phe
人SN Ser (Bzl) 人SN Ly
s (Z)Lys (Z)Lys (Z)personrg
(NOx)Asp (OBzl)Asp, (OB
zl) Phe Glu (OBzl) Lys (
Z) Leu Thr (Bzl) Person 5nTyr
(Bzl) Ser (Bzl) val Th
r (Bzl)人＄p(OBzl)Leu Asn
val Gln person rg (NO2)Lys
(Z) person 1a rle His (Bzl)G
lu (OBzl)Leu Ile GlnVa
lMet 人la Glu (OBzl)Leu
Ser (Bzl)Pr. AIa, AIa Lys (Z) Thr (Bzl
) Gly Lys (Z) Arg(NO2)Ly
' (Z) Person rg (NO2) Ser (B
zl) Gln MetLeu Phe person'g
(NO2) e3y person rg (NO2) person rg (NO2)Ala Ser (Bzl) Gl
n-resin, 3, BocMet Lys (Z)
Tyr (Bzl) Thr (Bzl) 5er(B
zl) Tyr (Bzl) Ile Leu
Ala Phe Gin LeuOys
(Bzl) Ile Vat Leu Gly S
er (Bzl) LeuGly Oys (Bzl)
l)Tyr (Bzl)Cys (Bzl)GIK
I Asp (OBzl) Pro Tyr (Bzl
) Mal Lys (Z) Glu (OI3z
l) Person 1a Glu (OBzl) Person sn
Leu Lys, (Z)Lys (Z)Tyr(B
zl) Phe Asn Ala Gly Hi
s (Bzl)Ser (Bzl)Asp (O
Bzl)-V, al person 1a Asp (OBz
l) 人SN Gly Thr(Bzl) Leu
Phe Leu Gly Ile Leu Ly
s (Z) AsnTrp Iys (Z) Gl
u (OBzl) Glu (OBzl) Ser
(Bzl)Asp (OBzl') Arg (NO2
) Lys (Z) Ile Met G1n5er
(Bzl) Gln rle Val Se
r (Bzl) Phe Tyr(Bzl)Ph
e Lys (Z)I, eu Phe Lys
(Z) person sn PheLys (Z)As
p (OBzl)人sp (OBzl)Gln
Set (Bzl)11e Gln Lys (
Z) Set (Bzl) Vat Glu (O
Bzl) Thr (Bzl) Ile Lys (Z
) Glu (OBzl) Asp (OBzl)Me
t Asn Val Lys (Z)Phc
Pbe Asn Ser (Bzl)Asi+
Lys (Z)Lys (Z)Lys (Z
) krg (NO2) Person 5p (OBzl) Asp
(OBzl) Phe Glu (OBzl) Lys
(Z)Leu Thr (Bzl) Asn Ty
r (Bzl) Set (Bzl') MalT
hr (Bzl)Asp (OBzl)Leu A
sn Vat Gln Arg(NOz) L
ys (Z)人la Ile His (Bz
l) Glu (OBzl)Leu Ile Gl
n Vat Met Ala Glu (O
Bzl) LeuSer (Bzl)Pro Person 1
a Ala Lys (Z)Thr (Bzl
)Gly Lys (Z) Arg (NO2) Ly
s (Z) Arg (NO2)Ser (Bzl)
Gln Met Leu Phe Arg (NO2
) Gly person rg (NO2) person rg (NO
2) Ala Ser (Bzl)Gln-resin, (
However, the symbols are the same as those defined above)
. Physical properties obtained in step 8: 1. Peptide molecular weight of IFN-γ: approximately 20,000 g 1 mole Amino acid composition (molti): histidine (1,4)) liptophan (o,3) lysine (13,6) arginine (s , s) Aspartic acid/Asparagine (116) Serine (7,6
) Glutamic acid/glutamine (11,3) Threonine ('3.4) Ri-nosine (3,4) Proline (1,4) Alanine (55) Valine (5,5)% Cystine (1,4) Methionine (17 ) Inleucif (4,8) Leucine (6,S) Phenylalanine (6,s) Tyrosine (3,4) Amino acid sequence: Cys Tyr C! ys, Gln Asp
Pro Tyr Val Lys Glu person 1
a Glu Asn, Leu Lys Lys
Tyr Phe Asn AlaGly
His Ser Asp Val Person 1a
Asp Asn Gly ThrLeu Phe
Leu Gly Ile Leu Lys Asn
Trp LysGlu Glu Ser Asp Ar
g Lys Ile Met Gln 5erGln
Ile Val 5erPhe Tyr Phe Ly
s Leu PheLys Asn, Phe L
ys, Asp Asp Gln Se'r Il
e GinLys Ser Vat Glu
'Thr Ile J, ys Glu Asp
Metλsn, Val Lys Phe Ph
e person sn Ser Asr+ Lys L
ysLys Arg Asp Asp Phe
Glu Lys Leu Thr AsnT
yr Ser Val Thr A&p Le
u Asn Val Gin ArgLys
Ala Ile His Glu Leu Ile G
in Valλ5etAla Glu Leu
Ser Pro Person 1a Ala Lys
Thr GlyLys Arg Lys Arg
Ser Gln Met Leu Phe
Person rgGly Arg Arg Person 1a Ser
GinZ IFN-γ-like pepti r Molecular weight: Approximately 20,000 g 1 mol Amino acid composition (mol%): Histidine (1,4) Tryptophan (0,3) Lysine (13,6) Arginine (5,4) 7 Sunozoragin Acid/Asparagine (116) Serine (7,5) Glutamic acid/Glutamine (112) Threonine (3,
4) Glycine (3,4) Proline (1,4) Alanine (s,4) Valine (5,4)'A cystine (
1,4) Methionine (3,4) Inleucine (
4,8) Leucine (6,s) Phenylalanine (6,8) Tyrosine (14) Amino acid sequence: Met Cys Tyr Cys Gln Asp P
ro Tyr Val LysGlu Ala G
lu Asn Leu Lys Lys T
yr Pbe A, in person 1a (:) Iy H
is Se,,r Asp Val Ala
A, sp Asn GlyThr Leu Phe
Leu Gly Ile Leu Lys Asn
TrpLys G1i+ Glu Ser Human sp Arg Lys Ile Met G
1n5erGIn Ile Val Ser Phe
Tyr Phe Lys LeuPhe Lys As
n Ph, e Lys Asp Asp Gln Se
r l1eGln Lys Ser Vat Glu
Thr Ile Lys Glu Asp: VIet
Asn Vat Lys Phe Phe
Asn Ser A, sn LysLys
Lys Arg Person sp Asp, Phe G
lu Lys Leu Thr Asn Tyr
Ser Vat Thr Person sp Lcu
Person sn Val Gin Person rgLysAlaI
IcHisGluLeuIleGlnVath(et
People la Glu Leu Ser Pro
Person 12 Person 1a Lys ThrGly Ly
s Arg Lys Person rg Ser Gi
n Met Leu PheArg Gly
Person rgArg Person 1a Ser G1n1 Pro I
Peptide molecular weight of FN-r: approximately 20,000 g/-Tyl amino acid composition (
molti): Histidine (1,2)) Liptophan (0,3) Lysine (xz7) Arginine (4,, g) Asno ξ Ragin e/As・ξ Ragin (1zo) Serine (7,8
) Glutamic acid/Glutamine ('9.4) Threonine (&6) Glycine (4,2) Proline (1
,2) Alanine (s, 4') Valine (5,4)
X12 Cystine (X,,S) Methionine (λ0)
Inleucine (S, 4) Leucine (8,4)
Phenylalanine (6,6) Tyrosine (4,2) Amino acid sequence: Met Lys Tyr Thr Ser Tyr I
Le Leu, Ala PheGln Leu C
ys Ile Vat Leu Oly S
er Lau GlyCys Tyr Cys
Gln A, sp Pro Tyr Mal
Lys Glu人1a Glu Asn'Le
u Lys Lys Tyr Phe As
n AlaGly His Ser Asp
Vat Person la Asp Asn Gly
ThrLeu Phe Leu Gly I
Le Leu Lys Asn Trp Ly
sGlu Glu Ser A4p human rg
Lys Ile Met Gln 5erG
ln Ile Val, Ser Phe T
yr Phe Lys Leu PheLys
Asn Ph-e-Lys Asp Person sp
Gln Ser IIe GlnLys S
er Vat Glu Thr Ile Ly
s Glu Asp MetAsn Val
Lys Phe Ph, e, Asn Ser
Asn Lys LysLys Arg A
sp Asp Phe Glu Lys L
eu Thr 人5nTyr Ser Val
Thr Asp Leu Asn Val
Glri ArgLys Ala Ile
His Glu Leu Ile G
la Vat Met person Ia Glu L
eu Ser Pro Person lx Ala Lys
Thr GlyLys Arg Lys
Arg Ser Gln Met Leu
Phe ArgGly person rg person r, g Ala
Ser Gin and Interfero 7 of the present invention
r (d) It has the following f1 quality. (1) Isoelectric point PH8,6 (2) Ultraviolet absorption shows a maximum peak at 278 mμ (Fig. 1). (31 SDS electrophoresis SDS PAGE Laemmli, U., (1
970) Nature (London) 227.
680-685, and then stained with Kuma-7-Brilliant Blue (R). The marker is Po5phorylase b (94k
), BSA (67k). Ovalbumin (43k), Carbon
ic anhydrase (30k). 5oybean Tri, psin Inhibito
r (20, Ik). Chilled with α-Lactalbumin (14,4k) (Fig. 2). In addition, the acute toxicity and formulation examples of interferon γ, a product of the present invention, are shown below. (1) Acute toxicity When the product of the present invention was subjected to an acute toxicity test in mice, the following results were obtained. No animals died and the LD5o It is impossible to calculate the numerical value. Mouse intravaginal 100 or more (2) Preparation example For intravenous injection Invention product 1 my7100ml 5
% glucose solution is used.

[Brief explanation of drawings]

Reference 82li I Step 1-5 Carry out in the same manner as in Example 1. Step 6 (BOC--<Butidyl resin) Repeat the same operation as Step 5 of Example 1 to extend the amine layer to obtain the following BOC-diagram butidyl resin DOC-HC' (0)-resin, Boa- HC' (-121, - mortar 26, -127, -
] 33, -136, -150, -151)-resin, Boc-HC' (-121, -126, -127, -
133, -136)-resin, T3oc-HC”
(-150, -151') -4ffJI+, T3
oc-11C*(-1-121, +126, +127,
+133, +136, +150, ++51)-resin, BOC-HC” (++21, +126, +127,
+133, ++36)-resin, Boc-HC' (++
50, +1'51)-resin, BOC-MC' (0)-
Resin, T3oc-MC*(-121, -126, -127, -
133, -136, -150, -151)-resin, Boc-MC" (-121, -126, -127, -
133, 13G)-(α1 fat, Boc-MO'(-15
0, -151)-resin, Boc-Met” (+121,
+126, +127, +133. +t3a, +ts
o, +ts1)→Sealing, Boa-MO” (+121,
+126. +127, +133. +136)-resin and Boa-MO" (+150, +151)-resin, where MO" represents methionyl HC", and HC" (0) represents Oys (Bzl) Ile Val Leu Gly
Ser (Bzl) LeuGly C70(Bz;
) Tyr (Bzl) Cys (Bzl) Gln
Asp (OBzl) Pro Tyr (Bzl) V
al Lys (Z) Glu (OBzl) A, Ia
Glu (OBzl) Asn Leu Lys (
Z) Lys (Z) Tyr(Bzl)Phe A
sn Ala Gly His (Bzl)S
er (Br3)Asp (OBzl)Vat
Ala Asp (OBzl) person sn G,
Iy Thr(Bzl) Leu Phe Leu
Gly Ile Leu Lys (Z) AsnTr
p Lys (Z) Glu (OBzl) Glu
(OBzl) Ser (Bzl) enter sp (OBzl)
l) Person rg (NO2)Lys (Z) I
le Met G1n5er (Bzl) Gln
Ile Mal Ser (Bzl) Phe Tyr
(Bzl) Phe Lys (Z)Leu
Phe Lys (Z)As,n PjheLy
s' (Z)Asp (OBzl) Aip (OBz
l) G, In Ser (Bzl)fle Gln
Lys (Z) Ser (Bzl) Mal Gl,
u (OBzl) Thr (Bzl) Ile Lys
(Z), GI! J (OB, ZI) Asp (0&
l) Met person sn val Lys (Z)
Phe Ph, e A, so Sar (Bzl
) Asn Lys (Z) Ly,s (Z)
Lys (Z) Arg (NO2) A, 5p (
OBzl) 人sp (OBzl), Pje G
lu (OBzl)Lys (Z)Leu Thr
(Bzl)Asn Tyr (Bzl)Ser
(Bzl) VatThr (Bzl) Asp
(OBzl) Leu Asn Val
Gln Ar, g(NO2) Lys (Z) A
ja -11e His (Bzl) Glu (OI
3zl) Leu Ile Gln val M
et person 1a Gfu (OBzl)LeuSe
r (Bzl) Pr, q, AIa Ala
Ly,s (Z) Thr (Bzl)Gly
Lys (Z) Person rg (NO2) Lys
(Z)Arg (NOx)Ser (Bzl)G
ln Met L, eu' Phe person rg
(NO2) Gly person rg (NO2) Arg (N
O2) represents Ala Ser (Bzl)Gin, however, the symbol is the same as defined above)
, a negative number within 0 indicates that the amino acid with that number is missing from the Boa-peptidyl resin, counting from the HC'' (0) nocarzexyl end, and a positive number within 0 indicates that the amino acid with that number is missing from the Boa-peptidyl resin Indicates that glycine is added to the amine group of the amino acid numbered from the end. Step 7 (cleavage of HC and Mc<zotide from resin, H
O” and MO”-J! While zotide has a protecting group, H
(C and MO represent Vepti 1 which does not have a protecting group) 1.4 g of Boc-HO” (0)-resin and 1-anine obtained in step 6 were mixed with tetrafluoroethylene in a stream. The reaction vessel was placed in a covered reaction vessel containing a stirring bar, and 20rnt of hydrogen fluoride was introduced into the reaction vessel while cooling with dry ice-acetone. After stirring at 0°C for 1 hour, the reaction vessel was placed under reduced pressure at 0°C. Hydrogen fluoride in the mixture is removed. A 1% by weight acetic acid aqueous solution is added to the resulting residue, and the peptide is extracted after stirring.
The extract is placed in a separatory funnel, washed with ether, and lyophilized. In this way, He(0) peptides without protecting groups are separated. Each Boc- obtained in step 6 by performing the same operation as above
<150 m9 each of the following iptides from butidyl resin
get. . He (-121, -126, -127, -13 people -13
6, -150, -1st), He(-121, -12ξ
-127, -13 people -136), He(-, tso, -
tSt), HC (+121, +126, +127, +13 +13
Den+150, +ts1), HC(+121, +12+
127, +133. +136), f(C(+150,
+ts't), Mc(o), MO(-121, -12 melons -127, -13 pieces -136
, -150, -1st), MO(-121, -12ξ
-127, -133% -136), Mc(-tso,
-151), h+c(+t2x, +12 teeth+127, +13 pieces+1
3 melon + tso, +1s1), MO (+121, +12
6, +127, +13 pieces +136), and MO (+15
0, +151) Step 8 (Physical properties of synthetic peptide) The molecular weight, amino acid composition, and amino acid sequence of each peptide obtained in Step 7 are measured. The molecular weight of each VeptiP is determined from the mobility measured by SDS gel electrophoresis. Amino acid composition (mol%) is 6N
Hydrolysis is carried out in an aqueous hydrochloric acid solution at 110° C. for 24 hours, and the resulting mixture is subjected to two-dimensional 1-8-chromatography for determination. Tricytophan, 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 analyzed using a new Hitachi automatic amino acid analyzer. 1. Peptide HC(O) Molecular weight: Approximately 20・ooog 1 mole Amino acid composition (mol%): Histidine (1,3)) Liptophan (0,3) Lysine (13,0:) Arginine (5,2) 7 ξlagic acid/asparagine (13,0) serine (7,8)
Glutamic acid/Glutamine (11,6) Threonine (3,2) Glycine (4,5) Proline (La
) Arani/(5,2) Nonorin (5,8) v2 cystine (20) Methionine (16) Inleucine (5,2) Leucine (7,8) Phenylalanine (as) Tyrosine (3,3) Amino acid sequence : C1ys lie Vat Leu G1. y Ser
Leu Gly Cys TyrC! ys Gln
Asp Pro Tyr Val Lys G
lu Ala GluAsn Leu Lyi Ly
s Tyr Phe Asn Ala Gly His
Sθr Asp Val Aha hap AO
n Qly Thr Leu PheLou
Qly Ilo Leu Lys AOn
Trp Lya Glu olu Ser A
op Arg LyG D1θ Met Gln
Ser ()In l1eVal Sor
Phe Tyr Phe Lys Leu
Phe Lys Asnphθ Lys As
p hap Gln Ser Ile Gl
n Lyo 5erVal Glu Thr
Ile Lys Glu Asp Met A
sn ValL7B rho phe Aan
ser Asn L7s I, ys Ly
G ArgAep hap Phe Glu
Lye Lou Thr Asn Tyr
5erVal 7hr ASP Lf3u A
On Val Gln Arg Lys Al
Alle Hls Glu Heu Ile
Gin Van Met Ala GluL
Ser r'ro Ala Ala
Lys Thr Gly Lye ArgLy
e Arg Ser Gin Met Leu
Phe Arg Gly Arg Arg Ala
Ser Gln Furthermore, the HC(0) of the present invention has the following properties. (II isoelectric point PH8,6 (2) Ultraviolet absorption spectrum shows maximum peak at 278 mμ (Fig. 1). (31 SDS electrophoresis 5I) s-PAGE fK: Laemmli, U,
(1970) Nature (London) 2
27,680-685 and then stained with Coomassie brilliant blue (R1. The marker is Po5phorylaae b (94k
), BSA (67k). Ovalbumin (43k), Carboni
c anhydrase (3nk). 5oybean Tripsin InhibitOr
(20,1k). α-Lactalbumin (14,4k) (Figure 2). 2, , <Putide HC (-121, -126, -1
27, -133, -136, -150, -151) Molecular bone: Approximately zO,000 g 1 mol Amino acid composition (mol %): [Above IC (0) -! ! Only amino acids with significantly different concentrations compared to peptides are shown. MCI below! The same applies to putide] Glycine (3,4) Alanine (3,4) Leucine (6,8) 3.4 putide HC (-121, -12ξ-
127, -133, -136) Molecular weight approximately 20,000
g1 mole amino acid composition (molti) Niglycine (4,0) Alanine (3,4) Leucine (7,4) 4, Peptide He (-150, -151)
Molecular weight: approx. 20・ooog 1 mole Amino acid composition (molti) Niglycine (3,9) Leucine (7-2) 5.4 butyF He (+121, +126. +127, +133
．． +136. +tso, +tst) Molecular weight: approx. 2
0・ooog1 mole Amino acid composition (molti) nigericin (8,7) 6, peptide HC (+121, +126, +127, +13 people+
136) Molecular weight: approx. 20,000g 1 mol Amino acid composition (molti) nigericin (7,5)? ,
-! ? Petide HC (+150, +ISt) Molecular weight: Approximately 20,000 g 1 mol Amino acid composition (Molti) nigericin (5,8) 8, Peptide Mc(o) Molecular weight: Approximately 20,000 g 1 mol Amino acid composition (mol%): Histidine (1, 3) Tricytophane (0,3) Lysine (IZ9) Arginine (5,2) 7spartic acid/Asnoξlagine (1z9) Serine (7,7)
Curtamic acid/Curtamine (116) Threonine (
3,2) Glycine (4,s) Proline (1,
1) Alanine (5,2) Valine (5,8)'A cystine (1,9) Methionine (3,2) Inleucine (s, 2) Leucine (7,7) Phenylalanine (6,5) Tyrosine (3 , 2) Amino acid sequence: Me) Cys Ile Val Leu Gly S
er Leu Gly, CysTyr Cys Gl
n Asp Pro Tyr Vat Lys Glu
AlaGlu A>n Leu Lys Ly
s Tyr Phe Asr+ 人IaGIyH
is Ser Asp val Ala As
p Asn Gly Thr LeuPhe La
u Gly Ile 'Leu Lys Asn Tr
p Lys GluGlu Ser Asp Person r
g Lys Ile Met Gln Se
r GlaI+e'Val Ser Phe Tyr
Phe Lys Leu Phe Lys Asn P
he Lys Asp Asp Gln Ser Il
e Gin LysSer Val Glu Thr
Ile Lys Glu Asp Met AsnVa
l Lys Phe Phe Asn Se
r Asn Lys Lys LysArg
Human sp Asp Phe Glu Lys
Leu Thr 人SN TyrSer V
al Thr Asp Leu Asn V
al Gln Arg LysAla Ile
His Glu Leu Ile Gln
Val Met AlaGlu Leu
Ser Pro Ala Ala Lys
Thr Gly Lys Arg Lys Ar
g Ser Gln MeL Leu Phe
Arg GlyArg Arg Ala
Ser G1n9, VeptiF'+vrc(-121
, to -12 -127, -134, -13 to -15 -1
51) Molecular weight: Approximately 20,000 g 1 mol Amino acid composition (Molti): [Only amino acids whose concentration is significantly different compared to the above MC(0) Vepti P are shown. The same applies to the following MC peptides. ] Chrysin (3,4) Alanine (3,4) Leucine (6,8) 10, PeptiF'MO (-121, -126, -12
7, -133, -136) Molecular weight: about 20,000g1
Molar amino acid composition (molti) Niglycine (4,0) Alanine (3,3) Leucine (7,3) 11, Pezothy rMe (-150, -ts
t) Molecular weight: approx. 20,000g 1 mol Amino acid composition (molti) nigericin (3°9) leucine (7,2') 12, peptide MC (++21, +126, +127,
+133, +136, +150, ++51) Molecular weight: approximately 20. ooog 1 mole amino acid composition (molti) nigericin (8,6) 13,
J Petit Mc (+12+, +126, +127,
+133, published 36) Molecule fir=about 20; Ooog 1
Molamino 1% l'& (1 (mol%) nigericin (
7,5) 14, R buti 1MC (+]50, ++5+)
6゛: approx. 20,000g 1 mol amino acid silk (
(mol%) nigericin (5', 7) The above peptides all have an isoelectric point of pH 8.6 and an ultraviolet absorption spectrum of 2.
78 mμ, SDS electrophoresis shows approximately α)K. Step 9 (Measurement of antiviral activity) Measurement of antiviral activity of each peptide obtained in Step 7°
by B.C. Merrigan - Evaluation of the inhibition of plaque formation by human interferon using monolayers of human neonatal dermal fibroblasts and bovine varicella varicella - "In vitro methods for cell-mediated immunity" by E. Edited by D.B.R. Bloom and B.R. Grade, Academic Press, Inc., New York 1971, Iβ, 48.
Page 9 [p, c, Yan Rigan. 1'jlaquo Inl+1bitionΔ5say
for Human InterferonEmpl
oying Human Neona+e 5k
in Fibroblast Monolayer
sk Bovine Vestical Storm
atitis Virus+'' In-vitro M
ethodin Ce1l Mediated Im
rr+unily'+edited by E-
D, B, R, Bloom& P, R, Grade+ A
Academic Press, N, Y, 1971,
It is measured by the same method as described in pp. 489). Specifically, each Veptibi obtained in step 87 is diluted variously and placed in 10 volumes of growth medium containing fetal microsera. FS-4 cells (human neonatal skin fibroblasts) are cultured in a monolayer in this medium. After 18 hours, the cells were infected with varicella stomatitis virus (VSV), which has the ability to form 20 plaques per cell, and incubated for an additional 1 hour.
Incubate at °C. Next, the cells are washed twice with the above growth medium, and then cultured again in the growth medium at 37°C for 24 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 following peptides exhibit antiviral activity at concentrations of 1 block/-: HC(0\HC(-121, -126, -127, -13 people-13
6, -150, -151), HC (-121, -126
, -127, -131, -136), 1-IC (-1s
o, -tSt), HC (+121, +12 melon +127, +133, +13
6, +150. +151), HC (+121, +126
, +127, +13 people +136), Hc (+tso, +
151), MO(0), MO(-1211-126,-127,-133,-1
36, -150, -151), MC (-121, -12
6, -127, -133, -136), +V+c(-1
50, -151), kIC (+121, +126, +127, +13
3. +136, +150. +151), MC
(+121, +126, +127, +13 people +136)
, and MO (+150, +151). Step 10 (Measurement of cell proliferation inhibitory effect) 2×105 FS-4 cells are transplanted into the growth medium described in Step 9 in a Nyare with a diameter of 6°. After 5 hours, remove the growth medium and replace it with the IC(0) peptide obtained in step 7 at 0 gg/mA' or Mc(o)-
! ! Pour fresh medium containing Zoti r into the above/yarns and continue culturing at 37°C for 18 hours. Reference example 2 Step 1 (benzoylation or isobutylation) d
Synthesis of bzA 50 mmol deoxyadenosine (dA) t=1s
Suspend omJ in dry pyridine. Next, 300 mmol of penzoyl chloride l'lk is added dropwise to this suspension while cooling, and the resulting mixture is allowed to warm to room temperature and stirred for about 1 hour to react. Completion of the reaction was confirmed by thin layer chromatography [developing solvent: chloroform-methanol (10:
1)]. After the reaction has ended, the reaction mixture is poured into a mixture of 500 ml of chloroform, 350 g of ice and 28 g of sodium bicarbonate. The resulting mixture is shaken in a branched funnel, left to stand, and the chloroform layer therein is separated. The aqueous layer is extracted twice with chloroform, and the chloroform layer is separated. The chloroform layers thus obtained are mixed and washed twice with water. Wash with water C
The chloroform in the σform layer was distilled off, and the resulting residue was
Add 50 grams of ethanol and Looml of Birikun to obtain a homogeneous solution. After cooling the obtained homogeneous solution to a temperature of <oC, 200 m/! 2NNaO
H. Add a mixture of the aqueous solution and 200 ml of ethanol while stirring to obtain a homogeneous solution. After stirring this homogeneous solution at room temperature for 5 minutes and cooling it, 200 ml of 2N aqueous hydrochloric acid solution is added, and the solution is further concentrated under reduced pressure to half the volume. Next, an equal amount of water is added to the concentrated solution, and the benzoic acid in the resulting solution is extracted with ether. The resulting aqueous layer is concentrated and azeotroped with water to produce N-benzoyldeoxyadenosine (abzA).
is precipitated. A mixture of the obtained N-benzoyldeoxyadenokune and a small amount of pyrinone is left in a refrigerator overnight, and then crystals precipitated from this mixture are collected. The obtained crystals are washed successively with a 5% by weight pyridine solution and with ether. As a result, 3 s mi') moles of abzA are obtained. The yield of dbZA is 70%. B. Synthesis of dbzC 100 mmol of deoxymethicone (dC) was dissolved in 500 mg of pyridine. Isoelectric trietheramine is added to this suspension, and after stirring for 30 minutes, 6 equivalents of inzoylchlori P are added while cooling with water. Next, the same operation as in section A above is performed to convert dC into 4-enzoyl. After the benzoylation reaction was completed, chloroform was distilled off and the resulting residue was
Dissolve in a mixture of 50 mZ of tetrahydrofuran, 1 t of methanol and 250 rnl of water. Add 250 portions of a 2N NaOH aqueous solution to the resulting solution while stirring under water cooling. After 10 minutes, the mixture obtained is neutralized with 250 ml of 2N aqueous hydrochloric acid solution. The following operations are the same as in Section A above. As a result, N-benzoyldeoxycytidine (dbzc)
is precipitated. Synthesis of 0d'ibG 100 mmol deoxyguanosine (dG) 500
mA' of pyridine, and 6 equivalents of inbutyl chloride are added dropwise to this suspension under water cooling. The mixture thus obtained is stirred in OC for 3 hours and then treated in the same manner as the benzoylation in Section B above to effect inobutylation of dG. After the completion of the inbutylation reaction, chloroform was distilled off from the chloroform layer, and the residue obtained was
Dissolve in 0 mg of ethanol, then add 50 mg to this solution.
Add 0 ml of 2N NaOH aqueous solution under OC and stir for 15 minutes. The resulting mixture was transferred to 1 t of ice-cold pyridinium-type cation exchange resin (Dowex
X 2, manufactured by Dak Chemical Co., USA) and neutralized. The thus neutralized mixture was added to a column packed with a small amount of the above dark fat, and 3
Wash with twice the volume of 10% (w/v) pyridine solution. The resulting eluate and washing solution are mixed and the resulting residue is
Recrystallized from a 5% (W/V) pyridine solution of
dIdG is obtained. Step 2 (5'-dimethoxytritylation) 100 mmol thymidine, 100 mmol dbzA% 100 mmol dbzo and 100 mmol dibQ in 200+++/, 400 rnl, 400 ml respectively
and 150ml of pyridine, and 1.1 equivalents of dimethoxytrityl chloride was added to each resulting mixture and reacted for 3 hours. The reaction is stopped by adding son/methanol. Each reaction solution obtained was concentrated, dissolved in 50 ml of chloroform, and then washed with water. In order to separate 5'-dimethoxytritylthymidine ((DMTr)
Dissolve in 500 rnl of benzene and heat. Next, add rhohexane until the solution becomes cloudy, and cool it until it reaches 4C (DMTr).
Obtain T crystals. (DMTr)dbzA , (DMT
r) Separate and separate dbzC and (DMT r ) d i b G. After distilling off the solvent of each reaction mixture of dbzA, dbzC and dIbG and dimethoxytrityl chloride P, dissolve the resulting residue in chloroform. ,t,s
Subjected to column chromatography using KP silica gel. 3% by weight methanol water is used as the eluent for column chromatography. Step 3 (Synthesis of dimer and trimer) 6 mmol of (
DMT r ) After azeotroping aG with pyridine, 10
Dissolve in mJ of pyridine. Meanwhile, 1.5XZ2 equivalents of triazole, 1.5Xλ2 equivalents of triethylamine and 2
p-chlorophenylphosphoric acid dichloride is added dropwise to a mixture of 0.0 g of dioxane while stirring under water cooling while avoiding moisture. After stirring the resulting mixture at room temperature for 1 hour, the triethylamine hydrochloric acid was removed. The obtained p-liquid was mixed with the above (D
MTr) Add to the pyridine solution of dG, avoiding moisture. Next, the solvent of the obtained solution is distilled off to an amount of about X, and then this solution is left standing at room temperature for 1 hour. This solution was then added with 4 equivalents of 1-methylimidanol and 8 mmol of 'd
Add 1bG and azeotrope with pyridine. The resulting residue was dissolved in 60 ml of pyridine and in this pyridine solution was added 9 mmol of triisopropylbenzenesulfonyl nitroimida:,/"J)' (JI under 1'-TPSN
(abbreviated as IJ). The resulting mixture is concentrated to X@ and left overnight at 30'C. The reaction is stopped by adding 6 ml of 50% strength by weight pyridine solution. After distilling off the solvent of the reaction-stopped mixture, the mixture was placed in a column filled with 150 g of type 60 silica gel (manufactured by Wako Pure Chemical Industries, Ltd.), and methylene chloride-P-methanol (30:1) containing 1% pyridine was added. Elute with a mixed solvent of Next, the eluate is concentrated, and the concentrate is added dropwise to n-hexane to extrude the desired dimer into powder. 2 mmol of the dimer thus obtained are phosphorylated in the same manner as described above and azeotroped with pyridine to obtain a residue. This residue was dissolved in 20 g of pyridine, then 3 g
ml of TPSNI was added and a condensation reaction was carried out in the same manner as above. After 21 hours, the reaction is stopped by adding a 50% by weight solution of Dugo in pyridine. Then add 20
After adding m1 glue oo form, 15rnl of 0.1 M
Tetraethylammonicum bromide (hereinafter referred to as 1'-TEABJ)
(abbreviated as ). After washing, the solvent of this reaction solution is distilled off, and the desired trimer is separated using a column filled with 60 g of phosphoric acid gel. The resulting overall yield of trimer is 60%. Step 4 (Removal of protecting groups from trimer) 20 m9 of trimer obtained in step 3 is dissolved in 3 ml of pyridine. Add 15 ml of concentrated aqueous ammonia to the resulting pyridine solution, seal it tightly, and leave it at room temperature for 19 hours.
Heat with SC for 5 hours to distill off the ammonia. Next 2
After adding 80% by volume of acetic acid, the acetic acid is distilled off. The resulting reaction mixture was azeotroped with toluene, and the residue was
After dissolving in 0.1 M TEAB solution, the solution is washed three times with 25 ml of chloroform and then once with 25 ml of ether. The resulting solution was evaporated and the residue was dissolved in 16
Dissolve in 7M urea solution. The mixture thus obtained was placed in a column packed with DEAE cellulose and
．． 05 M NaCt solution 500 +++ A' and 0.4
Elute with 7M urea-0.02M Tris-acetic acid (pH 8.0) solution with a salt concentration gradient using 500 mj of 0M Na1l solution. Of the resulting eluate, fractions having an absorption peak are collected to obtain the desired trimer. Step 5 (Synthesis of Olibinucleotide P) Oligonucleotide P is prepared by the same method as in Step 3, and the protecting group is removed from the resulting oligonucleotide by the same method as in Step 4. In this way, the oligonucleotide r described below is obtained. (1) GATCOATGTGTATCGTT(2
) T T G G G T T C! T T T
G (3) G G T T G T T A CT
G T (4) CA A G A COCA T
A C (5) GTTAAGGAAGCT (6) G A A A A CT T G A A
G (7) AAGTACTTTTAAC (8) GCTGGTCACTCT (9) GACGTTGCTGAO α01 AACGGTACTTTG (1℃ TTTTTGGGTATC (L5 TTGAAGAACTGG (131AAGGAAGAATCT α41 GACA GAAAGATO ti”i personTGCAATCTCAAA(161ATC
()TTTC! TTTTα7) TACTTTTAAG
TTG α110 TTTAAGAAOTTTαl AAG
GACGACCAA ■ TCTATCCAAAG Ql) TCTGTTGAAACTU ATC!
AAGGAAGAO 器人TGAACGTTAAG 1241 T T T T T T A A C!
T CT4 A A CA A G A A G
A A GW AGAGAOGAOTTT @GAAAAGTTGAOT @AAOTAOTCTGTT @ACTGACTTGAAC C30) GTTCAAAGAAAGL:3D G
CTA child ccAcGA person C33TTGATCCAAGTT C! ATGGCTGAATTG C341TO,TCCAGCTGOT C351AAGACTGGTAAG C311i) AGAAAGAGATCTC171C
AAATGTTGTTT (2) AGAGGTAGAAAG person C3Cj GCTTCTCCAAATAAG (4CII
TCGACTTATTGAGAAGcTCTTCT(
4υ ACCTCTAAAyCAA (43CATTT
GAGATCT (43CT T T CT CT T A C! C (
44) A, (3T CT T A G CA
G 0 (45) T G G A G A CA
AT T c+461 A (3C CA T A
A CT T () (47) GATCAATTCG
TG (48) GATAGCC! TTTG! T+49
1 T T G A A CG T T CA A
1501 GTCAGTAACAGAf:m'
GTAGTTAGTCAAfi CTTTTcAA
AGTO ff) G T CT CT CT T c T
to T) CTT'GTTAGAGTT (7) AAAAAAACTTAAO fi o'r'rcA, 'ra'rc'r'rcm
CTTGATAGTTTC (g) A A CA G A CT T T T
G (To)) OA, TAGATTGOTO (Middle)
GTCCTTAAAGTT 輯） CTTAAACAACTT (Foundation) A'AAGTAAAAAAGA (Foundation) AA
CGATTTGAGA ←l) TTGCATGATCTT. (fi' TCTGTCAGATTC(g) TT
CCTTCCA, GTT (fund) CTTCAAGAT
ACC (■ CAA, AAACAAAGT @ ACCGTTGTCAGC (To) AA, CGTCAGAGTG (3)' ACOAGC () TTAAA @ GT
ACTTCTTCAA 1735 GTTTTCAGCTTOCsuCT
T person λCGTATGG (To) GT'CTTGACAGTA (3) AC
AAOOC! AAAGAq ACCCAAAACGA
TACACATG step 6? Synthesis of lJ nucleotide P) Oligonucleotides (1) and (2) obtained in step 5
) 40 pmol each and 6.5 units of T j DNA kinase and 80 pmol of Cr ”p IATP (80;
/ Mi! JMo#), 100 μM spermidine, 20
mM D T T -, 10 m MMg C72,50
mM Tris-HCl (pH 9) and 0.1 m MEDT
25μ of the mixture containing A is placed inside. The reaction is 37C
for 30 minutes to combine the oligonucleotides F"(1) and (2) to form oligonucleotides P(1)-(2).
). To this reaction mixture, 2 to 5 times the amount of ethanol is added to precipitate the generated oligomer. This oligomer was subjected to electrophoresis using a 2o% polyacryloamide gel in 7M urea, and oligomer (1) -
(2) is obtained. (1) - (2> GATCCATGTGTATCGT
TTTGGGTTTTTTG Perform the same operation as above, oligonucleotide)' (
3) and (4) are combined to form oligomer (3)-(4
). Further, (1- (2) and (3) -(
4) by combining (1> −(2) −(3) −(4
). By repeating the above operations, the DNA described below is obtained. (1) −(2) −(3> −(4) −(5) −
(6) −(7) −(8) −(9) −(10) −
(u) (20) -(21) -(22) -(23)
, -(24) -(25) -(26) -(27'
) −((2)(37) −(38) −(39) . (40) −(41) −(42) −(43) −(
44) -(45)-(46) -(4?) -(wife)
-(49) -(50)-(51:l-,(52)-
(53) (54) (55)-(56)-(
57) - (58) - (59) ” (60) - (6
1) −(62) −(63) −(64) −(65)
DNAs (1) to (39) and (40) obtained above
(77) was mixed in 50 ml of TNE buffer,
65C, 45C. Culture at 37C and 20C for 1 hour each. This mixture was then mixed with 100 mM Tris-salt W (pH 7).
,5), 100 m M CaC/4 and l OOm
Add 20 μ of the mixture containing M MgC7z and cool on ice for 20 minutes. Step 7 (Cloning) Nie Noe Tokuinogu et al. (A, J, Twigg
e+al) is Netya, Vol. 254, pp. 34-38 (1
(975) (Nature, 254, 34-3
8 (1975). From this plasmid pAT 153, Michael D. Edge et al.
al) is published in Nature Vol. 292, pp. 756-762 (1
981) (:Na+ure, 292°756-76
2 (1981)'), Brahmid pPM5o having the promoter and operator of the lactose operon is prepared. 4 μg of the resulting plasmid pPM50 DNA was dissolved in 10 mM) lithium-hydrochloric acid (
pH7,6), 6mM MgCtz, 150mM
in a mixture of NaCt and 1mM DDT, and this mixture was treated with restriction enzymes BamHI and 5alK for 60 minutes with 37U.
The above pPM50 is cleaved by reacting for minutes. After stopping the reaction, remove the DNA from phenol-chloroform (
3:1 volume ratio), and the resulting DNA fragments were extracted with 40 mM Tris-HCl (pH 7, 8), 6 m
Large DNA fragments are recovered by electrophoresis using a 1% agarose gel in a mixture of M acetate++-1J cum and 1 mM BDTA. B obtained in this way
1 μg of amHISalI 3.2 kb vector fragment and the chemically synthesized gene obtained in step 6 were added to 20 mM
of Tris-HCl (pH 7,6), 10 mM MgCt
z and 10mM DTT in a mixture of 0.4 units of T'4 DNA lyase,
React at 12c for 16 hours. As a result, Shirasumi 1 is obtained in which the DNA containing the chemically synthesized gene obtained in Step 6 is bound immediately after the lactose operon. The plasmid thus obtained was transferred to E. coli X1776.
E. coli strain X1776 is transformed by contacting with the strain. After culturing the resulting transformant E bed, the DNA in the cells of this transformed strain was isolated, and the deoxynucleotide sequence of the resulting DNA was determined by N.M. Maxam et al.
axam et al) in Proceedings of the National Academy of Sciences America, Vol. 74, pp. 560-564 (1978) (Proc, Na.
Ilq Spring cad, Sci, USA, 74
, 560 564 (1978)). The resulting deoxynucleotide sequence is shown to match the theoretical sequence. The antiviral activity of the culture medium of the transformed strain is evaluated using FS-=4 cells and vSv as a virus to sensitize the FS- cells (F
S-cells and VSV were those used in the step of Example 1). As a result, the above culture solution exhibits antiviral activity. (From the operation of inserting Shirasumi r into E. coli X 1776 strain,
P of Asahi Kasei Corporation. This will take place in a level containment facility. ) Reference Example 3 Step 1 (Manufacture of mRNA) Add 1 rng of staphylococcal enterotoxin Δ (5FJA ) Rotate the lymphocytes at 37°C for 2 days at 37°C. After culturing, centrifuge the lymphocytes at a low speed of 800 rpm for 10 minutes. , 1.] A mixed solution containing 5r of Na2HP04H2H20 and 0.2 g of KH2PO4 (hereinafter referred to as "PB
(referred to as "P solution") and resuspend. While stirring the suspension, add 20mM) lithium-hydrochloric acid (pH 7,5) and 1mM to a 50t separating funnel.
Add 2 ml (W/V) to a mixture containing ITA (a mixture of Tris-HCl and EDTA is hereinafter referred to as 1"ze buffer").
of sodium dodecyl sulfate into mixture 17j. Next, pronase (Calbiosum, USA) is added to this mixture to a concentration of 200 μg/ml, and the mixture is stirred at room temperature for 1 hour. Furthermore, 1/20 amount of 2 M l-IJ susu-acid (pH 9) of the above mixed solution is added. The obtained mixture was heated to 15
Extract with t redistilled phenol for 20 minutes with vigorous stirring. Add another 3 tons of chloroform and stir vigorously for 10 minutes. After allowing the mixture thus obtained to stand for 1 hour to separate the phases, the resulting aqueous phase is re-extracted with phenol and chloroform. 60 g of SDS are added to the 18 t of hydration thus obtained. From this aqueous phase, 1/10 of the amount of 3M sodium acetate (p'Hs
, 5) and add twice the amount of ethanol to precipitate the nucleic acids in the mixture. After processing the above two steps and leaving the large mixture at -20C overnight,
Carefully remove the supernatant using a siphon. The remaining portion is centrifuged at -20C and 4000 rpm for 15 minutes to obtain a nucleic acid precipitate. The resulting nucleic acid precipitate was
M) Soo-hydrochloric acid (pH 7,5), 100mM NaC
6 and SmMEDT (hereinafter referred to as rTNEJ)
! : Abbreviation Suru) 0.5% (W/V) SDS was added to the mixture at 200 mA' and stirred. An additional 350 ml of TNE is added to the above mixture to dissolve the fibrous nucleic acid precipitate. Next, the obtained mixture was heated to 500
Centrifuge for 15 minutes at rpm to collect the precipitate. The collected precipitate is dissolved in 350 ml of THE with 0.5% [-W/V) SDS. This TNg solution and the above supernatant TNE solution are mixed and extracted 3 times with an equal volume of phenol, 3 times with a half volume of ether, and 3 times with an equal volume of ether. When the RNA recovered in this way is quantified using an absorbance meter at a wavelength of 260 nm, it is approximately 1
It can be seen that it is 00 Tn9. 21 g of type 7 oligo(dT) cellulose (Calbiochem, USA) was added to 500 wL of the RN-containing solution obtained above, and the mRNA was adsorbed onto the oligo(dT) cellulose by stirring at room temperature for 1 hour. let The oligo(dT) cellulose adsorbed with mRNA was centrifuged at 3000 rpm for 10 minutes, and the obtained oligo(dT) cellulose was separated into 50 rnl and 15 m
Rinse thoroughly with TNT. The mRNA is then eluted by five consecutive washes with 2 ml of water. Absorbance measurement shows that the yield of mRNA is 200 μg. Step 2 (Manufacture of cDNA) 40mM) Lis-HCl (pH 7,5), 30mM N
aCj. 5mM MgCl2.0.5mM D'rT (Calbiochem, USA), 20gg/gooligo (
dT)+2-+g (manufactured by P&L-ζ Iochemicals,
(USA), 5mM dGTP (Sigma, USA), 5mM dCTP (Sigma, USA), 5mM dTTP (Sigma, American Congregation '5J), 5mM"P'-dAT
P (manufactured by New England R Nuclear Co., American Congregation), 60gg/ml mRNA, 28
0 units of avian myeloblastosis virus (human MV) and 600
400 μl of a reaction mixture containing 100 units of reverse transcriptase (manufactured by Suda Research Laboratories, USA)
After incubating for 1 hour at 37G, 0.5 M
F, DTA and 20% (w/v) SDS are added to concentrations of 10 mM and 0.1% (w/v), respectively. This mixture was extracted with an equal volume of redistilled phenol and the phenol layer was extracted with 200mM) lis-hydrochloric acid (pH
7,5), 1 mM EDTA and 0.1% (W/V)
Wash with 200 μt of solution containing SDS. This washing liquid and the aqueous phase obtained above were mixed, and after extraction with an equal amount of ether to this mixed liquid, Sephadex G-
100 (manufactured by Armacia, Squeden) 3 ml capacity column (hereinafter referred to as "Sephadex G-100 column")
chromatography using the following methods: The fractions eluted from the column are o, 1 ml each, 0, 1 ml.
Collect at a speed of 7 minutes. Mix the radioactive fractions of both fractions, add 3M sodium acetate to this, and add 3ml of 3M sodium acetate.
This mixed fraction is then added to 25 times the amount of ethanol for both parts to precipitate the nucleic acids. This mixed fraction is left at -70°C for 10 minutes, and then centrifuged to remove the supernatant. The precipitated nucleic acid is dissolved in xoom/distilled water, and 20 μL of 5mNaOH is added thereto. The resulting mixture is left at room temperature for 40 minutes. Next, add 10 μL of 5M sodium acetate, 1 50 μL of distilled water, to this mixture.
Add L and 250μt of ethanol and heat at -70C for 10
After cooling for 10 min, the precipitate that precipitates out is heated with OC for 20 min.
Centrifugation at 000 x g yields 5 μg of cDNA. Step 3 (Manufacture of double-stranded cDNA) 5μg of single-stranded CDNA obtained in step 2 was added to 100μt
Dissolve in water and heat to 100C for 2 minutes. Add 0.1M heat-denatured potassium phosphate buffer (+) to the mixed solution after heating.
H6゜9), 10 m M MgClt, 10 m M D
TT, 1 mM dATP (Schwartz M), 1
mM dGTP (manufactured by Schwartz), 1rnM:
tc'rp ('/manufactured by Ywartz), 1mM3H-dTT
250 μt of a mixture containing New England Newk V7 (G, USA) and 150 units/E. coli DNA polymerase 1 (Boehringer Mannheim, Rooster P) was added to the P. tuber. The resulting mixture was heated to 15C
After incubation for 6.5 h at
Add TA and 20% (W/V) SDS to a concentration of 10 mM and 0.1% (W/V), respectively. The resulting mixture is extracted with 250 .mu.t of phenol (hereinafter this operation is often referred to as "phenol treatment"). Add the phenol phase to 20mM) Lis-HCl and 1mM ED.
Extract with 130 μt of 6TE buffer containing TA. The first obtained aqueous phase and the second obtained aqueous phase are combined and fractionated using a Sephadex G-100 column with a capacity of 3 ml. Of the 6 fractions obtained, the fractions showing radioactivity were collected, and the resulting mixed fraction was mixed with acetic acid.
Add 3M sodium acetate to make MK. Next, 2 to 5 times the amount of ethanol to this mixed fraction is added to this mixed fraction to precipitate the DNA. The mixture thus obtained is centrifuged to obtain 7 μg of I)NA. Next, add (D DNA, 0.2 M Mail,
50mM sodium acetate (pH 4,s) and 10mM
It is dissolved in 130 μt of a buffer containing zinc sulfate (hereinafter referred to as rs+ buffer) and kept at 37°C for 30 minutes. This mixture was then supplemented with 0.5 M EDTA and 20% (w/v) SDS at concentrations of 5 mM and 0, respectively.
．． Add to 1%. 130μ of the resulting mixture
The phenol phase was extracted with 50 μt of T phenol.
Wash with E buffer. This washing liquid and the aqueous phase obtained from the above phenol extraction are combined and fractionated using a Sephadex G-100 column to obtain 1 ml each of both fractions. Of the obtained fractions, both radioactive fractions were collected and 4 μg
A double jlcDNA is obtained. Step 4 (Production of dAMP-extended cDNA) Step 3
The double-stranded cDNA 150 m9 obtained in
ATP XI OOmM Sodium Caconolate (p
H7,2)'15mM CaC62,50tt g/
rLt bovine serum albumin (hereinafter abbreviated as sAJ)
and 8μ of a dATP solution containing 3 to 6 units of terminal deoxynucleotide transferase (hereinafter abbreviated as “T d TJ”) per DNAAlAg and incubated at 27'C.
After incubating for 8 minutes at -70C, the cells were frozen. Step 5 (Production of pBR322 cleaved with EcoRI and extended with dTMP) 20Ag (7) Plasmi I' pBR322 and 10 units of gcoRI (Bethesda Research Lazeratories, USA) were mixed with 10 mM) lith-hydrochloric acid (pBR322).
H7,5), 6 mM M gct2.50mM N
a046mM 2-mercaptoethanol and 200μ
Add 150μ of the mixture containing g/μt BSA and incubate for 2 hours at 37C. This mixture is extracted with an equal volume of ether and ethanol is added to form a precipitate. Then precipitate with 0.1mM sodium glycine (p)
((1,s), 5 m M MgCtz and 50 μg/
Dissolve in 100 μt of a mixture containing μtBSA. 17 units of λ-echin nuclease is added to the resulting mixture and cultured at 37C for 1 hour. After culturing, this mixture is treated with phenol and further ethanol is added to form a precipitate. The resulting precipitate (cleaved pBR322) was diluted to 100 mM
Sodium cacodylate (pH 7,2), 10 mM
N, a 2 HPO4, 5m M MgC1z, 1mM
dTTP, 50μg/rnl BSA and DNA
300 μt containing 173-6 units of TdT per μg
reaction mixture and incubate at 37C for 20 minutes. After the cultivation, the reaction mixture was treated with phenol and further Ig-
x Add tanol and collect plasmid P. Step '6 (annealing of pBR322 and cDNA)
dAMP-extended DNA product gng and EcoRI-cleaved and dTMP-extended plasmid l″pBR322.
20 ng were placed in 50 μt of TNE buffer and incubated at 6° cs 45C, 37[ and 20[, respectively] for 1 hour. Next, add 100mM to the culture mixture.
Add 20 μt of a mixture containing acid (1) H7,5), 100 mM C3LC1z, and 100 mM MgC4, and cool on ice for 20 minutes. Step 7 (Transformation of E. coli X1776) E. coli X17
100 colonies of 76 strains (Human TCC No. 31244)
μg/ml diaminopimelic acid, 10 μg/ml!
Inoculate 100 +++ l of Trisitone medium supplemented with nalidixic acid and 10 μg/ml ampicillin. In the resulting culture, E. coli strain X1776 is cultured at 37C, grown until the optical density (OD) at 650 μmK becomes 0.6, and cooled on ice for 30 minutes. Next, add 4
The cells are pelleted by centrifugation at 000 rpm for 10 minutes. The resulting cells are washed with 50 ml of I Q m M NaC4 and centrifuged. Dissociate cells into a 20 ml ioom
Suspend in MCaCtz and cool on ice for 30 minutes. Next, after pulverizing the cells by centrifugation, add 4 ml of 100mM C.
Resuspend in aC1z and leave overnight. The following experiments are conducted in Asahi Kasei Corporation's P-3 level containment facility under the approval of Asahi Kasei Corporation's Recombinant DNA Safety Committee in accordance with the Japanese Recombinant DNA Guideline. Annealed ρBR322 obtained in Example 3 Step 6 (
E. coli X17 treated with Ca-
Add 20 μt to 100 μt of the above suspension containing 76 strains.
After cooling with water for 1 minute, keep warm at 20C for 10 minutes. Then add 0.6 ml of tryptone medium to this mixture. The resulting mixture is inoculated onto tryptone medium agar plates supplemented with 100 μg/ml diaminopimelic acid, 10 pg/ml nadirixic acid and 10 lt g/ml anvinlin. Next, after culturing at 37C for 48 hours, each resulting colony is collected and suspended in 100 μt of tryptone medium placed in each hole of a micrometer plate. This micro measuring plate is 37cm
After overnight incubation at
Save to. In this way, 500,000 individual E. coli
A clone of the transformant of strain 1776 was obtained. Step 8 (Manufacture of mRNA probe) In the same manner as described in Step IK of Example 3, 200 μg of original mRNA derived from 38 people was obtained. Also, SE
38 people obtained 200 μg of the original uninduced m''RNA A by the same method as in Example 3 Step 1, except that the operation for induction with A was not performed. mM (r-32P IATP, 4
0mM Tris-HCl (pH 75), 30mM
@Solution 10 containing NaC/: and 5 mM MgCL2
Add 0 μt to 10 pg of SEA-induced mRNA- and incubate for 10 minutes at 37C. After inactivating the kinase in the culture by phenol treatment, 200 μg of non-SEA-induced mRNA is added to the resulting mixture. Add 3M sodium acetate to this mixture to a concentration of 0.3M. To the resulting mixture, 25 times the volume of ethanol was added to prepare 200 Ag consisting of SEA-induced 32p-labeled mRNA and non-SEA-induced mRNA.
A precipitate of . After centrifuging the resulting precipitate, -20
Save as 0. Step 9 (reduction of cDNA) The transformant of E. coli strain X1776 obtained in Step 7 of Example 3 is inoculated onto a tryptone medium agar plate containing 10 pg/rnt of amvirin and cultured overnight at 37C to form colonies. Next, Millipore filter-HAW
P (manufactured by Pharmacia, Sweden) to diameter 82
Cut a mm round filter and place it in an autoclave for 10 minutes. Sterile filter at 10μH/m
Place on another tryptone agar plate containing 1 ml of ampicillin. Each colony obtained above is inoculated onto this filter and cultured overnight at 37C to form two colonies on the filter. This filter is 0.5 N N
Soaked in aOH aqueous solution for 10 minutes, then 0.5 M)
Immerse in IJ susu-acid (pH 7,5) for 5 minutes. Next, the filter was soaked for 5 minutes in a mixture containing 1.5 M NRCt and 0.5 M) lithium-hydrochloric acid (pH 7.5). This filter was further treated with 0.3 M' NaC4 and 0.0
Solution containing 3M sodium citrate (pH 7) (hereinafter referred to as
This solution is referred to as the 1'-2XSsc solution. here,
SSC is 0.15 M NzC1 and 0.015M15
Soak for 5 minutes in 50% (V/V) formamide, 4X SSO (0,6M NaCL and 0 .06M
pH 7 solution containing sodium citrate) and SO
Pour 1 ml of a solution containing (mM) lithium-hydrochloric acid (pH 7, s) into a Petri dish, and add to this solution the mRNA mixture obtained in Step 8 of Example 3 (10 μg of 32p-labeled mRNA induced in 38 people and induced in SEA). mRNA that has not been
200 μg). The above filter is immersed in this solution and kept at 40C for 24 hours. The filter was then washed three times with chloroform for 15 minutes each at room temperature, and then washed with a solution containing 0.6 M NaCt and 0.06 M sodium citrate (pH 7) (hereinafter referred to as "4XSSc solution") for 15 minutes each time. Wash at room temperature for minutes. Incubate the bladder with 2X SSC solution for 15 min each at room temperature for 3
Wash twice. Next, put 2X SSC solution of 2' rat containing 20ml/golize nuclease into a petri dish, put the washed filter in it, and let it stand for 30 minutes at room temperature.
After standing for a minute, the filters are washed twice with 2X SSC solution and air dried. This filter is subjected to autoradiography. Thus, it can be seen that 7 colonies out of 500,000 colonies hybridize with the mFLNA described above. Step 10 (Production of recombinant plasmid t') Example 3 One of the colonies obtained in Step 9 was inoculated into 1 t of tryptone medium in 24 Erlenmeyer flasks, and the optical a
Culture with shaking at 37C until the temperature (OD) reaches 08 (() at 650 nm. Next, 1 t of tryptone medium is added to this culture, and then chloramphenicol is added at 170 μg/m
The solution was added to a concentration of 1 ml and cultured at 37c for 16 hours. Add 20ml of chloroporum to this flask and add 37C
Sterilize the culture by shaking for 10 minutes. After separating the culture from chloroform, 6000 rpm 4
Centrifuge for 15 minutes at 'Q to obtain 2 g of cells. Cells are suspended in 30 ml of 20 mM I-Lis-HCl (pH 7.5). This suspension was heated at 500Orpm 4C
Centrifuge the resulting cells for 20 min at 50 mM)
Resuspend in lithium-hydrochloric acid (pH 7.5). To this suspension, add 10 mg /
The lysozyme solution containing ml of lysozyme was added to the @
Add 8 mouths of cloudy liquid. After cooling the resulting mixture in OC for 10 min, this mixture was treated with 0.3 M
Add EDTA gently without stirring. After leaving the resulting mixture for 10 minutes at Oc,
2% (W/V)) Lido 7X-100 (surfactant, manufactured by Wako Pure Chemical Industries, Ltd.) is added. After 60 minutes, the mixture was heated to 10,000
Centrifuge for 60 minutes at rpm OC. Transfer the resulting supernatant to a beaker and add 3M NaOH to the beaker with stirring to adjust Ill to lλ5. Add this solution to 2
After stirring for 10 minutes at 0C, the pH is adjusted to 8.5. After stirring for a further 3 minutes, the volume of this solution was added to 5M NaC7.
and an equal volume of phenol are added to this solution, stirred vigorously for 5 minutes, and centrifuged at 110,000 rpm and OC for 10 minutes to separate the phases. The supernatant containing the circular double-stranded DNA is carefully separated from the medium phase containing the single-stranded DNA. The resulting supernatant is extracted three times with chloroform. Add sm9/ml pancreatic lysene nuclease, N to the extract containing circular double-stranded DNA to a concentration of 20 μg/ml. The resulting mixture is incubated at 37C for 60 minutes. To this mixture, add the volume of 5 M NaC4 of this mixture,
Thereafter, a sterilized 30% (W/V) aqueous solution of polyethylene glycol 6000 (manufactured by Union Carpair Co., USA) was added to a concentration of 7.5% (W/V). The resulting mixture is kept at -10'C for 14 hours, after which the mixture is centrifuged at 800 rpm, OT: for 20 minutes to collect the precipitate. This precipitate was treated with 0.075 M NaCt.
and 0.0075M sodium citrate so that the optical density (OD) becomes 20 at 650r+rr+. Next, add 20% (W/V) SDS to this solution.
．． Add to a concentration of 5% (W/V). Furthermore, pronase was added to this @ solution at a concentration of 0.5 μ/mi, and cultured at 37C for 30 minutes. This solution was then extracted three times with an equal volume of phenol, twice with an equal volume of chloroform, and heated at 15C at 20,000 rpm using a Beckman Model 5W-27 rotor.
Subject to sucrose density gradient centrifugation for 5 hours. This density gradient centrifugation involved 50mM) Lis-HCl (1)H7,5) and 1
A solution with a 5-23% (w/v) sucrose gradient containing mM EDTA is used. The resulting half of each half
Measure the optical density at 60 nm. Both parts containing DNA are collected, and the DNA is precipitated using sodium acetate and ethanol in the same manner as described above. Obtain 50 μg of circular double-stranded DNA by centrifugation. Step 11 (cleavage of plasmid 1) Ring-shaped double-stranded DNA 20 obtained in Step 10 of Example 3
μg hair, 10mM) Lis-hydrochloric acid (-pH7,5), 6mM MgC42,50
mM NaCl, 6mM 2-mercaptoethanol,
200μg/1ttBsA and 20 units of restriction enzyme Ec
Place in 150μ4 of the mixture containing oRI and incubate for 2 hours in j7C. Next, this mixture contains pronase, BDTA
and SDS each with a degree of 0.5 mo/rH
i, 10 mM and 0.5% (W/v). The resulting mixture was heated at 37C for 30 minutes at a humidity of 301Jt.
Extract with a mixed solvent of phenol-chloroform (1:1 g ratio). The resulting non-aqueous phase was diluted with 20mM
Wash with 50 μt of a solution containing Salt M (pH 7.5) and 1 mM EDTA fa. The washings and aqueous phase are combined and extracted three times with ether. Add a volume of 3M sodium acetate and 5 times glk of ethanol to the combined aqueous solution to form a precipitate again. Next, this mixture is cooled at -70C for 5 minutes and then centrifuged to obtain 9 μg of DNA. Step 12 (Degradation with restriction enzyme Ps + I) Use 5 μg of the plasmid cleaved with gcoRI obtained in Example 3 Step 811 instead of 20 μg of circular double-stranded DNA, and use the restriction enzyme pstI instead of the restriction enzyme gcoRI. Other than that, the same operation as in Example 3 Step 11 was carried out,
A mixture of Ps+I-digested DNA q pieces is obtained. This mixture was diluted with 50mM)') acetate buffer (pH
7,8) in 2% (W/W) horizontal agarose gel (10
Approximately 0.2 μg of DNA degraded with Ps+I is obtained. Step 13 (Partial digestion with restriction enzyme Bs+NI) Step 1
0.2 μg of Ps+I-decomposed DNA obtained in 2 days was added to 1
0mM) Lis-HCl (pH 7,5), 6mM MgC
12. Add 150μ of a mixed bath/ei containing 50mM NaC6, 6mM 2-mercaptoethanol, 200μg/μt BSA and 2 units of restriction enzyme Bs+NI, and incubate at 37c for 5 minutes to partially degrade the DNA. Next, the partially degraded DNA is treated with phenol-chloroform, ethanol, and electrophoresed in the same manner as in Step 11 of Example 3 to obtain 10 ng of BstNI partially degraded DNA fragments. Next 1 Example 2 Step 1
An oligodeoxynucleotide having the following formula prepared by the same method as in ~5: AATTCATGTGTATO GTOCTAGGCTCCCTC GGC! TGTTATTGTC TGACAAT personAyCAGCCGAGGGAGCO TAGGACGATACACATG. (However, A, G, O, and T are as defined above, and the left end and right end of each formula represent the 5'-hydroxyl group side and the 37-hydroxyl group side, respectively.) Anneal and bind to each other using T4 DNA IJ gauze in the same manner as in step 6. Step 14 (Expression of peptide in E. coli) David
Bui Gödel et al. Nature, Vol. 287, No. 411-
416 pages 1980 (David V, Goedde
leL al, Nature, Vol-287, pp
, 411-416 (x9go)), the DNA obtained above was inserted into the region of pBR322 cleaved with gcoRI and Pstl, and the E. coli tryptophan promoter and the E. coli tryptophan operator were inserted.
An EcoRI cleavage fragment consisting of 300 base pairs having a d-some binding site is created, and this fragment is ligated to pBR322 containing the above-mentioned target DNA. Plasmi P obtained in this way was used as Escherichia coli X1776.
E. coli strain X1776 is transformed by contacting with the E. coli strain. From the culture obtained by culturing this transformed strain, a peptide is separated using a monoclonal antibody that specifically binds to 4zotide HC(0). The above monoclonal antibody was prepared by David Niss Nanatsuba et al. in Nature, No. 285.
Volume 446, 1980 (David S, 5ech
er er al, Na+upe, Vat, 285
According to the method described in [P., 446 (1980)],
Peptide (Peptide) HC(0) obtained in Example I is used as an antigen. As a result of subjecting the obtained peptide to amino acid sequence analysis, the amino acid sequence of this peptide matched that of peptide MO(0). Reference example 4 D. V. Goeddel (D, V. Goeddel) at the International Conference on Interferon Research held over 2 years ago
We will conduct the following experiments using the method announced by Hindll Pv surrounding all origins of SV40 replication
uff cleavage fragment (R.M. Meyers et al., Flosseding-National Academy of Sciences America, Vol. 77, p. 6491, 1980 CR-M,
My er se+ al, Proc, J'J
at1. Ac5d-Sci, U-S, A,, 77
, 6491 (1980)] 6' EcoRI
Modify into a restricted fragment. That is, the Hindl11 cut end was made into an EC0RI cut end by binding a synthesized oligonucleotide, and p
vu■ The cut ends were treated with DNA polymerase [Klenak fragment (
KIen, ow fragment)] to fill in the vacant nucleotide part and then perform ligation.
Cut the end with EcoRI. The resulting EcoRI fragment containing all SV40 origins was inserted into plasmid pML-1 (pBR
322 and SV40 recombinant zolansmids with an ampicillin resistance marker and an origin of replication of E and Co11.
pBR322 and S V 40, which do not have sequences that prevent recombinant reproduction) (M. Luksy et al., Nature, Vol. 293, p. 79, 1981, C.M. Luksy)
eIal, Nature, 293.79 (19
81)')). Among the expressed genes obtained, those in which the SV40 late promoter is sequenced so that reading starts from the ampicillin resistance gene of pML-4 are selected. Next, EcoR closest to the ampinrin resistance gene of the selected fragment
The I restriction site was partially digested with EcoRI, and DNA polymerase [Klenow fragment
t) ': 1 fill and combine (K. Itakura et al., Science, Vol. 198, p. 1056, 1977 [K,
Itakura eIal, 3science, 19
8.1056 (1977)')). An EcoRI restriction fragment is prepared by adding an oligomer to the pst I fragment of Shirasumi P obtained in Example 2. The EcoRJ restriction fragment containing this cDNH is ligated to the ECORI cleavage site of the expression medium pML-8V40 obtained above. The resulting vector was transformed into the monkey cell line CO3-7 (Y. Gluzman, Cell, Vol. 23, p. 175, 1981).
Cell, 23.175 (1981)]. CO3-7 endogenously expresses the 5V-40 large T antigen and 1. Figure 2 shows that recombinant Cilasmi r containing pML-1 and 5V-40 origin sequences are allowed to grow. The presence of interferon in this CO3-7 cell culture medium is analyzed daily as described in Example 2. A yield of 100 units/ml of interferon was obtained 3-4 days after infection, indicating that interferon is excreted into the culture medium by C08-7 cells. Reference Reli 5 R.A. Hintzeman et al. Nature, No. 293
Vol. 717, 1981 (R, A, Hizzeman
et at. The following experiment was conducted according to the method described in Nature Engineering, 717 (1981). 10 μg of XhOI-cleaved pAcF301 was added to 20 ir+M) Lis-HCl (pH
8,1), 12 mM CaCtz \12 mM M
gCj2.0.2M NaCl, lmM EDTA
and 0.1 μg/μl BSA in an aqueous solution containing
．． Incubate for 15 seconds at 30C with 2 units of Ba131 nuclease (Bethesda Rithera Gelatori). During that time, approximately 50 base pairs of DNA are removed. A portion (1 μg) of the resulting DNA was incubated with DNA polymerase (Klenow fragment) and deoxylysed nucleon 1-triphosphate.
Both ends of A were filled with deoxylyzed nucleotides, followed by T4 DNA ligase and a synthesized EcoRI linker (manufactured by Colagelative Research).
(United States of America) for 12 hours at 14'C. Next, the DNA obtained above was digested with restriction enzymes EcoRI and 13amHI, and a mixture of various DNA fragments containing the base sequence of the ADH1 promoter was cut out from the resulting DNA fragments on a 1% agarose gel-water gel. Separate by preparative electrophoresis. The mixture of electrophoresis eluted fragments was ligated to the large EcoRI-BamHI fragment of pBR322, the individual clones were transformed into E. coli strain RRJ, and ampicillin-resistant colonies of the transformants were selected to separate the individual clones. select. DNA was extracted from each colony obtained using the quick screening method (H.C. 2-Nzeimula, Niekleitsk Assitu Research, Vol. 7, No. 151).
Page 3 1979 (H, O, Birnboim etat.
. Nucleic Ac1ds les,, 7.151
3 (1979)]). The length of each deleted portion is determined by the restriction enzyme E
37 of the DNA fragment obtained by cutting with coRI.
- DNA end, t? IJ merase [Klenow polymerase] and [α-
"P') dATP, and then the labeled DNA is cut with the restriction enzyme Aiu, and the resulting DNA fragments are measured using a urea-acrylamide gel. Plasmid pFRL4 is isolated from Shirasumi FYRp7.
[Plasmid YRp7 was described in Proceedings of the National Academy of Sciences of America, Vol. 76, p. 1035, 1979 (K.
5Luhl et at, Proc, Natl.
Acad, Sci. u, S, A, , 76, 1035 (1979
) of EcoRI of pBR322 according to the method described in
1.4 kb E, co containing the yeast gene TRPI at the site
It is created by inserting the li fragment. ) by the following method (No Noan ξ-ra, Sheen, Vol. 10, No. 15)
7 pages 1980 [I.G., Tschumper et.
al., Gene, 10.157 (1980)]. The two EcoR sites of plasmid YRp7 were cut with EcoRI, and the resulting sticky ends were digested with DNA polymerase
en+)] and connect the resulting pure ends (l
igalion), these two E c
o Delete the R,I cleavage site. Next, Shirasumi F″Y
of Rp7) (indl[l) to replace the two E
co HindI of Chilasmi r lacking the RI site [
By replacing it with the I small fragment, one EcoRI cleavage site is replaced, resulting in plasmid PFRL4. 20μ
Plasmid pIL4 of g is digested with BamHI and EcoRI and electrophoresed using a 1% by weight agarose gel-water. The resulting large fragment (500 base pairs) is excised from the gel, electroeluted, extracted twice with phenol-chloroform, and precipitated with ethanol. 3 μg of the resulting fragment is then separately isolated from the A that can be done
DN fragments with DH promoter and 20mM)
Lis-HCl (pH 7,5), 10 mM Mget,
, 10mM nothiothreitol, 0.5mM ΔTP and 0.5 units of T4 DNA ligase (1/It). The mixture was used to transform E. coli strain 294 (ATCC3+537), and cilasmids were transformed from the transformed mixture to 1 i'
; Manufacture. 50 μg of the plasmid obtained in Example 2 is digested with coRI, and 1.2 weight portions of the digested fragments are electrophoresed in low-scale water. A DNA fragment containing the genetic code of a pure peptide was cut out from the cage, electroeluted,
Extract twice with phenol-chloroform and precipitate by adding ethanol. A DNA fragment carrying the genetic code for R peptide was pre-cleaved with r2coRI and treated with bacterial alkaline phosphatase.
' Binds to the COR1 site. The obtained cilasmid is Bg
l if: Analyzed by 1 ill restriction analysis and used for yeast transformation. Recombinant Shirasumi P, in which DNA carrying the genetic code for 2-2 spotted r is placed between each ADH promoter fragment of the TRP I gene in the same or opposite direction, is transformed using a standard transformation method (Nie Hine/etc. , Producing National Academy of Sciences of America, Volume 75, Page 1929, 1978 (A, Hlnne
n et al, Proc, Natl, Acad
. scl, U, S, A, 75. ! 929 (
1978):] into yeast. Yeast reception method RH218 (tupl) (ATCC440
76) C Saccharomyces cerevisiae
A tryptophan-requiring mutant of L3 cerevisiae), Giini Miozari et al., Journal Bacteriology (, y, Bact, ), Vol. 134, No. 4
8 p. 1978] in spheroplasts (H. D. Alxe et al., Astrophysical, Tsuyanaru, aS 6, p. 325, 1981 [H, D, Alxe et a.
l, ABrOph7. 'J, , 86°325 C1
9s1) ) separately from the above cilasmid DNA.
Transformed colonies are selected by placing the culture mixture on a tryptophan-free agar medium. The cultures of each colony were grown under selective pressure in a tryptophan-free liquid medium, collected at mid-logarithmic phase, the cell walls of the cultured bacteria were separated, and the cells were lysed with guaninone hydrochloric acid to obtain cell extracts.・Made by 'J. -! ! Significantly high biological activity was observed in all transformants in which the plasmid containing the DNA with the genetic code for PETITTO was connected in the correct direction.

[Brief explanation of drawings]

Figure 1 shows the ultraviolet absorption spectrum of the HC(0) peptide obtained in Example I, and Figure 2 shows its SDS scale electrophoresis 1p.
J) is shown. As a marker, ovalbumin (molecule L:
'4', 1k'), nino) luponic anhirase (30k), large σ trypsin inhibitor (2
0,+k) and (χ-lactalbumin(]a,,ik)
was used. Asahi I' Figure VL Target 1 of r(c(o)) obtained in Reference Example 1!
The ultraviolet absorption spectrum of Fig. 2' is its SDS r'
Demonstrates electric chanting. As a marker, o-ζ-lubumin (
molecular weight 43k), carbonic anhydrase (30k
), canine putrid inhibitor (20,1k) and α-
Lactalbumin ('+71.l+k) was used. ! i, l,,,\"Vf1 person Asahi Kasei Corporation Ovalmine Carbonic Anhydrase Soybean Trypsin Inhibitor Purification IFN-γ Peptide Peptide Albumin Purification Unpurified Marker IFN-γ IFN-γ Peptide Peptide JI! □43 Figure 2 (B) Silk HC (0) Marker Peptide ovalbumin -43 at -30kO, -20, Ik, -- at -t4.4. F, to -30・α-lactalbumin□14.4・-Teitomerineho Seisho (Method) % formula % 1 Display of case 1985 Patent Application No. 206796 2, Name of invention Novel physiology Process for producing active peptides 3, relationship with the amended case Patent applicant address 1-2-6 Dojimahama, Kita-ku, Osaka-shi, Osaka Name (
003) Asahi Kasei Kogyo Co., Ltd. 4 Agent address: 3F Tamayagi Building, 3-1 Rokubancho, Chiyoda-ku, Tokyo Telephone: (261) 9636 3rd year of 1986
March 31st (shipment date) 7. Amendment details (1) The application form will be corrected as shown in the attached sheet. (2) H) In the fifth line from the top of page 102 of the specification, the statement ``Figure 1 is Example 1...'' was replaced with ``Figure 1 (8) is Example 1...''"..." he corrected. (2) On the 6th line from the top of page 102 of the specification: “...
, Figure 2 shows that...''.
・, Figure 2 (8) is corrected as ``...''. (3) In the 111th line from the top of page 102 of the specification, the statement ``Figure 1' is Reference Example 1...'' was replaced with ``Figure 1 (B) is Reference Example 1...''・” I corrected it. (4) Line 122 from the top of page 102 of the specification “...
, Figure 2' shows that...''.
. . . Figure 2 (B) shows that...". (3) Correct the drawing as shown in the attached sheet. that's all

Claims (4)

[Claims] (1) When synthesizing peptides using the solid phase method, a resin that has no functional groups other than those involved in peptide bonding is used as a support, and the reaction is carried out under strong acid conditions, and as the peptide chain becomes longer, A method for producing peptides characterized by easy washing with methylene chloride even in areas where molecules aggregate. (2) The peptide has the formula (I): [It has an amino acid sequence] (However, [It has an amino acid sequence] and Mot are cysteine, tyrosine, glutamine, aspartic acid, proline, valine, lysine, glutamic acid, alanine, asparagine, and leucine. , phenylalanine, glycine,
Claim No. 1 is a peptide of human interferon gamma having bioactive ability represented by residues of histidine, serine, threonine, isoleucine, tryptophan, arginine and methionine; and J represents a hydrogen atom. ) The method for producing the peptide described in section 2. (3) A peptide obtained by the production method described in claim (1). (4) A peptide obtained by the production method described in claim (2).