JPS62223199A - Novel peptide - Google Patents

Abstract

NEW MATERIAL:A compound shown by the formula. USE:An intermediate for synthesizing a low toxic novel cysteine-containing physiologically active peptide useful an antiviral agent, cytostatic agent and NK cell enhancer and an intermediate for synthesizing (pro)gamma-interferon, etc. Having improved shelf stability. PREPARATION:For example, a carboxylic acid group of alpha-amino-protecting group-containing amino acid is bonded to a substrate such as polystyrene resin crosslinked with divinyl benzene and the alpha-amino-protecting group is removed. Then, the deprotected free amino group is reacted with a second amino acid. Then, the deprotection and introduction of amino acid are repeated to give the aimed peptide chain and the covalent bond between the carboxylic acid group of carbon-end amino acid of peptide and the resin is cleft. The reaction is preferably carried out under a strongly acidic condition.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention provides antiviral effects, cell growth inhibitory effects, and
The present invention relates to a novel physiologically active takuputide intermediate that has excellent physiological activity such as K cell activity enhancement effect.

More specifically, the present invention has the following formula (II): Gln Asp Pro Tyr Val Lys
Glu Ala Glu Asn LeuLys Ly
s Tyr Phe Asn Ala Gly His
Ser Asp ValAha Asp Asn
Gly Thr Leu Phe Leu
Gly Engineering 1e LeuLys Asn
Trp Lys Glu Glu Ser
Person sp Arg Lys fleMet Gl
Ser Gin Ile Val Se
r Phe Tyr Phe LysLeu
Phe Lys Azn Phe Lys
Asp Asp Gin Ser l1eG
ln Lys Ser Val Glu T
hr IIe Lys Glu Asp Me
t person sn Vat Lys Phe Phe
Asn Ser Asn Lys Lys
Lys person rg Asp Asp Phe C
11u Lys Leu Thr Asn
Tyr 5erVat Thr person'I) L
eu person sn Val Gln Arg L
ys 人1a l1eHis Glu Leu
Ile Gln Val Met Ala
Glu Leu 5erProAla I
a Lj+s Thr Gly Lys A
rg Lys'人rg”5erGin Met
Leu Phe Arg Gly Arg
Arg Human 1a Ser Gin (Gln is a glutamine residue, human sp is an as-ξlagic acid residue, Pr
o is a proline residue, Tyr is a tyrosine residue, Val is /
ζ phosphorus residue, Lys harisine residue, Glu is glutamic acid residue, human 1a is alanine residue, Asn is asno ξ lagine residue, Leu is haleucine residue, Phe is phenylalanine residue, at-y is glycine residue , His is a histidine residue, Ser is a serine residue, Th is a threonine residue, 11e is an inleucine residue, Trp is a tryptophan residue, Arg is an arginine residue, and Met is a methionine residue). 7 regarding stain-free peptides.

The cysteine-free buty-P of the present invention has excellent storage stability and is useful as an intermediate for 7-stein-containing physiologically active peptides as described below.

That is, the present invention provides engineered FN-γ, IFN-γ-like 7-2 peptide, or the following formula (I): BCysX1CysX2CysX3-(1) (wherein B is a hydrogen atom or a methionyl group, Cys is a cysteine residue, Xl represents 5 to 9 amino acid residues, X2 represents 1 amino acid residue, and x3 represents 138 to 148 amino acid residues. This provides a stable and useful intermediate for peptides.

Conventionally, antimetabolites, alkylating agents, antibiotics, etc. have been used as antiviral agents or cell growth inhibitors, but these are highly toxic and are not preferred. Therefore, the development of antiviral agents or cell proliferation inhibitors with low toxicity is strongly desired.

Recently, interferon-α (hereinafter referred to as “FN-α”) and interferon-β (hereinafter referred to as “IFN-β”)
These studies have progressed due to the fact that virulence (referred to as ``virulence'') is a low-toxicity and effective antiviral agent. However, if
The cell proliferation inhibitory effects of N-α and IFN-β are not satisfactory. On the other hand, research on interferon-r (hereinafter referred to as "FN-1") has focused on IFN-α and IFN-1.
Compared to N-β research, not much progress has been made.

However, at the Second Annual International Conference on Interferon Research held in San Francisco, USA in October 1981,
ter-natiOnal Congress for
Although there was no report that IFN-r was isolated in the research conducted by David V.

Ckoeddel) [)
rF
Pro-IFN-r, IFN-γ, and IFN deduced from the deoxynucleotide sequence of the complementary DN (hereinafter referred to as 1'-cDNAJ) of N-r metsenger aNA (hereinafter referred to as "mRN person") The amino acid sequence of -r-like pepti 1 was announced.

These groups are IFN-r, IFN-1 and IF
The amino acid sequence of the NT-like peptide will be described later. Furthermore, at the aforementioned meeting, it was found that the culture solution of cells containing the gene containing rFV-r-like peptide r information has antiviral activity, but the culture solution of cells containing the gene containing pro-IFN-γ information has antiviral activity. It was also announced that they had not. On the other hand, production of a cell culture solution containing IFN-γ information 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. As a result, the present inventors surprisingly discovered that the iptide represented by the above formula (I) not only has extremely excellent all of the above-mentioned physiological activities, but also has low toxicity. . This invention was made based on this new knowledge.

Therefore, an object of the present invention is to provide a physiologically active peptide that not only has excellent antiviral activity but also has excellent cell proliferation inhibiting action and NK cell enhancing action.

Other aspects and advantages of the invention will become apparent from the detailed description below, along with the claims.

According to the invention, the following formula (I): BC7E'XIC7
Novel physiologically active R represented by 8X2C7SX3 (I)
(However, in formula (I), B is a hydrogen atom or a methionyl group, Cys is a cysteine residue, Xl is 5 to 9 amino acid residues, x2 is one amino acid residue, and X3 is 138
~148 amino acid residues) are provided.

Physiological activity of the invention-! ! Even though the amino acid sequence of this protein is similar to proeFN-γ, pro-FN-γ, and pro-FN-γ-like peptide r, it is significantly different from these in its physiological activity as shown below.

The amino acid sequence of proeFN-γ consists of 12 amino acid residues at the N-terminus of the novel physiologically active R peptide of the invention represented by the above formula (I) (wherein B is a hydrogen atom). Matches one of the amino acid sequences of the group added. However, while the novel physiologically active peptide of the present invention has excellent antiviral effects, cell proliferation suppressive effects, and NK cell enhancing effects, pro-IFN-r does not exhibit such effects.

The amino acid sequence of the engineered FN-γ-like peptide is
The novel physiologically active surfactant 12 of the invention represented by the formula (wherein B is a methionyl group). Matches one of the amino acid sequences except for The amino acid sequence of engineered FN-γ consists of the peptide residue represented by CysXl of the novel physiologically active peptide of the invention represented by the formula I1 (wherein B is a hydrogen atom) (however, Cys and Xl are as defined above)
Matches one of the amino acid sequences excluding . However, compared to the physiologically active peptide of the invention, IFN-γ-like peptide r and IFN-γ not only have inferior antiviral activity, but also inferior cell proliferation inhibiting action and NK cell enhancing action.

As is clear from the above, although PobutiP of the present invention has similar amino acid sequences to the known substances pro-FN-γ, pro-FN-γ, and IFN-γ-like peptide, it does not have physiological activity. This is a novel bioactive Peptide P with completely different abilities.

The bioactive peptide of the invention and the above-mentioned proeFN-γ.

Although the cause of the significant difference in physiological activity between IFN-γ and IFN-γ-like peptide r has not yet been completely elucidated, one of the causes is considered to be as follows. That is, in the formula (■) above, CysX1CysX
The three cysteine residues of the boptide residue represented by 2Cys (Cys, Xl and X2 are as defined above) play an important role in the excellent physiological activity of the butyptide residue of the invention. It is thought that this has been achieved. And Cys
XICysX2Cy day (However, Cys, X yo and
2 is as defined above) to CysXl (however, C
ye and Xl are as defined above), the biological activity decreases, and CysX1Cy
It is thought that when 12 amino acids are added to the N-terminus of sX2Cys (Cys, Xl and X2 are as defined above), physiological activity is lost.

As mentioned above, the novel physiologically active compound P of the present invention is represented by the following formula (I): BCysxlC:ysX2CysX3 (i)
(However, in the formula, B, CyS, X1. Xl has 7 amino acid residues and X3 has 143 amino acid residues.More preferable peptides include Xl caine leucyl valyl leucyl glycyl seryl leucyl glycyl heptaline residue and X2 as telonone. A particularly preferred peptide is one in which Xl is an inloinluvalyluroinruglycylserylleu/luglycine residue, X2 is a tyrosine residue, and X3 is the following formula (II): Gln Asp Pr
o Tyr Val Lys (nu Ala Glu
Asn LeuLys Lys Tyr Phe A
sn Aha Gly His Ser Asp Va
lAha Asp Asn Gly Thr Leu
Ph@Leu Gly Ile LeuLys As
n Trp Lys Glu Glu Se
r Asp Arg Lys IleMet
Gln Ser Gln Ile Mal Ser P
he Tyr Phe LysLeu Phe L
ys Asn Phe Lys Asp A
sp Gln Ser l1eGln Lys
Ser Val Glu Thr Ile
Lys Glu Asp MetAsn
Vat Lys Phe Phe Asn
Ser Asn Lys Lys Lys Ar
g Asp Asp Phe Glu Ly
s Leu Thr Asn Tyr 5e
rVal Thr Asp Leu Asn
Val Gln Arg Lys Ala
l1eHis Glu Leu Ile G
ln Vat Met Ala Glu L
eu'5erPro Ala Ala Lys Thr
Gly Lys A'rg Lys Arg 5er
Qln8(e+LeuPheArgGlyArgArg
AlaSerGln (however, Gln is a glutamine residue,
Asp is an asnoξlagic acid residue, Pro is a proline residue, Tyr is a tyrosine residue, Val is a /nosun residue, Ly
s rigidine residue, Glu is glutamic acid residue, Ala is alanine residue, Asn is asnoξlagine residue, Leu is leucine residue, Phe is phenylalanine residue, Gly
is a glycine residue, His is a histidine residue, Ser is a serine residue, Thr is a threonine residue, 11e is an isocoicine residue, Trp is a tryptophan residue, Arg is an arginine residue, and Met is a methionine residue) of the residues represented.

On the other hand, at the 2nd International Conference on Interferon Research in 2015, David Bui Gödel announced the above-mentioned
The amino acid sequences of FN-r, IFN-r, and IFN-r-like VeptiP are represented by the following formula (to).

2〇ID: J Cys Tyr Cys Gln Asp Pro
Tyr Val Lys GluAla Glu A
sn LL! u Lys Lys Tyr Phe A
An Ala GlyHis Ser Asp Vat
Ala Asp Asn Gly Thr Leu
PheLeu Gly Ile Leu Lys As
n Trp Lys Glu Glu 5erAsp
Arg Lys Ile Met Gln Ser G
in Ile Vat 5erPhe Tyr P
he Lys Leu Phe Lys A
sn Phe Lys AspAsp Gin
Ser Ile Gln Lys Ser Val G
lu Thr rleLys Glu Asp M
et Asn Val Lys Phe P
he Asn Ser person sn Lys Lys
Lys Arg Asp Asp Phe
Glu Lys Leu Thr Asn T
yr Ser Mal Thr Asp L
eu Asn Mal Gin person rg Lys
Ala Ile His Glu Leu
Ile Gln Val MetAla
Glu Leu Ser Pro Ala
Ala Lys Thr Gly Lys Ar
g Lys Arg Ser Gln Me
t Leu Phe Arg Gly Ar
gArg A1. a Ser Gln [However, C
ys, Tyr, Gln, Asp, Pro, Val, L
ys, Glu.

Ala, Asn, Leu, Phe, Gly, His, S
er, Ile, Trp, Arg.

Met and Thr are the same residues as defined above; and J is
Met Lys Tyr in case of P oT, FN-r
Thr Ser Tyr Ile Leu Ala Ph
e Gln Leu Cye 工１eVal Leu G
Ly Ser Leu Gly (However, Met,
Lys.

Tyr, Thr, Ser, Engineering 1e, Leu, Ala, P
he, Gln, Cys and Val are the same residues as defined above), hydrogen atoms in the case of IFN-r,
In the case of -r-like peptide r, it represents a methionyl group] Both the rupture peptides represented by the formula (Il) and the rupture peptides r represented by the formula (IIIl) are unstable. The formula (I) of the present invention obtained in the process
Any of the guptides represented by I) is stable and can therefore be a diluted intermediate of the guptide of the formula fI) or 20111).

In addition, Vepti r represented by formula (II) of the present invention can be expected to have pharmacological effects such as antiviral effects, cell proliferation inhibitory effects, and antirheumatic effects.

The novel protein of the present invention can be produced by a conventionally known method, namely, "Synthetic Chemistry Series - Veptyl Synthesis J.
Published by Maruzen Co., Ltd. in 1975 - Edited by the Japanese Biochemical Society "Biochemistry Experimental Course/Chemistry of Tan Q Lithium 1 - IVJ1977 Tokyo Chemical University Published by Tokyo Chemical Doujin Special Issue No. 26 of Acids and Enzymes
It can be produced by the method described in Volume 4, Genetic Manipulation J, published by Kyoritsu Publishing Co., Ltd., 1981; and Proteins, Nucleic Acids, and Enzymes Separate Issue No. 5, "Advances in Interferon Research J, Published by Kyoritsu Publishing Co., Ltd., 1981." Specifically, the novel method for producing peptides of the present invention can be classified into the following three types: (1) methods for producing peptides by chemical synthesis;

(2) A method in which a gene obtained by chemical synthesis is introduced into a host, and the host is made to produce a protein.

(3) Introducing biotechnologically obtained genes into the host,
A method for producing peptides in this host.

As one embodiment of the above first method, the solid phase method will be specifically described below.

In addition, when the peptide is synthesized by a solid phase method as a chemical synthesis method, as described in the Examples below, Vepti may have other functional groups in addition to the functional groups involved in binding. Good results can be obtained by using a non-reactive resin as a support, carrying out the reaction under strongly acidic conditions, and easily washing with methylene chloride even in areas where the molecules tend to aggregate as the butylated r-chain becomes longer. It will be done.

After the carboxylic acid group of an amino acid having an α-amine protecting group is bonded to a support such as a polystyrene resin crosslinked with divinylbenzene, the α-amine protecting group is selectively removed. 2 with an α-amino protecting group on the free amine group
React with the th amino acid. This removal of the α-amine protecting group and introduction of the amino acid having the α-amine protecting group are repeated to obtain the desired chain. Next, the C-
The covalent bond between the carboxylic acid group of the terminal amino acid and the resin is 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 above-mentioned solid phase method, all the reactions can be carried out in one container, so the production of the target compound is as simple as the 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 excess amount of reagents and byproducts used in the reaction can be completely removed by filtration, and the target peptide can be produced in a short time and with a high capacity.

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 reacting and bonding the resin with amino acids, functional groups are introduced into the resin to impart reactivity. Generally, in order to introduce a functional group into a resin, the aromatic ring of styrene is reacted with chloromethyl methyl ether by a Friedel-Krach reaction using 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 and easy to camp with a support and an amino acid having an α-amine protecting group without adversely affecting the side chain protecting group or Vepti P chain. It is preferable to use one that can be removed quickly. A preferred α-amine protecting group as described above includes a tert-butoxycardanyl (hereinafter abbreviated as "Boc") group.

This Boc group is
//Dioxane mixture or 50% (w/v) TfO
It can be easily removed from the peptide chain using H (tetrafluoroacetic acid) 10H2C/2 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 a benzyl group (hereinafter abbreviated as "B, +J") group is used to protect the side chain of as/ξlagic acid. However, for side chain protection of cysteine and glutamic acid, Bzl group or O-methylben-nor (hereinafter referred to as j'
-oB, j) group, Bzl group, benzyloxycarbonyl (hereinafter referred to as "
There are two groups for protecting the side chain of lysine, and two tosyl groups or diisopropylmethyloxycarbonyl (hereinafter referred to as DipmocJ) groups.
Sulfoxide groups for side chain protection of methionine and BZ for side chain protection of serine, threonine and tyrosine.
Examples include the I group.

The second method for obtaining the above-mentioned target material R of the present invention will be explained below. First, a genetic code corresponding to each amino acid in the amino acid sequence of the peptide of the present invention to be obtained is selected. Based on each of the selected genetic codes, a double-stranded DNA fragment having the genetic information of the waveptide of the present invention is chemically synthesized. The obtained double-stranded DNA fragment is incorporated into a vector, which is a replicable expression vehicle. Next, the vector containing the DNA fragment is inserted into the host.

The host is transformed by inserting the above vector incorporating the DNA fragment into the host, resulting in the formation of a transformant. Next, this transformant is isolated from the original host according to the presence or absence of the gene expression type by a conventional method. Vepti r of the present invention is produced by culturing the obtained transformant.

In this method, the non-butylene methionyl peptide (hereinafter referred to as Mc-s peptide) of the present invention is produced. This M
By partially hydrolyzing the C peptide with amine-determining butidase, the wavebutide of the present invention (hereinafter referred to as "HC-
) is obtained.

Examples of hosts used in the above method include microorganisms, salivary mammalian cells, and plant cells. Among these hosts, microorganisms and mammalian cells are preferably used. Desirable microorganisms include bacteria such as Escherichia coli, Bacillus subtilis, and thermophilic bacteria, actinomycetes, and yeast.

The replicable expression vehicle or vector used in the present invention is one that is capable of infecting the host described above.

Examples of such lids include plasmid P1 phage, viruses, etc. The vectors and hosts described above are also used when producing Veptir from a gene biotechnically obtained from an organism.

The circular genetic code corresponding to each amino acid residue shown below is used to synthesize the gene for the bioactive peptide of the present invention (however, in the genetic code shown below, human has a deoxyadenylic acid residue, G has a The deoxyguanylate residue is C
represents a deoxycytidylic acid residue, T represents a thymidylic acid residue, the left side of the genetic code represents the 5'-hydroxyl group, and the right side of the genetic code represents the 3'-hydroxyl group): Genetic for phenylalanine The secret match is TTT or TTC
, the genetic code for leucine is TT person, TTG.

CTT%CTC1CTA or CTG, the genetic code for isoleucine is ATT 5ATC or ATA, the genetic code for methionine is human TG, the genetic code for phosphorus is GTT, GTC! , GTA or GTG,
The genetic code for serine is TCT, TCC, TCA
1TCG, person GT or AGC.

The genetic code for proline is CCTlCCC, COA or CCa, and the genetic code for threonine is ACT.

ACC, A'C person or ACG, the genetic code for alanine is OCT%GCC1GCA or GCG, the genetic code for tyrosine is TAT or TAC, the genetic code for histidine is CAT or CAO, the genetic code for glutamine is CAA or CAG, for asparagine The genetic code is AAT or λAC, the genetic code for IJ Jin, AA person or human AG, the genetic code for aspartic acid is GAT or GAC, the genetic code for glutamic acid is GAA or GAG, the genetic code for cysteine is TGT or TGC1 the genetic code for tryptophan. is TGG, and the genetic code for arginine is CGT.

CGC, 00 people, CGG XAGA or AGG, and the genetic code for glycine is GGTSGGC, GGA
Or GGG is used. In addition to the above genetic code, ATG is the genetic code for methionine and is also used as the starting code for peptide synthesis, and TAA, TAG
and TGA are used as termination ciphers for peptide synthesis. The gene consists of the four types of deoxy IJ d nucleosides described above linked through phosphodiester bonds at the 3' and 5' sites. Therefore, peptide genes can be chemically synthesized using protecting groups and condensing agents. The protecting group is one that can selectively protect a predetermined position of deoxylyse nucleotide 1, is stable during the reaction of peptyl synthesis, and can be easily removed without breaking the bond between nucleotides. desirable. Furthermore, the reaction intermediates must be easily separated and purified. For the above reasons, the triester method described below is desirable for synthesizing the Pepti 1 gene.

That is, a trimer block of deoxyribonucleotide 0 corresponding to the amino acid code and, if necessary, a dimer block are first synthesized. The resulting blocks are then condensed to
This condensation reaction, which forms oligomers of about 12 to 23 nucleotide chains, is preferably carried out by a solid phase method using a support such as a polymer. By elongating the obtained oligomer using DNA ligase, a gene for the desired peptide is formed.

Examples of the protecting group for the amine group of the above-mentioned deoxyribonucleosides include benzoyl group for deoxycytidine and deoxyadenosine, and isobutyl group for deoxyguanosine. As a protecting group for hydroxyl group, trityl group,
Acyl groups, silyl groups and derivative groups thereof can be used. Among these hydroxyl group-protecting groups, trityl group derivative groups are preferably used. As a condensing agent,
2.4.6 - A strong condensing agent such as ``Iafurobylbenzenesulfonyltetrasilicate'' (hereinafter often abbreviated as ``TPST-'') is desirable. The protecting group for nucleotide r can be removed by the method described below. First, 3'-protecting groups such as amine protecting groups and acyl derivative groups are removed.
The hydroxyl protecting group was removed with aqueous ammonia, and then the 5'-hydroxyl protecting group, such as a trityl derivative group, was removed with 80%
Remove by volume of acetic acid.

The advantage of preparing genes by chemical synthesis is that it is possible to increase the productivity of the host P by using the genetic code that exists in large amounts within the cells of the host used to produce the host P.

A method of incorporating a chemically synthesized gene into a vector, a method of incorporating a vector into which the gene has been incorporated into a host to transform the host, and a method of culturing the transformed host to produce the novel physiologically active peptide of the present invention. , is the same as the method using natural genes described below.

A third method for producing the novel peptide of the present invention will be described below. First, a double-stranded DNA fragment having the genetic information of the novel dipeptide of the present invention is obtained by biotechnology. The obtained DNA fragment is incorporated into a vector, which is a replicable expression vehicle. A vector incorporating the DNA fragment is inserted into a unicellular host organism to transform the unicellular microorganism to obtain a transformant. The transformant is transformed into the DN
Depending on the phenotype of the vector incorporating the A fragment,
Isolate from the original unicellular organism. This is a method for producing the peptide of the present invention by culturing the isolated transformant.

In the third method described above, a specific type of polyptide is produced. That is, in the above method, in the general formula CI), Xl is an inleucylno2lylleucylglycylserylleucylchlysine residue, X2 is a tyrosine residue, and X3
The novel physiologically active peptide of the present invention is produced in which is a specific peptide residue represented by the formula (I). Furthermore, by this method, in the general formula (I), X and X2
is the same as above, and some of the amino acid residues of the target P residue of X3 represented by the above formula (ff) are changed to other amino acid residues or deleted, or other Those with added amino acid residues are produced by the method described above. An example of an implementation of this third method will be described in detail below. First, human peripheral blood lymphocytes are stimulated with mitogens to induce mRNA production, and then the lymphocytes are degenerated. The RNA containing mRNA is then centrifuged from the denatured lymphocytes. The RNA obtained next
mRNA is separated from among the cells using an oligo(dT) cellulose column. Using the obtained mRNA as a template, oligo(deoxylysesylthymine-phosphate) [hereinafter often abbreviated as rf IJgo(dT)J], deoxyadenosine triphosphate (hereinafter often abbreviated as "dATP"), Deoxycytosine triphosphate (sometimes referred to as dCTP)
In the presence of deoxyguanosine triphosphate (hereinafter often abbreviated as ['-aoTpj) and deoxylidacylthymine triphosphate (hereinafter often abbreviated as 1'-dTTPJ), by the action of reverse transcriptase, The above mRN
It creates a double strand of human and complementary DNA (hereinafter often abbreviated as "cDNA"). m obtained in this way
mRNA is removed from the RNA/cDNA duplex by alkali treatment or heat treatment to obtain cDNA. This c
Using DNA as a template, dATP, dcTP, cGTP
and in the presence of dTTP, double-stranded DNA with one end ligated is created by the action of reverse transcriptase or DNA polymerase 1. The ends of the DNA thus obtained are cleaved with nuclease St.

Addition of oligo (deoxyadenosine-phosphate) (hereinafter often abbreviated as "oligo (four)") to the 3'-end of the double-stranded DNA obtained above by the action of terminal transferase in the presence of dATP. let

On the other hand, plasmid pBR322 (F. Zeliper et al., [
"Construction and Characterization of New Cloning Systems", Norn, November (1977) [F, Bolivare
＋ al＋ ” cans! racjion and
Characterization of new c
loningvehicles I [; A muHi
purpose cloning system'
+ Gene *November (1977) is cleaved with the restriction enzyme EcoRI and then treated with λ-exonuclease. Subsequently, oligo(dr) was added to the 3'-end of the cleaved plasmid)' pBR322 by the action of terminal transferase in the presence of dTTPO.
Add. The double-stranded DNA added with the above oligos (6 people) and the linear plasmid l-'pBR322 added with OIJ-+' (dr) were mixed and annealed. The plasmid obtained by annealing was transformed into E. coli x177.
6 plants [American Type Culture Collection (ATCC), Rockville, Maryland, USA]
) and transform the strain.

The transformed strain is selected for resistance to drugs such as ampicillin. On the other hand, mitogen-induced mRNA was prepared in the same manner as described above, and the 5I-
At the end, T4 polynucleotide kinase and (r-32p
Label using 3hTp. The obtained 32p-labeled mRNA (hereinafter often referred to as r''+2p-mRNAJ
200 times the amount of unlabeled mRNA as this 32P-mRNA is added to the 32P-mRNA. The above-mentioned unlabeled mRNA was extracted from human peripheral blood cells that have not been stimulated by mitogens and therefore do not produce the physiologically active ButiP of the present invention. The transformed strain was evaluated by colony hybridization using the mRNA mixture obtained above as a probe. According to this method, "P-mRNA is present in cells induced with mitogens capable of producing the physiologically active peptide of the present invention, but is not present in cells not induced with mitogens," is used as a template. specifically hybridizes with the formed cDNA.The CDN that hybridizes with the above-mentioned 32P-mRNA.
Transformants containing A can be easily detected as shading on the autoradiogram. 32P-m RN mentioned above
A transformed strain containing C DNA that hybridizes with A is denatured, and the DNA in the cells of the transformed strain is recovered. Plasmid DNA containing the cDNA fragment is separated from the recovered DNA by ultracentrifugation.

The plasmid DNA thus obtained was treated with restriction enzyme P.
str, and then partially digested with the restriction enzyme Bstu,[. Next, a known method (Kei Itakura et al.,
Science, Vol. 198, p. 1056, 1976 CK, Takura et al: 5cien
ce, Vow, 193. p, 1Q55 (1976
)]): AATTCATGTGTATC GTCCTAGGCTCCTC GGCTGTATTGTC TGACAATA ACAGCCGAGGGAGCC TAGGACGATACACATG (where A, G, C and T are the same as defined above) Yes, the left side of each formula above is the 5'-hydroxyl group, and the right side is the 5'-hydroxyl group side. represents the 3'-hydroxyl side), that is, oligonucleotides determined according to the N-terminal amino acid sequence of the target amputation are annealed to each other to have the starting triplet λTG and have EcoRI and BsLNI cleavage ends. double stranded dna
Prepare oligomers. The double-stranded DNA oligomer thus prepared was ligated by the action of T4 DNA ligase.
It is ligated to the cDNA cut with restriction enzyme B, 5tNI and Pst'r. The DNA fragment thus obtained was inserted between the EcoRI-cleaved site and the Pstr-cleaved site of plasmid pBR322.

Next, a 300 base pair DNA fragment containing the Escherichia coli tryptophan operon promoter, ORator and lysesome binding site, and having EcoRI cut ends at both ends, was inserted into the plasmid pBR322.
It is connected to the EcoRI cleavage site of A and incorporated into the plasmid.

Next, the plasmid thus obtained is introduced into E. coli cells. According to the above method, if the cDNA fragment is correctly connected after the promoter in the direction of transcription into mRNA, the cDNA fragment will be
Humans produce mRNA (/
i (is transcribed.Furthermore, the transcribed #mRNA is translated into amino acids from the starting triplet code part, and the desired peptide is produced.In this way, from the cultured E. coli cells in the culture solution of ]t, The target peptide can be extracted in one volume.

Next, E. coli cells producing the desired protein are collected after culturing and lysed. After decomposing the nucleic acid in the solution containing the lysed cells with ribonuclease and deoxyribonuclease, the desired sterile protein is salted out with ammonium sulfate at 65% saturation. The obtained salting-out precipitate fraction was collected using chondral pore glass beads (Controlled pore glass beads).
(glass beads).

The method for determining the amino acid sequence of the novel peptide of the present invention is as follows. First, the methionyl bond of the novel veptyl of the present invention is cleaved with bromocyan. Cut 1
The peptide fragments obtained by analysis were separated using Sephadex a-100.
(manufactured by Pharmacia Fine Chemicals, Sweden), and the amino acid sequence of each fragment is determined sequentially from the N-terminus of each fragment using a known high-precision amino acid sequence analysis. On the other hand, the novel peptide of the present invention is partially digested with trypsin, and the resulting peptide fragments are separated using a Sephadex G-100 column. The amine chain 11 of each separated fragment is sequentially determined from the N-terminus of each fragment in the same manner as described above.

By comparing the amino acid sequence of each fragment of the peptide obtained by cleavage with promonoamine and the amino acid sequence of each fragment of the peptide obtained by cleavage with trypsin, the sequence of each fragment of the R-butyr is determined. In this way, the amino acid sequence of the novel physiologically active peptide of the present invention is determined.

When the purpose of suppressing cell proliferation, for example, suppressing the proliferation of malignant ulcer cells, is to use the peptide of the present invention as a raw material with further amino acid extensions, the peptide of the present invention is generally administered intravenously or intramuscularly. It can be administered intradermally or subcutaneously by injection. The daily dosage of the Invention D haptide varies depending on the age, medical condition, and weight of the patient, but it is usually administered by injection in an amount of txto' to Ix 109 units/mole per adult.

10 ointments to treat viral diseases? Vepti t' 1 x 10' to I x 109 of the present invention per
Soft agents containing units can be applied to the skin. Ointments containing the peptides of the invention can be prepared using conventionally known pharmaceutically acceptable ointment rules. The daily dosage of the ointment containing Vepti r of the present invention is:
Of course, it varies depending on the patient's age and medical condition, but it is usually possible to divide the amount and apply it in units of 1X10' to 1X10' based on the peptide of the invention.

EXAMPLES Hereinafter, the present invention will be explained in more detail with reference to examples, but the present invention is not limited to the following examples.

Explanation of abbreviations used in the examples ε-Z-lysine: A benzyloxycarbonyl group (Z-) bonded to the ε-amino group of lysine G-No2-Argini/: Guanidino group of arginine (
G-) with a nitro group (No2-) imBzl-histidine: Histidine imidazolyl group (im) with a benzyl group (BZI-) 0-Bz uniline: Serine with a pen attached to its hydroxyl group (o-) )
0Bzl: Ortho-methyl 4-benzyl group, which is one of the side chain protecting groups dA: Deoxyadenosine dC: Deoxycytidine dG: Deoxyguanosine dT: Deoxylidacylthymine dbzA: Deoxybenzyl Adenosine dibG: Deoxyisobutylguanosine (DMTr) A: 5
) Methoxytrityladenosine (DMTr)T
: 5'-dimethoxytritylthymidine dGTP:
Deoxyguanosine triphosphate dAMP: Deoxyadenosine-phosphate BSA: Bovine serum albumin SEA: Staphylococcal enterotoxin, a type of mitogen Human EDTA: Ethylenediaminetetraacetic acid SDS:
Sodium dodecyl sulfate DTT: ,,,7
Thiothreitol (Cleeland's reagent) Example 1 Step 1 (Washing of resin) Resin CBio-Bead S X2.2.00~'
400 MEC size 2-tidivinyl-4-benzene cross-linked styrene polymer trade name, manufactured by Pioran 1, USA)
] Add 150 g to 1e of distilled benzene with stirring. After 30 minutes, filter the resin through a glass filter and dry.

Next, add the dried resin to 11! The mixture was added to methanol with stirring, and after 30 minutes, it was dried using a glass filter. The resin obtained by drying is mixed with methanol, methanol/
Wash with water and water sequentially. Washed resin is 1e IN
N,', is added to the aOH 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 an aqueous IN hydrochloric acid solution of 1e and stirred for 1 hour in a boiling water bath. Next, the resin is washed with water.

The resin obtained by washing with water is suspended in 4e of distilled water and then left to stand. After 30 minutes of standing, floating fine resin was removed by decantation. In this way, the resin is obtained as a precipitate. The precipitated resin is placed on a glass filter and washed with methanol. The washed resin was added to a small amount of dimethylformamide (hereinafter abbreviated as "D'M F J") and stirred at 80°C for 30 minutes, and then the resin was washed several times with DMF and methanol. After stirring in methanol for 1 hour, the resulting resin was air-dried and further vacuum-dried at 100° C. for 2 hours.

Step 2 (Manufacture of chloromethyl resin) 25 g of the resin obtained in Step 1 and 100 g of distilled chloromethyl methyl ether were placed in a 1e round bottom two-headed flask, and stirred gently at 25°C for 1 hour to dissolve the resin. After swelling, it is cooled to 0°C. This swollen resin has 1.88-5
50-chloromethyl methyl ether containing nC14 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 was filtered through a glass filter, mixed with 500 g of dioxane-water (1:1) and then 500 g of dioxane-water (1:1).
Wash gently with rnt dioxane-3N hydrochloric acid (3:1). Further, the product is washed successively with water, dioxane-water and methanol, and then dried at 100° C. under reduced pressure.

Step 3 A, (ε-Z-lysine) 11 g of L-lysine copper salt, 10 g of Na HC!
A quarter of Z-CE of 03 and 12- was added every 10 minutes while stirring under water cooling, and the mixture was further stirred for 3.5 hours.

The precipitate thus obtained is separated and washed successively with water, ethanol and acetone/ether. The washed precipitate was suspended in 250-ml water, and then 41-ml water was added to this suspension.
2- The above precipitate is dissolved by adding 6N aqueous hydrochloric acid solution. Next, H2S gas is added to the resulting solution for 1~
Vent for 2 hours. The resulting CuS is removed using Iflosu ξ-Cell (Wako Tsumugi Co., Ltd., Japan),
``Wash this CuS with IN aqueous hydrochloric acid solution. The door liquid and washing liquid obtained in the above operation are collected, and air is passed through them to form H2S.
remove.

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, 10 g of ε-Z-lysine with a melting point of 254° C. is obtained. The yield of ε-Z-lysine produced is 65%. The recrystallization of the obtained ε-2I) gin is
After dissolving in dilute filler acid, it is neutralized with aqueous ammonia.

B, (G-No2-arginine) A mixture of oleum 120- and oleum 75- is cooled on ice,
110 g of L-arginine hydrochloride is gradually added to this mixture 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 mixed solution was adjusted to pH 6 with acetic acid, and then left in the refrigerator for about 4 hours.

The resulting precipitate is separated and recrystallized from hot water. The obtained crystals are washed successively with ethanol and diethyl ether and dried.

60 g of G-NO2-arginine with a melting point of 251 DEG C. is thus obtained. The yield of G-N o2-arginine is 50%.

C, (imBzl-histidine) 20 g of L-histidine hydrochloride H20 is dissolved in 200 g of liquid ammonia cooled to -30° C. in a dry ice-acetone bath, and a small piece of metallic sodium is added to this solution. Add while stirring until the blue color 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, NH3 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. The residue after NH3 removal is poured into 100-g ice water. After the resulting mixture is extracted with diethyl ether, the insoluble matter is removed.

The resulting solution was adjusted to pH 8 by adding dilute sulfurization, cold M,
Leave it in the refrigerator for 3 hours. The resulting precipitate is separated and recrystallized 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, (0-Bzl-serine) 237g of acetyl-L-serine was converted into 235-5NNa
Dissolve in OH aqueous solution and adjust p)I to 7.3. Add 4g of Takajustase (manufactured by Sankyo Co., Ltd.) to this solution.
5-〇〇, IM citrate buffer (pH 6,7, CaCl
Add a solution obtained by dissolving in 20.025M (containing),
After adding several drops of toluene, the resulting mixture was left at 36° C. for 10 days. After removing the resulting precipitate and concentrating the P solution, acetone is added to the concentrated solution to obtain crystals.

100 g of OBzl-serine are thus obtained. Following a similar method, 100 g of 0-Bzl-threonine is obtained from 240 g of acetyl-L-threonine.

E-(0-Bit-Tyrosine) 1.8g L-Tyrosine and 1.2g Cu S 04
- Suspend 5H20 in a solution of 5-2N NaOH aqueous solution and 10-water and stir for 2 hours. 60 to this suspension
- of methanol is added, then 1.2- of benzyl bromide and 0.7- of 2N NaOt (aqueous solution) are gradually added.

After 15 minutes 0.3 ml of benzyl bromide and 0.8-2
Add more NNaOH 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
The copper salt of 0-Bzl-tyro7 is obtained. This 0-Bzl
- The solid content is removed from the mixture obtained by kneading the copper salt of tyrosine and the 20 lN EDTA aqueous solution in a mortar, and the solid content is washed with water. Add a small amount of ED to the washed product.
By recrystallizing from hot water containing T, melting point 2
1 g of 0-Bzl-tyrosine having a temperature of 23° C. is obtained.

The yield of 0Bzl-tyrosine is 30%.

F, (S-Bit-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. The solution is then adjusted to pH 5 with acetic acid to form a precipitate. Collect and wash the formed precipitate with water. In this way, S Bzl-cysteine 1 with a melting point of 212 °C
60 g are obtained. The yield of 5-BzI-cysteine is 7
It is 0chi.

1 from 160 g of as/gragic acid in the same manner as above.
50 g of 0-Bzl-asnoξlagic acid and 150 g of 0-Bzl-glutamic acid are obtained from 160 g of glutamic acid.

G, (Boa-amino acid) Water is added to a solution obtained by dissolving 29 g of L-valine in a 250-1 N NaOH aqueous solution to make a 400-solution. 150m7 in this solution! of tetrahydrofuran, 100-
Add 1/5 of Boc-Cl every 10 minutes. Meanwhile, 2N NaOH is added to the solution each time the Boc-C1 is added to maintain the pH of the solution at i~9.

After 2 hours, the mixture is extracted with diethyl ether, the resulting aqueous layer is made acidic with a 0.5 M aqueous citric acid solution, and the precipitated oil is extracted with ethyl acetate. The resulting extract is washed with a small amount of water, dried over Na2S04 and concentrated under reduced pressure. Add petroleum ether to the concentrated extract,
Generate crystals left in the refrigerator. The resulting crystals are dried several times.

In this way, 30 g of Boc-/' phosphorus with a melting point of 78 DEG C. are obtained. The yield of BOC-/' phosphorus is 55%.

H, (Other Boc-amino acids) By performing the same method as above, Boc-glycine 15g1 Boc-alanine 16g, Boc-leucine 15. g, Boa-inleucine 15 g, Boc-serine (Bzl) 14 glBoc-threonine (Bz I
) 14 g, Boc-cystine (Bzl) 15 g,
Boc-methionine 15 g, 'Bo'c-proline 12 g, Boc-asnoξlagic acid (OBzl)
15 glBoa-glutamic acid (osz+) I
S g, Boc-glutamine 15 g1 Boc-histidine (Bzl) 12 g, Boc-leucine (Z)
13 g, Boa-arginine (No2) 15
g.

Boa-Phenylalanine 15g 5 Boa-Tyrosine (Bzl) 15g and Boa h Lipdoan Lo
g is 30 g of glycine, 30 g of alanine, and Cf, respectively.
f Isine 30g1 Isoleucine 30g1 Serine (B, I
) 30g1 Threonine (Bzl) 30g, Cystine (Bzl) 30gl Methionine 30g1 Proline 3
0g1 Asnoξragic acid (oBz+) 30 g, glutamic acid (OBzl) 30 g, glutamine 30
g1 histidine (Bzl) 30 g, lysine (
z) 3og, arginine (NO2) 30g,
Phenylalanine 30g 1 Tyrosine (Bzl) 30g
and 30 g of tryptophan.

Step J (Coupling of Boc-glutamine and resin) A mixture of 2 g of chloromethyl resin (total CE content of 4 mmol), 2 mmol of BocL-glutamine, 1.8 mmol of triethylamine and 12 g of dimethylformamide P was heated to room temperature. 24 hours up.

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, 50 cm of the product is weighed and 1
The mixture is placed in a 1:1 volume ratio mixture of 2N hydrochloric acid and dioxane 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.

Step 5 (Boc-8er-Gln-Resin) 2 g of Boc-glutamine obtained in Step 4 (hereinafter 1'-Boa-
The resin (abbreviated as Glnj) 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.
Boa groups are removed from the resin by shaking in 1 nt IN aqueous hydrochloric acid/acetic acid at room temperature for 30 minutes.

The product thus obtained is separated and washed three times in sequence with acetic acid, ethanol and dimethylformamide. The washed product is placed in 160-10% diethylamine/dimethylformamide and shaken for 10 minutes to effect 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 Boc-serine (BZI) in 12-methylene chloride and shaken. After 10 minutes, a solution of 20 mmol of dicyclohexyl carbodiimir dissolved in 6Q ml of methylene chloride is added to this mixture, and the mixture is 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-peptidyl resin) The same operation as Step 5 is repeated to elongate the amino acid to obtain the following Boc --<butidyl resin. That is, first, cysteine-free Boa--! A butidyl resin is obtained and then used as a raw material to obtain a cysteine-containing BOc-wave butidyl resin. : BOC-HC(Q)-resin, BOc-HC' (-121, -126, -127, -
133, -136, -150, -151)-resin, BOc-HC"(-121, -126, -127, -1
33, -136> -Resin, Boc-HC” (-150,151) -Resin, Boc
-HC” (+121, +126, +127, +133
, +136, +150, +151)-resin, Boc-HC"(+121, +126, +127, +1
33, + 136) - Resin, Boc-HC" (+150, + 151) - Resin, B
oc-MC"(0)-resin, Boc-MC' (-121, -126, -127, -
133, -136, -150, -151)-resin, Boc-MC" (-121, -126, -127, -
133, -136') -resin, Boc-MO" (150,151) -resin, Boc
-MC'''(+121, +126, +127, +133
．． +136, +150, +tst) - [fat, Boa
MC' (+121, +t26. +t27, +133.
+136)-resin and Boc-MC” (+15
0. +151)-Resin However, MC" is methionyl H
HC” (0) represents Cys (Bzl) Il
e Vat Leu Gly Ser (Bzl) L
euGly Cy5 (Bzl) Tyr (Bzl)
Cys (Bzl) Gln Asp (OBzj:l
Pro Tyr (Bzl) Val Lys (Z
) Olu (OBzl)Ala Glu (OBzl
) Asn Leu Lys (Z) Lys (Z)
Tyr(Bzl) Phe A, sn Ala Gl
y His (Bzl) Ser (Bfl) person sp
(OBzl)Va! Ala Asp (OB
zl) 人SN Gly Thr(Bzl)
Leu Phe Leu Gly Ile
Leu Lys (Z) Asnjrp Lys (
Z) Glu (OBzl) Glu (OBzl)
Ser (Bzl)Asp (OBzl) personrg
(NO2) Lys (z> Ile Met
GIr+Ser (Bzl) Gln Ile V
al Ser (Bzl) Phe Tyr(Bzl)
Phe Lys (Z)Leu Pbe Lys
s (Z) person sn PheLys (Z), A
sp (OBzl) Ajp (OBzl) G, Ir
+ Ser (Bzl)fle Gin Lys (Z
) Ser (Bzl) Vat Gl, u (OBz
l) Thr (Bzl) 1. IeLys (Z) G
lu (OBzl) Asp (OBzl) Met A
sn Val I;ys (Z) Phe Ph,e
A, sn Se1 (Bzl) As! I Lys
(Z)Ly,s (Z)Lys (Z)Arg
(NO2) People! (OBz l) person sp
(OBzl)Phe Glu (OBzl)Lys
(Z)Leu Thr (Bzl) Asn Ty
r (Bzl) Ser (Bzl) ValThr
(Bzl)Asp (OBzl)Leu Asn
Val Gin Ar,g(NO2)Lys
(Z) Person 1a IIe His (Bzl)
Glu (OBzl)Leu, Ile Gln
Vat Met Ala Glu (OBzl
)LeuSer (Bzl)Pr, o Ala A
la Ly,s (Z)Thr (Bzl)Gl
y Lys (Z)Arg (NO2)Lys
(Z) Person rg (NO2>Ser (Bzl)
Gin Met Leu'Phe Arg (NO2
) GlyArg (NO2) Personrg (NO
2) represents Ala Ser (Bzl)Gln, however, the symbol is the same as defined above),
A minus number within 0 indicates that the amino acid with that number is missing from the Boc-peptidyl resin, counting from the calxyl terminus of HC'' (0), and a plus number within 0 indicates that the amino acid with that number is missing from the Boc-peptidyl resin.
This indicates that glycine is added to the amine group of the amino acid numbered from the cardaxyl terminal of C'' (0).

Step 7 (cleavage of HC and MO iptide from resin, however H
C" and MC""VeptiP have protecting groups whereas HC"
and MC represent 4 peptides without a protecting group) 1.4 g of Boc-HC”(0)-resin obtained in step 6 and 17!
of anisole was placed in a reaction bath containing a stirring bar covered with polytetrafluoroethylene, and placed in a dry ice bath.
Hydrogen fluoride 20- is introduced into the reaction vessel while being cooled with acetone, and after stirring at 0°C for 1 hour, the hydrogen fluoride in the reaction vessel is removed under reduced pressure at 0°C. A 1% by weight acetic acid aqueous solution is added to the resulting residue, stirred, and then Vepti P is extracted.The extract is placed in a separating funnel, washed with ether, and freeze-dried. In this way, Hc(o)-? Separate the putido.

Each Boc- obtained in step 6 by performing the same operation as above
From the peptidyl resin, 150 μm of each of the following peptides were obtained.

HC (-121, -126, -127, -133, -1
36, -150, -151), HC (-121, -12
to -127, -13 pieces -136), HC (-150, -
151), HC (+121, +126, +127, +13 +13
+150, +151), HC (+121, +12ξ+
127, +13 +136), HC (+150, +15
'l), MO(0), MC! (-121, -12 to -127, -13 books -13
6, -150, -151), MO (-121, -12 to -127, -133, -136), MC (-150, -
151), MO (+121. +12 to +127, +133. [
36, +150, +151), N mouth C (+121, +1
26, +127, +13 people +136), and MO (+1
50, +tSt) Step 8 (Physical properties of synthetic peptide r) The molecular weight, amino acid composition, and amino acid sequence of each synthetic peptide r obtained in step 7 are determined.

The molecular weight of each pepti r is determined from the mobility measured by SDS gel electrophoresis.

Amino acid composition (mol %) 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 Penograph chromatography for determination. Tryptophan, cystine and cysteine are not detected by this method. Therefore, the composition of tryptophan and cystine or cysteine is determined by decomposing another fixed amount of each peptide with a protease. Note that glutamic acid and glutamine, and aspartic acid and asparagine cannot be distinguished.

The amino acid sequence of each peptide was analyzed using a new automated amino acid analyzer manufactured by Hitachi.

1,-! Pted HC (0) Molecular weight: Approximately 20,000 g 1 mol Amino acid composition (molti): Histidine (1,3) Tryptophan (0,3) Lysine (13,0) Arginine (s, 2) Asno ξ lagic acid/Asno ξ lagin (13,0) serine (
7,8) Glutamic acid/Glutamine (11,6) Threonine (3,2) Glycine (4,5) Proline (1,3) Alanine (5,2)--phosphorus (s, s) V
Cystine (20) Methionine (Z6) Inleucine (5,2) Leucine (7,8) Phenylalanine (6,5) Tyrosine (3,3) Amino acid sequence: Cys Ile Vat Leu G1. y Ser
Leu Gly C! ys TyrCys Gin A
sp Pro Tyr Val Lys Glu Al
a GluAsn Leu Lys Lys
Tyr Phe Asn Person 1a Gly
HisSer Asp Val Ala Asp As
n Gly Thr Leu PheLeu Gly
Engineering 1e Leu Lys Asn Trp L
ys Glu GluSer Asp Arg L
ys 工le Met Gln Ser Gln 工 1eVayu Ser Phe Tyr Ph8
Lys Leu Phe Lye AsnPh
e L'ys Asp Asp Gln Ser 工１e Gln Lys 5erVal Glu T
hr Ile Lys Glu Asp Met
Asn ValLys Phe Phe As
n Ser Asn Lys Lys L7s
ArgAsp Asp Phe Glu Lys
Leu Thr Asn Tyr 5erVal Th
r Asp Leu Asn Val Gln Arg
Lys Allalle His Glu Le
u 11e Gln Val Met Ala
GluLeu Ser Pro Ala
Ala Lys Thr Gly Lys A
rgLys Arg Ser Gln Met L
eu Phe Arg Gly ArgArgAla
Ser Gln Furthermore, HC(ol) of the product of the present invention has the following properties.

(1) Isoelectric point pH8,6 (2) Ultraviolet absorption spectrum A very thick peak is shown at 278 mμ (Fig. 1).

(3) SNS electrophoresis SDS-PAGEic Laemmli, U., (
197Q) Nature (London) 2
27. 680-685, and then stained with Coomassie Brilliant Blue (R).

The marker is Po5phorylase b (94k
), BSA (57k).

Ovalbumin (43k), Carbon
ic anhydrase (3Qk).

5oybean Tripsin InhibitOr
(20,1k).

α-Lactalbumin (14,4k) (Figure 2).

2. Aptide HC (-121, -126, -127, -
133, -136, -150, -151) Approximately 20.000 to 1 mole amino acid composition (molti) per molecule: [HC(0)A above! Only the amino acids that have a significantly different concentration compared to Zobuti are shown.

The same applies to the following HC peptide P] Glycine (3,4) Alanine (3-:4) 'Leuci 3, a peptide HC (-121, -126, -1
27, -133, -136) Molecular weight: approximately 20,000g
1 molar amino acid composition (mol%) niculisine (4,0) alanine (3,4) leucine (7,4) 4, peptide HC (-150, -151)
Molecular weight: Approximately 20,000 g 1 mole Amino acid composition (mole ratio) Nikulisine (3,9) Leucine (7,2)5, Peptide Hc (+t2r, +126. +127, +1'3
3. +13ξ+15 to +151) Molecular weight: approximately 20,00
0g 1 mole Amino acid composition (molar ratio) Nigericin (,8,7) 6.4
Petit HC (+121, +126, +127, +133
%+136) Molecular weight: Approximately 20,000g 1 mole Amino acid composition (mole ratio) Nigericin (7,5) 7, Peptide HC (+150. +1st) Molecular weight: Approximately 2 o
+o OO'g 1 mol Amino acid composition (molar ratio) Nigericin (5,8) 8, peptide Mc(o) Molecular weight: Approx. 20.000g 1 mol Amino acid composition (molar ratio): Histidine (1,3) Tryptophan (0,3) ! Ju
7 (IZ9) Arginine (5,2) 7Snoglagic acid/Asnoξlagin (119) Serine (7,7) Glutamic acid/Glutamine (11,6) Threonine (
3,2) Glycine (4,5) Proline (1,1
) Alanine (5,2) Noguri/(5-8) ≠ Cystine (1,9) Methionine (3,2) Isoleucy:y (s, 2) Leucine (7,?)
Phenylalanine (+, s) Tyrosine (3,2
) Amino acid sequence: Met Cys He Vat Leu Gly S
er Leu Gly CysTyr Cys Gln
Asp Pro Tyr Val Lys Glu
AlaGlu Asn Leu Lys Ly
s Tyr Phe Asn Person Ia Gl
yHis Ser Asp Val Ala Asp
Asn Gly Thr LeuPhe Leu Gl
y Ile Leu Lys Asn Trp Lys
GluGlu Ser Asp Arg Lys I
le Met Gln Ser G1nl1e Vat
Ser Phe Tyr Phe Lys Leu
Phe Lys Asn Phe Lys Asp As
p Gin Ser Ile Gln LysSer
Vat Glu Thr l1eLys Glu As
p Met AsnVat Lys Phe Phe
Asn Ser Asn Lys Lys Lys Ar
g Asp Asp Phe Glu Ly
s Leu Thr Asn TyrSer
Vat Thr Asp Leu Asn
Val Gln Human rg LysAla I
le His Glu Leu IIs G
ln Mal Met AlaGlu Leu
Ser Pro Ala Ala Lys
Thr Gly Lys Arg Lys
Arg Ser Gln Met Leu
Phe Arg Gly Arg Arg Al
a Ser G1n9, target r MC(-12
1, -126S-127, -13 people-13ξ-15-
151) Molecular weight: Approximately 20,000 g per mol Amino acid composition (mol %): [Only amino acids with significantly different concentrations compared to the above MC(O) peptide are shown. The same applies to MC Vepti P below. ] Chrysin (3,4) Alanine (3,4) Leucine (6,8) 10, Pepti)'MC (121, -126, -127
, -133, -136) Molecular weight ° approx. 20,000 g 1 mole Amino acid composition (mol%) Nikulisine (4,0) Alanine (3,3) Leucine (7,3) 11, Peptide MC (-150, -1s
l) Molecular weight: about 2o, ooog1 mole Amino acid composition (mol%) Nigericine (3,9) Leucine (7,2) +2. Wavebutido MC (+121, +126, +12
7, +133, +136, +150, +151) Molecular weight: approx. 20.000? 1 mole amino acid composition (mole ratio) nigericin (8,6) +3.
Takupuchido MC (+121, +126, +127, +1
33, +136) Molecular weight, approximately 20.000? 1 molar amino acid composition (mole ratio) nigericin (7,5) 14,
Aptide MC (+150, +151) Molecular weight ゛ approx. 20
．． 00OL? 1 mole amino acid composition (mole ratio) nigericin (5, 7) The above peptides all have isoelectric points of pH 8, 6, ultraviolet absorption wavelength maximum width of 278, and SD8 electrophoresis of about 20K.

Step 9 (Measurement of antiviral activity) The antiviral activity of each wave obtained in Step 7 was measured by comparing a monolayer of human neonatal skin fibroblasts with bovine varicella stomatitis virus by P.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 P.R. Gray 1, Academic Press, New York 1971, p. 489 ( P
, C, Ms-rigan, Plaque Engineering nhibibi
tion Aesay for Human Engineering
rferon Employing Human Neona+e 5
kin Fibroblas+ Monolayers
＆ Bovine Ves+1cular Stoma
titis Virus%' In-vi+ro M
ethodin Cell-Mediated I+n
muniLy” + edited by E, D,
B, R, Bloom& P, R, Grade+ Aca
demic Press, N, Y, 1971,
I) l+489) Measured in the same manner as described in I).

Specifically, each test subject 1 obtained in step 7 is diluted variously and placed in 10 volumes of growth medium containing fetal microsera. FS-4MJ 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) capable of forming plaques at a rate of 20 per cell, and incubated for an additional 1 hour.
Incubate at 7°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 subject F
shows antiviral activity at a concentration of lμg/-: HC(0).

HC(-121,-1,26,-127,-133,-
136, -1so, -tst), HC(-121, -1
26, -127, -13 pieces -136), HC (-150
, -151), He(+121, +126. +127, +13a,
+136, +150, +151), HC (+121, +
126, +127, +13 +136), HC (+15
0, +ts1), MO(0), MO(-t21, -126, -127, -133.-1
36, -tso, -151), MO (-121, -12
6, -127, -133, -136), MO (-150
, -151), MO (+121, +126, +127, +133, +1
36, +150. +151), MO (+121, +12
6, +127, +13 people +136), and MC (+15
0, +151).

Step 10 (Measurement of cell proliferation inhibitory effect) 2X105FS-4 cells are transplanted into the growth medium described in Step 9 in a Petri dish with a diameter of 60+m++. After 5 hours, the growth medium was removed and replaced with HC(0) peptiP obtained in step 7 at a concentration of 0.1 μg/mj but still MO(
0) Pour fresh medium containing the peptide into the above-mentioned petri dish and continue culturing at 37°C for 18 hours.

After culturing, remove the medium and add 3H at a concentration of 5 μCi/ml.
- Add a medium containing thymidine and further incubate 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 an i-body scintillation counter (manufactured by Packer F, USA). For comparison, Davi P.
dv, Gocdel)
Pebuti P having the same amino acid sequence as N-r, IFN-'r, and IFN-7 homogeneous Pebuti P is prepared by the solid-phase synthesis method described above, and the cell proliferation inhibitory effect is measured by the method described above. . The results are shown below.

(Hereinafter, blank space) From the above, it is clear that the vepti l″HC(0) and MC(O
) is very effective in suppressing cell growth, whereas pro-IFN-r has no cell growth suppressing ability and
It can be seen that r and IFN-γ uniform peptides have low cell growth suppressive ability.

(The following is a blank space) Example 2 Step 6 (/Stin-free Boc-Beptinol resin) BOc-8er-Gln- obtained in Step 5 of Example 1
The same operation as in step 5 of Example 1 was repeated using a resin to elongate the amino acid to obtain the following cysteine-free Boa.
- Obtain a corrugated butidyl resin.

Boc Gln Asp (OBZI) Pro Ty
r (BZI) Val Lys (Z) Glu (O
BZI) Ala Glu (OBZI) Asn L
eu Lye (z) Lys (phrase Tyr (Bzl)
Phe A8n Aha Gly His(Bzl)
Ser (BZI) Asp (OBzl) Val
Ala Asp (OBZI) Asn Gly Th
r (BZI) Lou Phe Leu GlyLy
s Leu Lys (Z) Asn Trp Lye
(Z) Glu (OBZI) Glu (OBZI)
Ser (Bzl) Asp (OBZI) Arg
(No.2) Lys (Z) Engineering 1e Met Gln S
er (Bzl) Gln 工 1e ValSer (B
zl) Phe Tyr (Bzl) Phe Lys
(Z) LeuPhe Lys (Z) Asn P
he Lys (Z) Asp (OBzl) Asp
(OBzl) Gln Ser (BZI) 工1e G
ln Lys (Z) 5er(Bzl) Val G
lu (OBzl) Thr (Bzl) Ile L
ys (Z)Glu (OBZI) Asp (OBZ
I) Met Asn Val Lys (z)Phe
Phe Asn Ser (Bzl) Asn L
ys (Z) Lys (Z)LyS(Z) A
rg (No,) Asp (OBzl) Asp (
OBZI) PheGlu (OBzl)Lys (
Z) Lsu Thr (BZI) Asn Tyr (Bz
l) Ser (Bzl) Val Thr (BZ
I) Asp (OBZI)Leu Asn Val
Gln Arg (NO2) Lys (Z)
Ala l1eHis (Bzl)Glu (OB
zl) Leu Ice Gln Val MetAla
Glu (OBzl)Leu Ser (BZI)P
ro Ala AlaLys (Z)Thr (B
zl)G17 Lys (Z)Arg (No□)L
ys (Z)Arg (No2)Ser (Bzl
)Gln Met LeuPhe Arg(No□)
Gly Arg (No. 2) Arg (No.)
AlaSer (Bzl) Gln-resin The same operation as Step 5 of Example 1 was further repeated using the cysteine-free BOc-steroid resin obtained here to obtain engineered FN-γ, IFN-r-like glutinide, and HC.
BOc-a butidyl-resin for cysteine-containing peptides such as (0) and MHC (0) is obtained.

Step 7 (cleavage of sterptide from resin) Cystine-free Boc of 1,47 obtained in Step 6
- Heftidyl resin and 1rfl anisole were placed in a reaction vessel covered with polytetrafluoroethylene and equipped with a stirrer, and hydrogen fluoride was introduced into the reaction vessel while cooling with dry ice-acetone.
After stirring at °C for 1 hour, hydrogen fluoride in the reaction vessel was removed under reduced pressure at O''C. 1 wt% acetic acid aqueous solution was added to the resulting residue, and after stirring, butylene chloride was extracted and the extract was separated. It is placed in a liquid funnel, washed with ether, and lyophilized.In this way, the cysteine-free waveputide, which has no protecting group, is separated.

This trick is stable. When necessary, the N-terminal amine group and the side chain functional group were protected again using the above-mentioned protecting groups, and then reacted with ao-methyl resin to restore the original cysteine-free Boc-wave butidyl resin. The desired physiologically active wave peptide can be produced by repeating the same operation as step 5 of 1.

Step 8 (Physical Properties of Synthetic Waveputide) The molecular weight, amino acid composition, and amino acid sequence of the waveputide obtained in Step 7 are measured.

The molecular weight of Takubutide is determined from the mobility measured by SDS gel electrophoresis.

The amino acid composition (mol %) is determined by hydrolyzing a certain amount of Niputide in a 6N aqueous hydrochloric acid solution for 24 hours at 110'C and subjecting the resulting mixture to two-dimensional -.♀-chromatography. Tryptophan, cystine and cysteine are not detected by this method.

Therefore, the composition of tryptophan and cystine/stine is determined by decomposing another fixed amount of each putide with a protease. In addition, glutamic acid, glutamine, and Asuno? It is difficult to distinguish between lagic acid and as-glagin.

The amino acid sequence of each Takubuti r is analyzed using a new automatic amino acid analyzer manufactured by Hitachi.

Molecular weight: approx. 20,000?1 mole Amino acid composition (molti): Histidine (1,4)) Lyptopha:y (0,3) Lysine (13,6) Arginine (5,5) Asnoξlagic acid/Asparagine (13 ,6) Serine (7
,6) Glutamic acid/glutamine (11,3) Threonine (3,4) Glycine (3,4) Proline (1,4) Alanine (5,5) Valine (5,5)%
Cystine (0,0) Methionine (2,7> Inleucine (4,8) Leucine (6,8) Phenylalanine (6,8) Tyrosine (2,7) Amino acid sequence: Cr1n Asp Pro Tyr Val Lys
Glu Ala Glu AsnLeu Lys Ly
s Tyr Phe Asn Ala Gly His
5erAsp Val Ala Asp Asn G
ly Thr Leu Phe LeuGly Ile
Leu Lys Asn Trp Lys Glu
Glu 5erAsp Arg Lys Ile Me
t Gln Ser Gln Ile ValSer
Phe Tyr Phe Lys Leu Phe L
ys Asn PbeLys Asp Asp Gin
Ser Ile Gln Lys Ser ValG
lu Thr Ile Lys Glu Asp Me
t Asn Val LysPhe Phe Asn
Ser Asn Lys Lys Lys Arg A
spAsp Phe Glu Lys Leu Thr
Asn Tyr Ser Val Thr Asp L
eu Asn Val Gln Arg Lys Al
a l1eHis Glu Lau Ile Gln
Val Met Ala Glu Leu Ser Pr
o Ala Ala Lys Thr Gly Lys
Arg LysArg Ser Gin Met L
eu Phe Arg Gly Arg ArgAla
Ser Gln Isoelectric point PH8,6 The ultraviolet absorption spectrum shows a maximum peak at 278 mμ (Fig. 1).

SDS electrophoresis SDS-PAG [Laemmli, U, K,
(1970) Nature (London) 22
7. After carrying out the method of 680-685, Coumasieve I
Stain with J IJ Ant Blue (R).

The marker is Po5phorylase b (94k
), BSA (671c).

Ovalbumin (43k), Carboni
c anhydrase (30k).

5oybean Tripsin inhibitor
(20,1k).

α-Lactalbumin (14,4k) (Figure 2).

Reference example 1 Step 1 (benzoylation or isobutylation) A,
150r of millimoles of deoxyadenokune (dA), a synthetic compound of dbzA.
n7! of dry pyridine. Next, 300 mmol of benzoyl chloride is added to this suspension under water cooling, and the resulting mixture is returned 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 (+0:1
)) to confirm. After the reaction is completed, the reaction mixture is
/! Pour into a mixture of 3507 parts of chloroform, 350 parts of ice, and 282 parts of sodium bicarbonate. The resulting mixture is shaken in a separatory 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. Distill the chloroform from the water-washed chloroform layer,
Add 150 liters of ethanol and 100 ml to the resulting residue.
Add eQ of lysine to obtain a homogeneous solution. After cooling the obtained homogeneous solution to <OC, 2
Add a mixture of 0'Oml of 2N NaOH aqueous solution and 200ml of ethanol with stirring to obtain a homogeneous solution. This homogeneous solution was stirred at room temperature for 5 minutes, cooled, and then
Add 00ml of 2N aqueous hydrochloric acid solution and further concentrate 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 precipitate N-benzoyldeoxyadenosine (dbzA). A mixture of the obtained N-benzoyldeoxyadenosine and a small amount of pyridine is left in a refrigerator for one night, 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, 35. :IJ mole of db2A is obtained. The yield of dbzA is 70%.

B. Synthesis of dbzo 100 mmol of deoxincytidine (dc) was added to 500 m
1. Suspend in pyridine. An equal amount of triethylamine is added to this suspension, and after stirring for 30 minutes, 6 equivalents of benzoyl chloride are added while cooling with water. Next, the same operation as in section A above is performed to convert dC into benzoylation. After the benzoylation reaction was completed, chloroform was distilled off and the resulting residue was
Dissolve in a mixture of 50 ml detrahydrofuran, 1 t methanol and 250 ml water. Add 250 portions of a 2N NaOH aqueous solution to the resulting solution while stirring under water cooling.

After 1.0 minutes, the resulting mixture 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-pendiyldeoxycytidine (dbzC
) is precipitated.

Synthesis of C, dibQ 100 mmol of deoxyguanosine (dG) was added to SOO
6 equivalents of inbutyl chloride are added dropwise to this suspension while cooling with ice. The mixture thus obtained is stirred with OC for 3 hours and then treated in the same manner as the benzoylation in Section B above to effect isobutylation of dG. After the completion of the inbutylation reaction, chloroform was distilled off from the chloroform layer, and the resulting residue was collected for 500 m
/ in ethanol, then add 500 m
Add a 2N NaOH aqueous solution of / in OC and stir for 15 minutes. The resulting mixture was transferred to 1 t of ice-cold pyridinium-type cation exchange resin (Dowex
2. Pour into Dow Chemical Co., USA) and neutralize. The thus neutralized mixture is loaded with a small amount of the above resin into a column and washed with 10% (w/v) pyridine solution in an amount three times the amount of packed resin. The resulting eluate and washing solution are mixed and concentrated to produce a residue of 50%
Recrystallized from a 5% (w/v) pyridine solution of 0 m(d)
ldG is obtained.

Step 2 (5'-dimethoxytritylation) 100 mmol thymidine, 100 mmol dbzA N100 mmol dbzc and 100 mmol dibQ in 200 rnl, 400 ml, 400 m, respectively.
1 and 750 m/m of pyridine, and 1.1 equivalents of )methoxy)IJ chloride r are added to each resulting mixture and allowed to react for 3 hours. The reaction is stopped by adding 50 g of methanol. Each reaction solution obtained was concentrated and 50 m
1. Dissolve glue in aroform and wash with water. In order to separate 5'-dimethoxytritylthymidine C (DMTr)T), the solvent of the reaction mixture of thymidine and dimethoxychloriP was distilled off, and the residue obtained by azeotroping with toluene was dissolved in 1500 rue benzene. Heat it up.

Next, n-hexane was added until this solution became cloudy, and it was left to cool until the temperature reached 4C.
Obtain crystals of o (DMT r ) dbz A %
(DNITr)dbzC and (DMTr)dib
To separate Q, dbzA.

The solvent of each reaction mixture of dbzC and dib() and dimethoxytrityl chloride is distilled off, and the resulting residue is dissolved in chloroform and subjected to column chromatography using tsKp glue gel. 3% by weight methanol water is used as the eluent for column chromatography.

Step 3 (Synthesis of dimer and trimer) 6 mmol of (
DMTr) After azeotroping dQ with pyridine, 10 ml
Dissolve in pyridine. On the other hand, LsX'2.2 equivalents of triazole, 1.5X2.2 equivalents of triethylamine and 2
p-chlorophenylphosphoric acid dichloride r is added dropwise to a mixture consisting of 0 ml of dioxane while stirring under ice-cooling while avoiding moisture. After stirring the resulting mixture at room temperature for 1 hour, the triethylamine hydrochloric acid was removed. The obtained solution was mixed with the above (
DMTr) Add to the pyridine solution of dQ, 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-methylimidazole and 8 mmol of d
Add ibG and azeotrope with pyridine. The resulting residue was dissolved in 60 ml of pyridine and the pyridine
9 mmol of triisopropylbenzenesulfonyl nitroimidazolide (hereinafter i'-TPSN'lJ
) is added. The resulting mixture is concentrated to X volume and left overnight at 30C.

The reaction is stopped by adding 6 ml of 50% strength by weight pyridine solution. After distilling off the solvent of the mixture that had stopped the reaction, the mixture was placed in a column packed with 150 g of type 60 silica gel (manufactured by Wako Tsumugi Pharmaceutical Co., Ltd.), and methylene chloride-methanol (30%) containing 1% pyridine was added. Elute with the mixed solvent of 1). Next, the eluate is a-condensed, and the a-condensate is dropped into i-hexane to precipitate the desired dimer in powder form.

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 ml of pyridine and then 3
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 2 ml of 50% strength by weight pyridine solution. Next, after adding 20ml of chloroform to the reaction solution, 15ml of 0.1M
Tetraethylammonium 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 silica gel. The resulting overall yield of trimer is 60%.

Step 4 (Removal of protecting group from trimer) 20 cm of trimer obtained in step 3 was dissolved in 3 ml of pyridine.

Add 15ml of concentrated ammonia water to the resulting pyridine solution, seal it tightly, and leave it at room temperature for 19 hours.
C. for 5 hours to distill off the ammonia. then 20
After adding 80% by volume of acetic acid based on m1, the acetic acid is distilled off.

The resulting reaction mixture was azeotroped with toluene, and the residue was
After dissolving in 0.10, I M TBAB solution, wash three times with 25 ml of aqueous solution, followed by once with 25 ml of ether. The resulting solution is evaporated and the residue is dissolved in 16rnl of 7M urea solution containing 20mM Tris-acetic acid (pH 8,0). The mixture thus obtained was placed in a column packed with DEAE cellulose and mixed with 500 ml of 0.05 M NaCt solution and 0.00 ml of 0.05 M NaCt solution.
7M urea-0.02M) lis-acetic acid (pH 8) with a salt gradient using 500 rnl of 40M NaCt solution.
,0) Elute with solution. Of the resulting eluate, fractions having an absorption peak are collected to obtain the desired trimer.

Step 5 (Synthesis of oligonucleotide r) An oligonucleotide is prepared in the same manner as in Step 3, and the protecting group is removed from the obtained oligonucleotide in the same manner as in Step 4. In this way, the oligonucleotides described below are obtained.

(1) 0ATOOATGTGTATCGTT(2)
TTGGGTTCTTTG (3) G G T T G T T A CT G
T(4) CAAGACCCATAC (5) GTTAAGGAAGCT (6) GAAAAAAOTT()AAG(7) AA
GTAC! TTTAAC(8) GOTGC)TCA
OTOT (9) GACGTTGCTGAC! 0 ■
AAC! GGTACTTTG α1) TTTTTGGGTATC (12TTGAAGAACTGG α Prisoner AGGAAGAATCT Q4) GACAGAAAGATC (151ATGCAATCTCAA (16) ATCGTTTCTTTT (17) T
ACTTTAAGTTGα8) TTTAAGAAC
TTT α9 AAGGACGACOAA C■ TCTATCICAAAAG (21) TCTGTTGA, AT of AAOT
CAAGGAAGAC ATGAACGTTAAG 124) T T T T T T A A CT
CT25) AACAAGAAGAAG(2■
AGAGACGACTTT (27) GAAAAGTTGACT ball AAC
TACTCTGTT (291AOTGACTTGAAO (To) GTTOAAAGAAAG ol) GCTATCCACGAAC32)
TTGATCOAAGTT ATGGCTGAAT
TG C34) TOTOCAGOTGC! TC35)
AAGAC:TGGTAAGfi A()AA
AGAGATCTG7) CAAATGTTGTT
T■ AGAGGTAGAAGA GI GCTTCTCAATAAG (4■ TCG
ACTTATTGAC)AAGCTC:TTCT(41
) ACCTCTAAAACAA(42) CA
TTTGAGATCT(43) CT T T C
T CT T A CC (4 滲AGTCTTTAGC
AGO (4■ TGGAGACAATTC (46) AGCCATAACTT() (4η
GATCAATTCGTG (48) GATAGCCTTTCT (4 mortar T
TGAACGTTCAA 61 G T CA, G TA person 0 AOA (51)
GTAGTTAGTCAA (Foundation) CTTTTC
AAAGTC @ GTCTCTCTTCTT) CTTGTTAGAGTT (E) AAAAAAACTTAAC (E) GTTCATGTCTTC (9) CTTGATAGTTTC (G) AACAGAOTTTTG @ GATAGATTGOTO ( C)TCCTTAAAGTT) CTTAAACAACTT (Foundation) AAAGTAAAAAAGA (To) AACGATTTGAGA ←ri TTGCATGATCTT (To) TCTGTC! AGATTC (To) TT
CCTTCOAGTT (67) CTTCAAGATACO (4) C
AAAAAAyOAAAGTfA ACcGTTGTC
AGO CA AAOGTCAGAGTG (71)' ACCAGCGTTAAA@GT
ACTTCTTCAA (To) C) TTTTCAGCTTC) CTT
AACGTATGG @ GTOTTGAOAGTA (to) ACAACOCAAAGA @ ACOOAAACGATACACATG process 6
(Synthesis of ,t?Rinucleote P) 40 pmol each of oligonucleotide P(1) and H(2) obtained in step 5 and 6.5 units of T4 DNA kinase were added to 80 pmol of (r
"p') ATP (8Cr/mmol), 100 μM
spermidine, 20mM DTT, 10mM MgCt
2.50mM) Lis-HCl (pH 9) and 0.1mM
Place 25μ of the mixture containing DTA. The reaction is 3
The oligonucleotide P(
1)-(2) are obtained. In this reaction mixture? - Add 5 times the amount of ethanol to precipitate the generated oligomer.

This oligomer was dissolved in 20% boriacryloasP in 7M urea.
Oligomers (1) are subjected to electrophoresis using a gel.
-(2) is obtained.

(1)-(2) OATCCATGTGTATOGTT
TGGGTTTTTG Perform the same operation as above to attach oligonucleotide 1″ (3
) and (4) to form oligomers (3)-(4)
k get.

Furthermore, (1) - (2) and (3) - obtained above
By combining (4), (1> −(2) −(3) −(
4) is obtained. By repeating the above operations, the following DNA can be obtained.

-(12) -(13) -(14) -(ts) -
(16) -(17) -〇g) -(19) -(3
7) -(38) -(39).

-(66) -(67) -(68) -(69) -
(To) -(71) -(72) -(73) -DNAs (1) to (39) and (40) to (
77) in 50ml of TNE buffer and mix.1.
Culture at 65C, 45C, -37C and 20C for 1 hour each.

This mixture was then treated with 100 mM) lis-hydrochloric acid (pH 7).
,5), 100 m M CaC1z and 100 m M
Add 20 μ of the mixture containing M Mg0Lz and cool on ice for 20 minutes.

Step 7 (Cloning) Nie Noe Twigg et al.
etal) is published in Nature, Vol. 254, pp. 34-38 (1
975) CNature, 254, 34-3
8 (1975)], plasmid)'pAT153 is prepared.

From this plasmid pAT 153, Michael D.
t) is published in Nature Vol. 292, pp. 756-762 (19
1981) (Nature, 292°756-762
(1981)], plasmid l' pPM50 containing the promoter and operator of the lactose operon is prepared. Obtained plasmid rp
4 μg of PM50 DNA was added to 10 mM) Lis-HCl (p
H7,6), 6mM MgCt2.150mM N
aC1 and 1 mM DDT (7) in a mixture, and this mixture was treated with restriction enzymes BamHI and Sci.
By reacting at C for 60 minutes, the above pPM50
cleave. After the reaction was stopped, the DNA was extracted with a mixed solvent of phenol and chloroform (3:1 volume ratio), and the resulting DNA fragment was extracted with 40 rruV-) lith-salt! (pH7
, s), large DNA fragments are recovered by electrophoresis using a 1% agarose gel in a mixture of 6rnM sodium acetate and 1mM EDTA. BamHI-8alI 3.2 kb vector fragment 1 thus obtained
μg and the chemically synthesized gene obtained in step 6,
20mM Tris-HCl (PH7,6), 10mM
30μ of MgCl2 and lQmM DTT (7) mixture
t and 0.4 units of T4 DNA IJ gauze, and reacted at 12°C for 16 hours.

As a result, a plasmid is obtained in which the DNA containing the chemically synthesized gene obtained in step 6 is fused immediately after the lactose operon.

The plasmid thus obtained was transferred to E. coli
E. coli X1776 strain is transformed by contacting with the E. coli strain X1776. After culturing the resulting transformed strain, the DNA in the cells of this transformed strain was isolated, and the deoxynucleotide sequence of the resulting DNA was determined by N.M.-7 Kissam et al.
M., Maxam et al., Proceedings of the National Academy of Sciences of America, Vol. 74, pp. 560-564 (1978) (Proc.
Natl, Acad, Sci, USA, 74.
560-564 (1978)).

The resulting deoxynucleotide sequence id is shown to match the theoretical sequence. The antiviral activity of the culture solution of the transformed strain is evaluated using VSV as a virus that sensitizes FS-4 cells and their FS-cells (FS
- Cells and VSV used in the step of Example 1). As a result, the above culture solution exhibits antiviral activity. (The operation of inserting cilasmid into Escherichia coli strain 1 mg of Staphylococcal enterotoxin A (SKA) was added to the culture solution 101 of 9772 spheres, and cultured with rotation at 37°C for 2 days. After culturing, the lymphocytes are centrifuged at a low speed of 80° rpm for 10 minutes. The 9772 spheres thus collected were mixed with a mixed solution (hereinafter referred to as "PB") containing 87 NaCL, 0.22 KCj, 1.155' Na2HPO4.
1 (referred to as "P solution") and resuspend. While carefully stirring this suspension, 20 frlM to J Soo-hydrochloric acid (pH 7.5) was added to a 50 t separating funnel.
and a mixture containing 1 mM EDTA (Tris-HCl and E
A mixture of DTA (hereinafter referred to as "TE buffer") at 2%
Mixture 17 containing sodium dodecyl sulfate (, W/V) is added thereto. Next, pronase (Calbiochem, 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 2M) IJ susu-acid (pH 9) of the above mixed solution is added. The obtained mixed liquid was
Extract with 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. 60H SDS is added to the 18t aqueous phase thus obtained. From this aqueous phase, add 1/10 amount of 3M acetic acid (IJcum) (pH 5).
, s) and twice the volume of ethanol to precipitate the nucleic acids in the mixture.

After leaving the mixture treated as above 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) Lis-HCl (pH 7,5), 100mM NaCl
Add 0.5% (W/V) SDS (hereinafter abbreviated as "TNBJ") containing 5mM EDTA and 200mM EDTA.
mJ into the mixture and stir. Another 350m1!
of TNE is added to the above mixture to dissolve the fibrous nucleic acid precipitate. The resulting mixture was then heated at 50 rpm for 15 min.
Centrifuge for a minute and collect the precipitate. The collected sediment is
THE 350ml with 0.5% (W/V) SDS
dissolve in This TNE solution and the above supernatant TNE solution are mixed and extracted three times with an equal volume of phenol, three times with a half volume of ether, and three times with an equal volume of ether. The RNA thus recovered was measured at 260 n using an absorbance meter.
When quantified at a wavelength of m, it is found to be approximately 100 m9.

To 500 ml of the RNA-containing solution obtained above, 21 g of type 7 oligo(dT) cellulose (manufactured by Calbiochem,
(USA) and stirred at room temperature for 1 hour to adsorb the mRNA onto oligo(dT) cellulose. The oligo(dT) cellulose adsorbed with mRNA was centrifuged at 3000 rpm for 10 minutes, and the resulting oligo(dT) cellulose was divided into 50 ml and 15 ml.
of 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 Tris-HCl (pH 7,5), 30mM N
a07.

5mM MgC1z, 0.5mM DTT (Calbiochem, USA), 20μg/goolibi (d
'l')+2-18 (manufactured by P&L-ζ Iochemicals, USA), 5mM dGTP (manufactured by Sigma, USA), 5mM dCTP (manufactured by Sigma, USA), 5mM dTTP (manufactured by Sigma, USA)
(United States of America), 5mM32P-dATP (= Near England Nuflare, United States of America)
, 60 μg/M mRNA, 280 units of avian myeloblastosis virus (AMV), and 600 units of reverse transcriptase (Bethesda Research Lazeratory, USA) were incubated at 37 C for 1 hour. Add 0.5 M EDTA to this reaction mixture.
and 20% (W/V) SDS, each at 10 mλ
Add at concentrations of 1 and 0.1% (W/V).

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);
2 containing 1mM EDTA and 0.1% (W/V) SDS
Wash with 00 μt solution. This washing liquid and the aqueous phase obtained above were mixed, and after extraction with an equal amount of ether to this mixed liquid, a 3 ml column of Sephadex G-100 (manufactured by Pharmacia, Sweden) packed with TNE was added. (hereinafter referred to as "Sephadex G-100 column"). The fractions eluted from the column are collected in 0.1 ml portions at a rate of 0, 1 rnt 7 min. Mix the two radioactive parts and add 3M acetic acid (IJ) to this until the concentration is 0.3M. Next, this mixed fraction is placed in 25 times the amount of ethanol for both fractions to precipitate the nucleic acids. This mixed fraction is left at -70C for 10 minutes, and then centrifuged to remove the supernatant. The precipitated nucleic acid is dissolved in 100ml of distilled water, and 20μ of 5mNaOH is added thereto. The resulting mixture is left at room temperature for 40 minutes. Then add to this mixture
Add 10 μm of 5M sodium acetate x 50 μm of distilled water and 250 μt of ethanol and cool at -70C for 10 minutes.
Centrifugation at g gives 5 μg of cDNA.

Step 3 (Manufacture of double-stranded cDNA) 5μg of single-stranded cDNA obtained in Step 2 is 100μt
Dissolve in water and heat to 100C for 2 minutes. After heating, add Q, 1M heat-denatured potassium phosphate buffer (pH
6゜9), 10mM MgCtz, 10mM DT
T-! mM dATP (manufactured by Niwarno), 1
mM dGTP (manufactured by Schwartz), 1mM dCTP
(manufactured by Boehringer Mannheim, Inc.), 1mM 3H-dTTP (manufactured by Boehringer Mannheim, Inc., USA) and 150 units of C. coliformis DNA polymerase (manufactured by Boehringer Mannheim, Inc., USA).
Add 0 μt. After incubating the resulting mixture at 15C for 6.5 hours, the mixture was supplemented with 0.5 M EDTA and 20%
(W/V) SDS 10mM and 0.1% (W
/V). The resulting mixture is
Extract with 50 μt of phenol (hereinafter this operation is often referred to as “phenol treatment”). The phenolic phase is further extracted with 130 μt of TE buffer containing 20 mM) Lis-HCl and 1 mM EDTA. The first and second aqueous phases are combined and fractionated using a Sephadex G-100 column with a capacity of 3 ml.

Collect the fractions showing radioactivity from both obtained fractions, and add 3M sodium acetate to the obtained mixed fraction so that the concentration of acetic acid is 0.3M.

Next, ethanol in an amount 2 to 5 times the amount of this mixed fraction is added to this mixed fraction to precipitate the DNA. The mixture thus obtained is centrifuged to obtain 7 μg of DNA. Next, this DNA was mixed with 0.2 M NaC! , 50
mm1! i Sodium yate (pH 4,5) and 10mM
It is dissolved in 130 μt of a buffer containing zinc sulfate (hereinafter referred to as rs+ buffer) and kept at 37C 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 0.1 ml of each fraction.

Of the obtained fractions, the radioactive fractions were collected and diluted with 4μ
g double-stranded c DNA is obtained.

Step 4 (Production of dAMP-extended cDNA) Step 3
The double-stranded cDNA 150 m9 obtained in
ATP, 100mM sodium cacodylate (pH 7.2
) 15 mM CaC1z, 50 tt g/at
Bovine serum albumin (hereinafter abbreviated as "BSAJ") and 3 to 6 units of terminal deoxynucleotide P transferase (hereinafter abbreviated as "TdTJ") per 1 μg of DNA were added to 8 μt of dATP solution and incubated at 27C for 8
After incubation for minutes, freeze at -70C.

Step 5 (Production of pBR322 cleaved with EcoRI and extended with dTMP) 20 gg of plasmid ρBR322 and 10 units of Eco
RI (manufactured by Hesesda Research Lazeratories, USA) was dissolved in 10mM HCl (pH 7).
,5), 6mM MgC1z, 50mM Na046
mM 2-mercaptoethanol and 200μg/μt
Put 150μ of the mixture containing BSA in it and
Incubate at C for 2 hours. This mixture is extracted with an equal volume of ether and ethanol is added to form a precipitate. Next, this precipitate was mixed with 0.1mM glycine sodium (pH 9.5).
, 100 μtK of a mixture containing 5 m M MgC1z and so μg/μt BSA. 1 to the resulting mixture
Add 7 units of λ-echin nuclease to 1 at 37C'.
Incubate for hours. 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
az HPOa, 5mM MgCl2.1mM
aTTP, 50 μg,/ml BSA and 30 μg containing 3-6 units of TdT per μg of DNA
Add to 0 μt reaction mixture and incubate at 37C for 20 minutes.

After the culture is completed, the reaction mixture is treated with phenol, and ethanol is further added to collect Plasmi P.

Step 6 (annealing of pBR322 and cDNA) d
80 g of AM-extended DNA product and plasmid F″pBR3222 cleaved with EcoRI and extended with dTMP.
0 ng were placed in 50 μt of THE buffer and incubated for 1 hour at 60IZ'', 45tT, 37C and 2OC, respectively.Then, the culture mixture was incubated with loOmM)'J sous-
acid (pH 7,5), 100 m M CcC6z and 1
Add 20 μt of a mixture containing 00 mM MgCl2 and cool on ice for 20 minutes.

Step 7 (Transformation of E. coli X1776) E. coli X17
100 colonies of 76 strains (ATCC No. 31244)
μg/ml diaminopimelic acid, 10 μg/ml
Inoculate tryptone medium 1'OOme 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 nm becomes 0.6, and cooled on ice for 30 minutes. Next, add 40
Centrifuge at 00 rpm for 10 minutes to pellet cells. The resulting cells are washed with 50ml of 10mM NaCl and centrifuged. Dissociated cells were added to 20ml of 100mMC.
Suspend in aCl2 and cool on ice for 30 minutes. The cells were then granulated by centrifugation and then 4 ml of 100mM CaC.
Resuspend in 1z 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 1) BR322 obtained in Reference Example 2 Step 6
Escherichia coli X (recombinant DNA molecule) treated with Ca
The mixture was added to 100 μt of the above suspension containing strain 1776, cooled with water during addition, and then kept at 20°C for 0 minutes. Next, add Qj, ml of 1-1) Pton's medium to this mixture.

The resulting mixture was mixed with 100 μf/ml diaminopimelic acid, 10 μf/ml nasilixic acid and 10 μf/ml
Inoculate tryptone agar plates supplemented with ml ampicillin.

Next, after culturing at 37°C for 48 hours, each resulting colony was collected and suspended in 100 μt of tryptone medium placed in each hole of a micrometer plate. This micro measuring plate is 37
After overnight incubation at °C, 100 μt of 40% (w/v) aqueous glycerin solution is added to each well, and the microplates are stored at °C. In this way, 500,000 individual E. coli strain X1776 transformant clones are obtained.

Step 3 (Manufacture of mRNA probe) Reference example 2 Step I
A sample of 200 μ7 of IA-induced mRNA was obtained using the same method as described in . In addition, 200 μ7 of a sample of mRNA not induced by SEA is obtained in the same manner as in Step 1 of Reference Example 2, except that the inducing operation in step 81 is not performed. lO year unit T 4 ;+? ! J nucleotide kinase, l
mM C1-”P) ATP, 40mM Tris-HCl (+) H7,5), 30mM NaC6 and 5mM
100μt of solution containing MgC42 was added to 10μ7 of SEA.
Incubate at 37°C for 10 minutes. After inactivating the kinase in the culture by phenol treatment, 200 μ7 of mRNA not induced with SEA is added to the resulting mixture. Add 3M to this mixture
Add sodium acetate to a concentration of 0.3M.

To the resulting mixture, 2.5 times the volume of ethanol was added to the SEA-induced 32p-label/l/m
200μ consisting of RNA and mRNA not induced by SEA
A precipitate of 2 is obtained. After centrifuging the resulting precipitate, -n
Save with 'c.

Step 9 (Selection of cDNA) The transformant of Escherichia coli
Form the knee. Next, Millipore filter-HAWP
A circular filter with a diameter of 82 mm was cut from (Squaden, manufactured by Pharmacia) and sterilized in an autoclave for 10 minutes. Sterile filter 10μg/ml
Place on another tryptone agar plate containing ampicillin. Each colony obtained above is inoculated onto this filter and cultured overnight at 37C to form colonies on the filter. This filter was heated to 0.5 N Na
Soaked in OH aqueous solution for 10 minutes, then 0.5 M)
Immerse in IJ susu-acid (pH 7,5) for 5 minutes. Next, the filter was treated with 1.5 M NaCL and 0.5 M)
Soak for 5 minutes in a mixture containing hydrochloric acid (pH 7.5). This filter was further treated with 0.3 M NaCl and 0.03 M
A solution containing sodium citrate (pH 7) (hereinafter, this solution is referred to as "2x SSC solution". Here, SSC is a solution containing 0.15 M NaC7 and o, o i 5 M sodium citrate, e.g. ) for 5 minutes and then heated at 70c for 3 hours.

per filter, 50% (V/V) formamide, 4X SSO (0.6M NaCl and 0.06M
pH 7 solution containing sodium citrate) and 50
Pour 1 ml of a solution containing (mM) lithium-hydrochloric acid (pH 7,5) into a Petri dish, and add to this solution the mRNA mixture obtained in Step 8 of Example 3 (32p-labeled mRNA induced with SEA, 10 μg of NA and 10 μg of SEA-induced mRNA). 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 with chloroform three times for 15 minutes each at room temperature, and then washed with a solution containing 0.6 M NaC4 and 0.06 M sodium citrate (pH 7) (hereinafter this solution is referred to as "4x SSC solution"). ) for 15 minutes at room temperature. Subsequently, incubate with 2×SSC @ solution for 15 minutes at room temperature for 3 times.
Wash twice. Next, put 2 ml of 2XSSC solution containing 20m97m1 lysene nuclease into a petri dish.
The above washed filter was placed in this and left at room temperature for 30 minutes, then the filter was washed twice with 2XSSC solution.
Air dry. This filter is subjected to autoradiography. In this way, 7 out of 500,000 colonies
It can be seen that several colonies hybridize with the mRNA described above.

Step 10 (Manufacture of recombinant plasmirr) Reference example 2 Step 9
One of the colonies obtained in step 1 is placed in a 2 t Erlenmeyer flask, inoculated into 1 t of tryptone medium, and cultured with shaking at 37 r: until the optical degree (OD) of the culture solution reaches 08 at 650 nm. Next, 16 tryptone medium was added to this culture solution, and then chloramphenicol was added at 170 μg/
ml aKK and culture at 37C for 16 hours.

Add 20rnl of chloroform to this flask and
Sterilize the culture by shaking for 10 minutes at C.

After separating the culture from chloroform, 6000rprr+
Centrifuge for 15 minutes at 4 C to obtain 2 g of cells.

Cells were grown in 30ml of 20mM) Lis-HCl (pH 7.5).
Suspend in This suspension was centrifuged at 500 rpm for 20 minutes at 4'C, and the resulting cells were collected in 30 ml of 50 mM).
'Resuspend in J susu-acid (pH 75). To this suspension, add 1 omtp of 50mM) Lis-HCl (pH 7,5).
A lysozyme solution containing /rnl of lysozyme is added to X volumes of the above suspension. After cooling the resulting mixture in OC for 10 minutes, 0,3
Add M BDTA gently without stirring. After leaving the resulting mixture in the OC for 10 minutes, 2% of the X volume of this mixture
(W/V)) Add Lydon X-100 (surfactant, manufactured by Wako Tsumugi Pharmaceutical Co., Ltd.). After 60 minutes, the mixture was
Centrifuge for 60 minutes at mOC. Transfer the resulting supernatant to a beaker and add 3M NaOH to the beaker with stirring to adjust the pH to 12-5. After stirring the solution for 10 minutes at 20C, the pH is adjusted to 8.5. After stirring for an additional 3 minutes, an amount of 5 M NaC7 and an equal volume of phenol are added to the solution, stirred vigorously for 5 minutes, and centrifuged at 110,000 rpm and O'p for 10 minutes to cause phase separation. 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. To the extract containing circular double-stranded DNA, add 5 mg/ml of pancreatic acid nuclease to a concentration of 20 μg/ml. The resulting mixture is incubated at 37C for 60 minutes. Add Z volume of 5 M NaCl to the mixture followed by 1 M 30% (w/v) polyethylene glycol 6
000 (Union Car) (manufactured by Zeta Co., USA) aqueous solution is added to give a concentration of 7.5% (W/V). The resulting mixture is kept at -10C for 14 hours, after which the mixture is centrifuged at 800 rpm, Q7:: for 20 minutes to collect the precipitate. This precipitate was dissolved in 0.075 M Na
Dissolve in a solution containing C1 and 0.0075M sodium citrate to an optical density (OD) of 20 at 650 nm. Next, add 20% (W/V) SDS to the Kono solution.
．． Add at a degree of 5% (W/V). Further, pronase is added to this solution at a concentration of 0.5ml/ml, and cultured at 37C for 30 minutes. This solution was then extracted three times with an equal volume of phenol and twice with an equal volume of chloroform to extract Beckman 5W-27
The cells were subjected to sucrose density gradient centrifugation using a molded rotor at 20,000 rpm and 15C for 15 hours. This density gradient centrifugation involved 50mM) Lis-HCl (pH 7.5) and 1
A solution with a 5-23% (w/v) sucrose gradient containing mM EDTA is used. 2 of each resulting fraction
Measure the optical density at 60 nm.

Fractions containing DNA are collected, and the DNA is precipitated using sodium acetate and ethanol in the same manner as described above. 50 μg of circular double-stranded DNA is obtained by centrifugation.

Step 11 (cleavage of plasmid) Diagnosis fi12 Circular, double-stranded DNA obtained in step 102
0 μg, 10 mM) Lis-HCl (IllH7,5),
6mM MgC12, 50mM NaCt, 6mM
2-mercaptoethanol, 200μg/μt BSA
and 150 units of a mixture containing 20 units of the restriction enzyme EcoRI.
Place μ in the tube and incubate at 37C for 2 hours. Next, pronergy, EDTA and SDS were added to this mixture at respective concentrations of O-5%/ml 10mM and O-5% (W/ml).
V).

The resulting mixture was incubated at 37C for 30 minutes and then extracted with 30 μt of a mixed solvent of phenol-chloroform (1:1 volume ratio). The resulting non-aqueous phase was dissolved in 20mM Tris-HCl (
1) Solution 50 containing H7,5) and 1 mM EDTA
Wash with μt. Combine the washings and aqueous phase and dilute with ether for 30 minutes.
Extract times. Add 10,000 volumes of 3M sodium acetate and 15 volumes of ethanol to the combined aqueous solution to form a precipitate again. Next, this mixture was cooled at =70t for 5 minutes,
Centrifuge to obtain 9 μg of DNA.

Step 12 (Degradation with restriction enzyme pst1) Reference sequence 2 5 μg of plasmid cleaved with EcoRJ obtained in step 11 is used in place of 20 μg of circular double-stranded DNA, and restriction enzyme ps+I is used in place of restriction enzyme EcoRIO. Other than that, the same operation as in Example 3 Step 11 was carried out,
A mixture of DNA fragments digested with Ps+I is obtained. This mixture was diluted with 50mM)') acetate buffer M (pH 7,
g) 2% (W/W) horizontal agarose gel (10X20
X0.7CJn) and separated by electrophoresis, ps
Approximately 0.2 μg of DNA degraded with +■ is obtained.

Step 13 (partial digestion with restriction enzyme 13s INI) 0.2 μg of Ps + I-degraded DNA obtained in step 12
, 10mM) Lis-HCl (pH 7,5), 6mM
150μ of a mixed solution containing MgC42, 50mM NaCL, gmM 2-mercaptoethanol, 200μg/μt BSA, and 2 units of restriction enzyme 13stNI was added and incubated at 370 for 5 minutes to partially degrade the DNA. Next, the partially degraded DNA was subjected to phenol-chloroform treatment, ethanol treatment, and electrophoresis treatment in the same manner as in Step 11 of Example 3 to obtain II) B51NI.
10 ng of partially degraded DNA fragments are obtained. next,
Olibodemanxynucleotides having the following formula prepared by the same method as in Example 2 steps 1-5: AATTCATGTGTATC GTCCTA, GGCTCCTC GGCTGTTATTGTC TGACAATA ACAGC! 0GAGGGAGCC TAGGACGATACACATG (However, A, G, C, 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) and the above partially decomposed DNA fragments were used as examples. 2. Anneal and bond to each other using T4 DNA ligase in the same manner as in step 6.

Step 14 (Expression of peptide in E. coli) David
Bui Ether et al. Nature, Vol. 287, No. 411-
416 pages 1980 (1) avid v, Goedd
ele+at, Na+ureXVat, 287,
Ec of DNA tl-pBR322 obtained above according to the method described in pp. 411-416 (1980))
It is inserted into the region cleaved with oRI and I'SII, and contains an E. coli tryptophan promoter, an E. coli tryptophan operator, and a lysene binding site.
An Eco cut fragment consisting of 0 base pairs is extracted and this fragment is ligated to pBR322 containing the above-mentioned target DNA. The plasmirr obtained in this way was used as Escherichia coli
76 strain to transform E. coli strain X1776. From the culture obtained by culturing this transformed strain,
Pepti 1 is isolated using a monoclonal antibody that specifically binds to Pepti 1'HC(0). The above monoclonal antibody was developed by David Niss Nanatsuba et al.
Volume 285, Page 446, 1980 (1) avid s,
5acher eL at,.

Na+u? e, Vo 1.285 p, 446
(1980)] by using the peptide HC(0) obtained in Example 1 as an antigen according to the method described above. As a result of subjecting the obtained PeptiP to amino acid sequence analysis, the amino acid sequence of this peptide was found to be that of peptide MO
Matches the amino acid sequence of (0).

Reference example 3 At the 2nd International Conference on Interferon Research, D. V. Goedde I.
) The following experiment was performed using the same method as published by.

Hindl[[-Pvu
ri cleavage fragment (R. M. Myers et al., Proceedings National Academy of Sciences of America, Vol. 77, p. 6491, 1980 [几, M., My.
ersej a I, Proc,, Na+ 1.
Acad, 'sc i, U, S, A,, 77
, 649'1 (1980)')
Convert to I restriction fragment.

That is, the Hin d m cut end is made into an EcoRI cut end by binding the synthesized oligonucleotide P,
The pvu■ cut end is made by filling in the vacant nucleotide part using DNA polymerase I (Klenow (ragment)) and then ligating it into Ec.
Make an oRI cut end. The resulting Ec-engineered fragment containing all the SV40 origins was isolated from Shirasumi)' pML-1 (pBR3
22 and 5V40 (7) Recombinant pransmilad with an ampicillin resistance marker and an origin of replication of E and Co11, but without sequences that block the replication of pBR322 and SV40 recombinants) (M Rafshi et al. Nature, Vol. 293, p. 79, 1981 CM, Luksy
et at, Nature, 293.79 (19
81))) binds to the BcoRI cleavage site. Among the expressed genes obtained, those in which the SV 40 late promoter is arranged so that reading starts from the ampicillin resistance gene of pML-1 are selected. Next, the EcoRI closest to the ampicillin resistance gene of the selected fragment
The restriction part was partially digested with BcoRI, and DNA polymerase Klenow fragment (Klenow fragment
] Fill and combine (K, Itakura et al., Science, Vol. 198, p. 1056, 1977 (K, Itak)
ura etal, 5science, 198.10
56 (1977)]). pst of Shirasumi 1 obtained in Example 2. 1. By adding oligomers to the cut pieces. Create an EcoRI restriction fragment. This cD
The EcoRI restriction fragment containing NH is ligated into the EeoRI cleavage site of the expression medium pML-8V40 obtained above. The monkey cell line C08-7 transformed with the resulting vector (Y. Gullaman, Cell, vol. 23, p. 175, 1981 [Y, Qluzman nc.
23, 175 (1981))).

008-7 endogenously expresses the 5V-405-)T antigen and 1. It is shown that propagation of recombinant plasmids containing pML-1 and 5V-40 origin sequences is allowed. The presence of interferon in the cell culture medium of 008-7 is analyzed daily as described in Example 2. A yield of 100 Q/ml of interferon was obtained 3-4 days after infection, indicating that interferon is excreted into the culture medium by C08-7 cells.

Reference example 4 R.A. Hitzeman et al. Nature, No. 293
Volume 717 1981 CR, A, Hitzeman
eIal.

The following experiment is conducted according to the method described in Nature 293.717 (1981)]. 10 μg of X h
o ■ Cleaved pAcF301, 20mM) Lis-HCl (pH 8,1), 12mM CaC1z, 12mM
MgCjz, 0.2 M NaCl XL mM B
Incubate for 15 seconds at 30°C with 0.2 units of 3a131 nuclease (Bethesda Research Laboratory) in an aqueous solution containing DTA and 0.1 part g/ttt BSA. During that time, approximately 50 base pairs of DNA are removed.

A part (1 part g) of the obtained DNA was treated with DNA polymerase I [Klenow fragmer+t
)] and deoxylyze nucleon)'' triphosphate to fill both ends of the DNA with deoxyribonucleotides, followed by T4 DNA ligase and the synthesized EcoR.
12 at 14C with I linker (l 1nker) (manufactured by Colagelatape Research, USA).
Incubate for hours. Next, the DNA obtained above was digested with restriction enzymes EcoRI and BamHI, and a mixture of DNA fragments containing the base sequence of the ADH1 promoter that had been variously excised from the resulting DNA fragments was placed on a 1% agarose gel-water gel. Separate by preparative electrophoresis. The mixture of electrophoretic eluted fragments was converted into pBR, 322 EcoRI-B.
The large fragment cut with amHI is ligated, individual clones are transformed into E. coli strain RRI, and ampicillin-resistant colonies of the transformants are returned to select individual clones.

DNA from the individual colonies obtained was collected using the quick screening method (Eite C. Pernzim et al., Ninny Klein Research, Vol. 7, No. 1513).
Page 1979 (H, C-Birnboim et at
.

Nucleic Ac1ds Res,, 7.151
3 (1979))). The length of each deleted portion is determined by using restriction enzyme Ec for each plasmid obtained above.
37- of the DNA fragment obtained by cutting with oRI.
Cut the ends with DNA polymerase [Frenara fragment (kleno fragment)]
w polymerase )) and [α-”P ′1d
ATP and then this labeled DN
A is digested with restriction enzymes A+uI, and the resulting DNA fragments are measured using urea-acrylamide gel. plasmid)' pFRL4 and plasmid PYR117 [Plasmid YRp7 was published by Kaystall et al., Pro7-Ge National Academy of Sciences of America, Vol. 76, No. 1o3s! 1979 (K, 5tu
hl etat, Proc, Natl, Acad
, Sci.

U.S.A., 76, 1035 (1979)
EcoRI of pBR322 according to the method described in ':]
1.4kb E and C containing yeast gene TRP 1 at the site
It is created by inserting the o11 fragment. ) by the following method (Zi Tsuan ξ- et al., Gene, Vol. 10, No. 1)
57 pages 1980 (G, Tschumper et al.
Gene, 10.157 (1980)). The two EcoRI cleavage sites of plasmid YRp7 were cut with EcoRI, and the resulting sticky ends were digested with DNA polymerase 1 [klenow frame
nt)] and connect the resulting pure ends (li
ga+ion), these two E co
Delete the RI cleavage site. Next, Plasmi)”YRp
Replace the Hindm small fragment in 7 to make 2 pieces (7) B
co RI region (Hind of the plasmid lacking fL)
[Replacement with Q small fragment replaces one EC0RI cleavage site, resulting in plasmid PFRL4. 20
μg of plasmid pFRL4 was added to B a m HI and E
Digest with coRI and electrophoresis using 1% by weight agarose gel-water. The resulting large fragment (500
0 base pair) is excised from the gel, electroeluted, extracted twice with phenol-chloroform, and precipitated with ethanol. Next, 3 μg of the obtained fragment was separately added to the DN & various fragments having the ADH promoter obtained above,
20mM) Lis-HCl (pH 7,5), 1 (1mM M
gCl2.10mM dithiothreitol, Q, 5mMA
Ligation is achieved by reacting 50μ of a mixture containing TP and 0.5 units of T4 DNA ligase for 12 hours at 15°C. This binding mixture of ampicillin-resistant Escherichia coli K-1,2 strain 294 (ATCC 31537)
and purify plasmir r from the transformation mixture.

50μ7 of the plasmid obtained in Example 2 is digested with EcoRI, and the digested fragments are electrophoresed on a 1.2% by weight agarose gel-water. A DNA fragment carrying the genetic code for aptide was excised from the gel, electroeluted, extracted twice with phenol-chloroform, and precipitated by adding ethanol. The DNA fragment carrying the genetic code for aptide is ligated into the unique KcoRI site of plasmid pFRP, which has been previously cut with EcoRI and treated with bacterial alkaline phosphatase. The resulting plasmid is analyzed by BgIH restriction analysis and used for yeast transformation. A recombinant plasmid in which the DNA carrying the genetic code for waveptide is placed between the GADH promoter fragments of the TRPI gene in the same or opposite directions was transformed using standard transformation methods (Nie Hinen et al., Proceedings National. Academy of Science America, Vol. 75, p. 1929, 1978 (A.

Hlnnen at al, Proc, Natl.
Acad, Sci. U.S.A., 75°1929 (1978):).

Yeast reception method RH218 (tupl) (ATCc
44076 ) (: Satucharomyces cerebinae (
SaccharOm7Ce8 ce -revisia
e) tryptophan-requiring mutant of Nomiozari et al.
Journal Bacteriology (J, Bact,) No. 13
Vol. 4, p. 48, 1978] spheroplasts (H. D. Alle et al., Astrophysical Journal,
Vol. 86, p. 325, 1981 (H, D, A11e
etal, Astrophy, J., 86.32
5 (1981))) is separately cultured with the plasmid IF DNA described above, and the culture mixture is placed on tryptophan-free agar to select transformed colonies. A culture of each colony is grown under selective pressure in a tryptophan-free liquid medium, collected at mid-logarithmic phase, the cell wall of the culture is degraded, and the cells are lysed with guanidine hydrochloride to prepare a cell extract. Significantly high biological activity was observed in all transformants that had integrated the plasmid in which the DNA with the genetic code for aptide was connected in the correct direction.

[Brief explanation of drawings]

Figure 1 shows the ultraviolet absorption wavelength of f(C(o) obtained in Example 1), and Figure 2 shows its SDS gel electrophoresis.The marker used was ovalbumin (molecular weight 43k).
, carbonic anhydrase (30k), soybean triventh inhibitor (20.1k) and α-lactalbumin (14.4k). Figure 1' shows the ultraviolet absorption spectrum of the cysteine-free takuputide obtained in Example 2, and Figure 2' shows its 8DS gel electrophoresis. Markers include ovalbumin/(molecular weight: 13k), carbonic anhydrase (30k), soybean trypsin inhibitor (20.1k), and α-lactalbumin (14k).
, 4k) was used. Patent applicant: Asahi Kasei Industries, Ltd. Wavelength (m, u) Wavelength Cm) An Figure 2 (A) Purified HC (0) Marker Bebutitovalbumin -431〈・Ka2. Boy, 4717 pα- to Duse-30
Lactalbumin □14.4 Figure 2 old. Marker Purified onorumin □43・Carbonic anhydrase-30k-eapmil soybean trypsin inhibitor 20.1@purified IFN-γ
Peptide □ -α-lactalbumin ~14.
41 (0 Procedural amendment (written by the Commissioner of the Patent Office on April 9, 1988) 1) Mr. Yu Akira 1, Indication of the case 1986 Patent Application No. 206797 2, Title of invention Novel peptide 3° amendment Relationship with the case of the person who filed the patent application Address of the patent applicant 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) 96:16 March 31, 1986 (shipment date) 7. Contents of amendment ( 1) Correct the application form as shown in the attached sheet. (2) +11 In the fourth line from the top of page 115 of the specification, “1st
The figure shows Example 1...'' instead of ``Figure 1 (A)
is corrected to ``Example 1...''. (2) On page 115 of the specification, line 5 from the top: “...
, Figure 2 shows that...''.
・, Figure 2 (A) shows that...''. (3) At the top of page 115 or line 699 of the specification, replace the phrase "Figure 1 is Example 2..." with "Figure 1.
Figure (B) is Embodiment 2...'' is corrected. (4) Line 111 from the top of page 11.5 of the specification “...
・, Figure 2 shows that...'' instead of ``...''
. . . Figure 2 (B) shows that...". (3) Correct the drawing as shown in the attached sheet. that's all

Claims (1)

[Claims] Formula (II): [Amino acid sequence] (However, in the formula, Gln is a glutamine residue, Asp is an aspartic acid residue, Pro is a proline residue, Tyr is a tyrosine residue, and Val is a valine residue. residue, Lys is a lysine residue, G
lu is a glutamic acid residue, Ala is an alanine residue, As
n is asparagine residue, Leu is leucine residue, Phe
is a phenylalanine residue, Gly is a glycine residue, Hi
s is a histidine residue, Ser is a serine residue, Thr is a threonine residue, He is an isoleucine residue, Trp is a tryptophan residue, Arg is an arginine residue, and Met is a methionine residue).