JPS6133200A - Gamma-interferon - Google Patents

Abstract

NEW MATERIAL:A polypeptide having a human immune interferon activity, wherein an amino acid is at the 144th position from N terminal end, which is the first amino acid in a usual human immune interferon, is glycine USE:An antitumor agent, antiviral agent and immunological activator. PREPARATION:A culture of a microorganism transformed with a replicative expression vehicle having a polynucleotide sequence which codes a polypeptide having glycine as an amino acid at the 144th position from N terminal end which is the first amino acid in a usual human immune interferon, for example, is multiplied to produce a polypeptide having a human immune interferon activity, followed by recovering thereof.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to the field of recombinant DNA technology, and in particular to microbial cultures transformed by the introduction of novel human immune (γ) cilasmids having γ-interferon activity and
- Concerning the expression of the interferon axis me44≠1tepolytezotide sequence.

Nature, Vol. 284, p. 593 (1980) and Sonnenfeld et al., Cellular Immunol.
40, p. 285 (1978)] and that interferon has antitumor activity [Pralonoc (B
Cellular Immunoronau (Cellu Lalock) et al.
lar Immunology), Volume 49, Page 390 (1
'980) and Rubin et al., Prop, Natl, Aca.
d.

Sci, ) (USA) Vol. 77, p. 5928 (198
Recently, active research activities have been carried out regarding human immune interferons, as there are research papers suggesting that This interest has led to attempts to determine and synthesize DNA sequences encoding human immune interferons. In fact, Gray et al.
Human γ-
We have isolated and characterized a recombinant plasmid containing a cDNA sequence encoding an IFN species, and have reported success in expressing this sequence in E. coli and cultured monkey cells. Nature magazine volume 295 50
3 (1982)).

It is an object of the present invention to provide novel polydiazotides having interferon activity that is considered to be a γ-IFN species. The object of the present invention is also to provide grasmids containing DNA encoding the novel γ-IFN species, transformants obtained by introducing these plasmids into microorganisms or cell cultures, and transformed microorganisms. The present invention provides a polypeptide having human immune interferon activity, wherein the amino acid at position 144 is glycine, and the method for producing the novel γ-IFN poIJ d peptide, which comprises the final expression of γ-IFN species in cells and cells. Ru.

According to another aspect of the present invention, there is provided a DNA sequence comprising a sequence encoding the above-described polytaupeptide.

Further in accordance with the present invention there is provided a replicable expression vehicle capable of expressing the above-described polyhazotide in transformed microorganisms or cell cultures; A microbial cell culture is also provided.

Furthermore, according to the present invention, a therapeutically effective amount of the above-mentioned human immune interferon activity is obtained. Pharmaceutical compositions are provided comprising a υgutide and a pharmaceutically acceptable carrier or diluent or excipient.

Still further in accordance with the invention, the method comprises growing a culture of the transformed microorganism with a replicable expression vehicle to produce a protein having human immune interferon activity as described above, and recovering said polypeptide. A method for producing human immunointerferon polypezotide is provided.

Various aspects of the present invention are realized by the methods described below, but the present invention is not limited thereto.

Induction of r-rFN mRNA Cell fractionation (cytapher
Human mononuclear cells are obtained from healthy adult volunteers by the following method. This cell 81 is treated. Mononuclear cells are further separated by centrifugation on Linnocyte Separation Medium (LSM, Litton Bionetics). Sterile 5
Blood products (
25me) and centrifuge at approximately 600X for 15 minutes. Mononuclear cells are harvested from the interface and washed twice with saline. A total of 7.6 x 109 mononuclear cells are collected.

The washed cells (approximately 10 cells/m13) were treated with 10 days of heat-inactivated calf fat serum (Irvine 5Ccientifi).
c) and Section 1 tissue culture antibiotics-antifungals
ycotin) mixture (Gibco) and 5 n
Roswell Park Memorial Institute supplemented with g/mA of tetradecanoylphorbol acetate (Sigma; 10.000x stock in DMSO).
al In5 position, RPMI) 164
Resuspend in 0 medium. A total of 7.51 cultures were divided into 7 273 Corni'ng roller bottles and filled with 5% CO□ (5% CO2 in the air).
). This culture was placed on a roller apparatus at 37°C.
pre-incubated for 90 minutes with staphylococcal enterotoxin A (SEA) (0.1 μg/r
ug) and 2-mercaptoethanol (final concentration 10-”
Add M). Add biotinylated SEA to 51 out of a total of 7.54 and unmodified SEA to 2.51. Preliminary studies suggest that they are equivalent for γ-IFN induction.

Cultures are incubated for approximately 40 hours, after which cells are harvested by centrifugation at 4°C. The cell pellets are stored on ice until used for mRNA isolation.

A, Isolation of RNp. 7 x 10 mononuclear cells prepared as above were centrifuged at 1000x'7 for 5 min and washed once in phosphate buffered saline (PBS) (PH 7,4). Wash, 10
mM NaCA and 10 mM) lithium hydrochloride (pH 7)
,4) and a solution containing 5mM NgC12 50TLe
Resuspend in 0.5% NP40 (ShellOll) [
Berger et al., Biochemistry (
Biochemistry) Volume 18, page 5143 (1
979)]. After leaving it at 0℃ for 5 minutes,
Nuclei are also removed by centrifugation at 0X, 9 for 5 minutes. Extract cell cytoplasmic RNA and extract RNA containing poIJA.
389 a9 by the method by Singet et al. [J, Mol.

Biol., Vol. 78, pp. 321-334 (1973)].

According to the phosphotriester method using nucleotide monomers, a desired DNA oligomer is prepared by the following method.

To a 500 μl stainless steel reaction vessel with a stainless steel filter on each end, add ~20 ~ of polystyrene resin to which nucleotides (2.0 μM) are attached by succinate linkage. The resin was treated with a solution of zinc bromide (IM) in dichloromethane-Ishiguro-Yanol (18:15) to remove the nomethoxytrityl (DMT) protecting group, and to remove the nomethoxytrityl (DMT) protecting group. Wash with acetonitrile and dry with a stream of nitrogen. To the dry resin, add 200 μl of birinone to DMT-nucleotides (
20 μM) and mesitylenesulfonylnitrotriazole (60 μM) are dissolved, a solution is added, and a camping reaction is performed at 45° C. for 20 minutes.

This cycle of deprotection and coupling is repeated until the desired DNA oligomer is assembled on the resin. Next, this resin was treated to remove DNA oligomers, and the resulting DNA was analyzed by Ito et al. [Nuc, Ac, Res, Vol. 10, 1755]
(1982)].

γ-IFN except for the three underlined nucleotides
A synthetic NA primer/mutantly induced gene strand (complementary to mRNA and prepared by the above method)
5'GCAGGΣCGACCATTACTGGGATC
3') (4.4 μg) and polyA-containing mRNA 80
tt9 was treated with 1O tris hydrochloride (pH 8,3) and 0
．． In a reaction mixture of 135μβ containing 5mM EDTA,
Heat at 55°C for 3 minutes, then cool quickly in ice water.

50mM) Lis hydrochloride (pH 8,3), 10mM
gC12, 2mM nothiothreitol (DTT),
40mM KCI, each (INTP O,,5rnM
and 120-L = 7 ribonucleacin (RNa
sin) [: 7-chi17 (Martynoff)
Layosu, Biodam. pieoffice,res,com,
(Biochem, Biophys, Res, Co
93, pp. 645-653 (1980) as ``Synthesis of Total DNA Complementary to Thyroglobulin 33S Methsenger RNA'', published by Biotec, Inc., Mediaon.
Wiaconsin) containing 300 μl
Specially primed complementary DNA (
cDNA) is synthesized.

Boil the reaction mixture for 3 minutes. Then] 8000 x
Centrifuge for 2 minutes at , 9C to remove denatured proteins.

The second synthetic NA glycer strand (5'AGTCCTTT
GGACCTGATCAGC3' ) (also prepared by the phosphotriester method described above) 30 μg and 100 μg
75 μl of mM Tris hydrochloride (PH8,3), IM
70.5 μl of KCl.

Mix 100 rnNi MgCL212 N with the supernatant and add water to make 550 μl. The mixture was boiled for 3 min, then quickly cooled in ice water and diluted with 100 mM p
- Add 45 μl of Mercagtoethanol.

Synthesize. 0.2M EDTA 91tl and 5M
The reaction is stopped by adding NaCt21 All. The reaction mixture is extracted twice with phenol and once with chloroform. - Precipitate the DNA using ethanol.

C. Digestion of cDNA with 5alI and BclI The DNA precipitate prepared as above was dissolved in 51 μl of water.
) Liss hydrochloride (pH 7,5) and 6mM MgC
t2 + 70μl containing 1.50mM NaC/S
endonuclease 5 for 2 hours at 37°C in a mixture of
Digest with 20 units of alI. Precipitate the DNA with ethanol and then dissolve in 32.2 μl of water. Next, 50
) Lis hydrochloride (PH8,0) and 10 mM M
gC/-2, 50mM NaCl and 100 μg/m
l of bovine serum 7/L/40 mixture containing Bumin (BSA)
Digest with 21 units of endonuclease BclI in μβ at 50° C. for 15 hours.

6% polyacrylamide gel electrophoresis was performed to size-fractionate the product and electrophoretic elution [Yang et al., Methods Enzymol
68, p. 178 (1979)], double-stranded DNA molecules between 400 and 700 bp in length are eluted from the cell. cDNA using ethanol
precipitate and then dissolve in 6 μl of water.

D. Cloning of cDNA The cDNA digested with 5all and Bcl I was transformed into pBR3.
27 [Oberon et al., Gene 9, pp. 287-305 (1980),
“Construction and characteristics of a new lonning vehicle■, p.
BR322 and a deletion derivative of pBR325] and 66 mM Tris-HCl (pBR325).
H7,5) and 6.6 rn) VL MgC62,0,5
Digest with 5alI and BamHI for 16 hours at 4°C in a 10 μl reaction mixture containing mM ATP and 2 units of T4 DNA IJ gauze. Escherichia coli MC1061
Transformation was performed using the Kushner method [Genetic gn
gineering) (1978), Elsevier, Amsterdam (Elsevier.

Amsterdam, page 17]. As a result, 1128 ampicillin-resistant colonies were obtained.

32p-labeled γ~IFN gene-specific oligonucleotide (5'CCATTATCCGCTACATCTGA
In-situ colony high-solidization using three ATGACs [Wallace (Wallace)
) et al, nuku, aku, res.

(Nuc, Ac, Rea, ) Volume 9 879-89
4 (1981)), γ-IFN gene clones are selected from the transformants. Five colonies are hybridized using this zorope. cDNA inserts were prepared from these five colonies, a genetic map was constructed using restriction endonucleases, and a depth (Devo)
[Nuc, Aku, Res, (Nuc.

Ac, Res, ) Vol. 10, p. 2487 (1982)
] pGI-10 from one of these clones, which was found to be almost identical to the described γ-IFN gene! The complete nucleotide sequence of the cilasmid γ-IFN locus, designated as
11bert) Chemical methods [Methods in Enzymology]
), Vol. 65, pp. 499-560 (1980)], it was found to be the same as that of Devos et al., except for the presence of n). Thus, the alanine codon at position 144 of Deps et al.'s γ-rFN, the second amino acid from the C-terminal amino acid, is pG
In I101, it is changed to glycine.

Two deoxyoligonucleotides, (5'TGGCA
GTAGCACATATA3') (1) and (5'G
ATCTATATGTGCTACTGCC3'Hn) is synthesized by the phosphotriester method described above.

500 n,9 oligonucleotide I was mixed with 60mM Tris-HCl (pH 8) and 10mM MgCl2.7m
MDTT.

30 min at 37°C in a 10 μl mixture containing 0,5 mM ATP and 1 unit of T4 polynucleotide kinase.
Phosphorylate for minutes. Boil for 3 minutes to stop the reaction and cool quickly. Add oligonucleotide ■ and incubate at 0°I
M
Make it into μl. After boiling for 3 minutes, the reaction mixture was gradually warmed to 37°C.
Anneal by cooling to 4°C and store at 4°C.

In 100 μl of reaction buffer, 10 μl of cilasmid p
ci -101 using 21 units of BstNl, 65
Digest for 3 hours at °C. After extraction with phenol and chloroform, DNA fragments are recovered by precipitation with ethanol. These fragments were treated with 66-mM) lithium hydrochloride (PH7,
5) and 6.6mM MgCl2, 0.5mMAT
P and ``55 single T 4 DNA IJ contains -ze 2
Combine with 7207L, 9 annealed oligos 8 and 1 in a 0 μl reaction mixture for 20 hours at 4°C.

Precipitate the DNA with ethanol and add 20% of the above reaction buffer.
In all, 10 units of Bgllll and 4 units of 5all
Digest for 3 hours at 37°C. After size fractionation by electrophoresis on a 6% polyacrylamide gel, 45
Fragment (A) containing 4 base pairs (bP) is isolated by electrophoretic extraction.

Construction of Vector DNA Containing the C9 Synthetic Promoter pBR327 (10 Lg
) is digested with 20 units of Pstl and 20 units of 5all at 37°C for 3 hours, and subjected to low-scale electrophoresis. The method of Parker et al.
zymol, Vol. 65, p. 358, 1980), 2.8 μg of an approximately 187.3 bp fragment is isolated from the Glu slice.

Further synthesis requires the use of psRBs.

For the preparation of pSRBS, P+rt((position 3372)
and Hind[l (position 91) to pYEJOOl (
ATCC37185) and intervening 8 38 b
Isolate the fragment of p.

Sma pMc1034! Cut with Pstl, 1a
The intervening 5500 bp fragment containing the cZ gene is isolated. These two fragments are synthesized with a linker - AGCTTGATCGATAAGGAGATCTATA
ATGACTAGCTATTCCTCTAGATATT
Coupling with AC yields the cilasmid shown below.

GAATTAATTG TTATCCGCTCACA
ATTAATT GTTATCCGCTCACAAT
TAAT TCTTGACAAT TAGTTAA
CTA TTTGTTATAATGTATTCATA
AGCTTGATCG ATAAGGAGAT
CTATA ATGET GGG GAT CC,CGTCGTT TTA
CAA CGT CGT GACTGGGl
y Asp Pro Val Val Leu
Gln Arg Arg Asp TrpGAA A
ACCCT GGCGTT ACCCAA CTT
AAT CGCCTTGlu Asn Pr
o Gly Val Thr Gin Leu
Asn Arg LeuGCA GCA C
AT CCCCCT TTCGCCAGCTGG
CGT AATAla Ala His Pro
Pro Phe Ala Ser Trp
Arg AsnAGCGAA GAG GCCCG
CACCGAT CGCCCT TCCCAASe
r Glu Glu Ala Arg Thr Asp
Arg Pro Ser G1nCAG TTG
CGCAGCCTG AAT GGCGAA
TGG CGCTTTGln Leu 'Arg
Ser Leu Asn Gly Glu T
rp Arg PheGCCTGG TTT CC
G GCA CCA GAA GCG GT
G CCG' GAAAla Trp Phe
Pro Ala Pro Glu Ala Va
l Pro GluAGCTGG CTG G
AG TGCGAT CTT CCT GAG
GCCGAT Ser Trp Leu Gl
u Cys A! IP Leu Pro G
lu Ala AspAla Phe His Leu
Trp Cys Asn Gly Arg Trp
ValSer Leu Trp Gin Gly G
lu Thr Gin Val Ala 5erGly
Thr Ala Pro Phe Gly Gly
Glu Ile Ile, AspGlu Arg
Gly Gay Tyr Ala Asp Arg
Val Thr Leu Arg Leu Asn
Vat Glu Asn Pro Lys Leu
Trp 5erGCCGAA ATCCCG AAT
CTCTAT CGT GCG GTG GT
Tala Glu Ile Pro Asn.
Leu Tyr Arg Ala Val
Va1104.5 10
6Q 1075GAA
CTG CACACCGCCGACGGCACG
CTG ATT GAAGlu Leu H
is Thr Aha Asp Gay Thr
Leu Ile GluGCA GAA G
CCTGCGAT GTCGGT TTCCGCG
AG GTGAla Glu Ala Cys A
sp Val Gly Phe Arg Glu
Va1CGG ATT GAA AAT
GGT CTG CTG CTG CTG A
ACGGC Arg Ile Glu Asn
Gly Leu Leu Leu Leu
Asn GlyAAG CCG TTG CT
G ATT CGA GGCGTT AACC
GT CACLys Pro Leu Lue
Ile Arg Gay Val Asn
Arg HisGAG CAT CAT CC
T CTG CAT GGT CAG GTC
ATG GATGlu His His Pr
o Leu His Gly Gln Val
MET Asn220
1225 1.240
GAG CAG ACG ATG GTG CAG
GAT ATCCTG CTG ATGGlu Gi
n Thr MET Val Gin Asp
, IIe Leu METAAG CA
G AACAACTTT AACGCCGTG
CGCTGT TCGLys Gin Asn A
sn Phe Asn Ala Val A
rg Cys 5erCAT TAT CCG
AACCAT CCG CTG TGG
TACACG CTGHis Tyr Pro
Asn His Pro Leu Trp
Tyr Thr LeuTGCGACCGCTAC
GGCCTG TAT GTG GTG GA
T GAACys Asp Arg Tyr Gl
y Leu Tyr Val Val As
p GluPne Pro Ata Val
Pro L7B Trp Ser Ile Lys
LysM18 Gtn Uln Gln Phe
Phe Gin Phe Arg Leu
5erA1a Ala Ser )iis A
ha rle Pro His Leu T
hr Thr'l'rp Gln His G
ln Gly Lys Thr Leu P
he SerAsp Thr Pro
His Pro Ala Arg Ile Gly
Leu AsnCys Gln Leu Ala
Gin Val Ala Glu Arg Va
lAsn2800'
2815TGG CTCGGA TTA GGG
CCG CAA GAA AACTAT CCCT
rp Leu Gly Leu Gly Pro G
in Glu Asn Tyr Pr.

Asp Arg Leu Thr Ala Ala C
ys Phe Asp Arg TrpAsp L
eu' Pro Leu Ser Asp MET
Tyr Thr Pro TyrGTCTTCCC
G AGCGAA AACGGT CTG CG
CTGCGGGGVal Phe Pro Ser
Glu Asn Gly Leu Arg
Cys GlyThr Arg Glu Leu
Asn Tyr Gly Pro His Gin
TrPArg Gly Asp, -Phe Gin
Phe Aan Ile Ser Arg TyrA
GT CAA CAG CAA CTG AT
G GAA ACCAGCCAT CGC8er
Gln Gln Gln Leu MET G
lu Thr Ser His Arg302
5 3040'
3055CAT CTG
CTG CACGCG GAA GAA GGCA
CA TGG CTGHis Leu Leu
His Ala Glu Glu Gly
Thr Trp LeuAAT ATCGACG
GT TTCCAT, ATG GGG ATT
GGT GGCAsn Ile Asp Gl
y Phe His MET Gay Ile
Gly Gly3100,
311. ! 5GACGACTCCTGG AGCC
CG TCA GTA TCG GCG Z
ZZAsp Asp Ser Trp Ser P
ro Ser Val Ser Ala? ZZ
Z CAG CTG AGCGCCCGGT CG
CTACCAT TACCAG? Gin Le
u Ser Ala Gly Arg Tyr
His Tyr G1nTTG GTCTGG
TGT CAA AAA TAA TAA
TAALeu Val Trp Cys G
in Lys・--319132013
211, 3221 CCGGGCAGGCCATGTCTGCCCGTAT
TTCGCGTAAGGAAATCCATTATGTA
CTATTTAAAAAAAAACAAAACT
TTTGGATGTTCGGTTTATTCTTTTT
C'rTTT ACTTTTTTTAT CATGG
GAGCCTACTTCCCGTTTTTCCCG
AT TTGGCTACAT GACATCAAC
CATATCAGCAA AAGTGATACG cc
TATTiTTTTTGCCGCTATGTGCGC
ATGA (Note) Nucleotides 3119:3124 are the first EcoR4 sites present in the sequence from which this portion is derived from Casadapan, Chou and Cohen (Ca5adaban
+(::hou and Cohen), Jay,
Nonokterioguchino (J, Bacteriology
) Since it has been changed as described in Vol. 143, p. 971 (1980), it is expressed as ZzzZzzZ. 1
The LaC2 sequence at 26:3181 was derived from Kalnins (Kal
nins) et al.
J, ), vol. 2, p. 593 (1983)], 22+30.
The sequence 3182:3434 was derived from the sequence part 78 of Buechel et al.
(Nature) Volume 333, page 541 (1980)
] 61:315 of the sequence.

The remaining nucleotide sequence from position 3435 to position 6369 is 5utcliffe [
Cold Spring Harbor Symposium Oncological Biology
HarborSymposium on Quanti
tative Biology) Volume 43, Page 77 ('
1'979)] is numbered according to the method of cilasmid pB.
It originates from the 1425:4339 position of R322.

Synchronous promoter sequence [Rossi la,
Zoroku, Natl, Akado, Sai (Proc, Natl
, Acad.

Sci,) Vol. 80, pp. 3203-3208 (1983)
pSRBS containing a synthetic promoter with a
Cilasmid (ATCCA 39514, in E. coli) (2
0 μg) and synthetic IJ, J? The seam joining site was isolated with 100 I with 40 units of BglII and 40 units of PstI.
Digest in the reaction mixture of LL for 3 hours at 37°C. From this digestate, approximately 8. ．． 3.4 μg of a 53 bp fragment are isolated.

6 fragments of approximately 1873 bp and 6 approximately 853 bp
6mM Tris Hydrochloride (PH7,5), 6.6mM
MgCl2.0, 5mMATP and 5 units of T4
Ligation is performed in 20 μl containing DNA ligase at 4° C. for 20 hours.

Precipitate the DNA using ethanol and add 10 units of Bgl II and 4 units of Sal in 20 μt of reaction buffer.
Digest with I for 3 hours at 37°C. 6 products
% polyacrylamide gel. By electrophoretic elution, a fragment (B) of approximately 2722 bp is isolated.

Fragments A and B, prepared as above, were ligated in a 10 μt reaction mixture using 2.5 units of T4 DNA IJ gauze at 4°C for 20 hours, and then E. coli MC
[Casadaba 7, (Ca5a
dabλn) et al., Jay 1 mole, Paiol.

(1980)). 167 ampicillin resistant colonies are obtained. Grasmid DNA was prepared from three ampicillin-resistant colonies and Sal I
, PstI, and BglI, and subjected to electrophoresis. This method shows that all of these cilasmids are identical and contain the desired glue, NA fragment. This is designated pGs I-102 and its partial sequence is shown below.

Met IJxn oer tle 1Jln Lys Ser
Val Glu Thr Ile LysGAA
GACATG AAT GTCAAG TTT
TTCAAT AGCAACGlu Asp Met
Asn Val Lys Phe Phe A
sn Ser AsnAAA AAG AAA
CGA GAT GACTTCGAA AAG
CTG ACTLys Lys Lys Ar
g Asp Asp Phe Glu Lys
Leu ThrAAT TAT TCG CTA
ACT GACTTG AAT GTCCAA C
GCAsn Tyr Ser Val Thr A
sp Leu Asn Val Gln ArgA
AA GCA ATA CAT GAA CT
CATCCAA GTG ATG GCTLys
Ala Ile His Glu Leu
Ice Gin Val Met AlaGAA
CTG TCG CCA GCA GCT
AAA ACA GGG AAG GCA
Glu Leu Ser Pro Ala Al
a Lys Thr Gly Lys Ar
gAAA AGG AGT CAG ATG C
TG TTT CGA GGT CGA AGA
Lys 'Arg Ser Gln Met L
eu Phe Arg Gly Arg Arg
Gly Ser Gln GAGCCTTCAA CCCAGTCAGCTC(
The nucleotide sequence from position 562 to position 3181 of the residue is such that position 1 is the first T of the specific EcoRI site.
The number material is 651:32 of pBR327 according to the system.
(derived from the nucleotide at position 73) D, E. coli MC
Expression of γ-IFN in 1061 (pGSII-102) 30μfl/+++A! L- containing ampicillin of
E. coli MC1 with pGSII-102 in gross
An overnight culture of 061 is diluted 1:50 in fresh L-broth containing 30 μfi/ml ampicillin. Grow cells for 5 hours at 37°C. Culture 10m
/? Centrifuge and resuspend the pellet in 2rnl of phosphate buffered saline. Crush the cells with ultrasound, 3
Centrifuge at 1000x9 for 0 min to obtain supernatant. The obtained supernatant was
[Simple analytical evaluation of interferon (Co
nvenient As5ay for Interf
erons), Jiei, /<Eoogy (J, V
irology) Vol. 37, pp. 755-758 (198
Interferon activity is analyzed using the human amniotic cell line (WISH). Escherichia coli MC106
1 (pGSII-102) Extract recovered from 7 punishments-
It has IFN activity and is useful in a variety of possible uses for γ-IFH.

In another embodiment of the invention, the nucleotide G (guanine) at position 536 is A (abdi)) and the corresponding encoded amino acid thus occurring is glutamine at position 9 of arginine. Plasmids are prepared in the same manner as above, except for the following. This cilasmid and the corresponding polyzotide which can be expressed by the above-mentioned E. coli and exhibit γ-IFN activity are shown below.

Ser Asp Val Ala Asp Asn G
ly Thr Leu Phe LeuGly Ile
Leu Lys Asn Trp Lys Glu
Glu'Ser AspArg Lys Ile
Met Gin Ser Gln Ile
Val Ser PheTyr Phe Ly
s Leu Phe Lys Asn Phe Lys
Asp AspLys Arg Ser Gin M
et Leu Phe Gln Gly Arg Ar
gGly Ser Gin (The nucleotide sequence at positions 562 to 3181 of the remainder 9 is derived from the nucleotide sequence at positions 651:3273 of pBR327 according to the numbering scheme where position 1 is the first T of the specific EcoR1 site) The nucleotide sequence of the γ-IFN locus on the plasmid contains a G (cytosine) rather than a C (cytosine) at position 548.
Gray (guanine) is present.
) et al. ("Expression of human immune interferon cDNA in Escherichia coli and monkey cells", Nature 295, pp. 503-508 (1,982)). With respect to certain embodiments of the invention, in which the novel γ-IFN species are expressed in transformed E. coli cultures,
Those skilled in the art will recognize that other microorganisms and cell cultures may also be used in this expression method.

In addition to the useful γ-IFN species of the present invention and its important pharmaceutical use as an alternative to interferon administration for the treatment of the various clinical conditions mentioned above, the present invention also provides the cilasmid pGS
It is also advantageous that 1-101 and pGSII-102 can be combined with pGZ 72 to produce a hybrid γ-IFN/β-galactosidase protein or a grasmid containing an NA sequence. - As an example, mention may be made of the cilasmid pGZ72, which is prepared by the following method.

1. Cut Grasmid pGSII-102 using BamHI (BamHI site) (position 547) and close the ends. This product is then cut with PstI (position 2430). Isolate the P st I −+BamHI fragment (1298 bp).

2, pMLB1034 (Berman)
, Biotechniques
1, p. 178 (1983)) was cut with EcoRI and the ends were closed. This product is then cut with PstI.

3. The BamHI→PstI fragment obtained in step 20 is obtained in step 1, and the resulting Ps t I −+ Ba
mHI fragment and ligation at the Pstl ends. The schizolasmid is closed by blunt end ligation of the blocked BamI (I and EcoRI ends).

Plasmid pMLB1034 was derived from Casadavan.
aban) et al.
Bacteriol).

143, p. 971 (1980)] β-gal reconstructed at the eight amino-terminal codons described
It has genes. In this reconstruction, originally β-gal
Cal? of the gene (position 3559:3564)? The EcoRI position near the xyl terminus is also removed by mutation, resulting in an unidentified sequence. Therefore, these positions are 2. Plasmid pGz7
The partial nucleotide sequence of 2 is shown below.

Ala Glu Asn Leu Lys
Lys Tyr Phe Asn Ala
GlyHis Ser Asp Val Al
a Asp Asn Gly Thr Leu
PheAsp Gin Ser Ile G
in Lys Ser Val Glu Tbr
IleAsp Arg Pro Ser Gin
Glri Leu Arg Ser Leu AsnG
GCGAA TGGCGCTTT GCCTGG TT
T CCG GCA CCAGly Glu Trp
Arg Phe Ala Trp Phe Pro A
la Pr.

GAA GCG GTG CCG aAp, AGCT
GG CTG cAc TGCCATGlu Ala
Val Pro Glu Ser Trp Leu G
lu Cys AspAsn Trp Gln MET
His Gly Tyr Asp Ala Pro
IlePhe Asp Gly Val Aan Se
r Ala Phe His Leu TrpAsp
Tyr Leu Arg Val Thr Val S
er Leu Trp GlnPt+e Gly G
ly Glu Ile IIe Asp Glu
Arg C1y GlyTAT GCCGAT CG
CCTCACA CTA CGT CTG AACG
TCTyr Ala Asp Arg Val
Thr Leu Arg Leu Asn
ValGlu Asn Pro Lys Leu
Trp Ser Ala Glu Ile Pr
.

Asn Let+ Tyr Arg Ala
Val Val C1u Leu His
ThrAla Asp Gly Thr Leu
Ile Glu Ala Glu Ala
CysGTG CAG GAT ATCCTG
CTG ATG AAG CAG AACA
ACVal Gin Asp Ile Lau
Leu MET Lys Gln Asn AsnT
TT AAC, GCCGTG CGCTGT T
CG CAT TAT CCG AACPhe
Asn Ala Val Arg Cys
Ser His Tyr Pro AsnCA
T CCG CTG TGG TACACG
CTG TGCGACCGCTACHis Pr
o Leu Trp Tyr Thr Leu
Cys Asp Arg TyrGGCCTC
TAT GTG GTG GAT GAA GC
CAAT ATT GAAGly Leu Ty
r Val Val Asp, Glu Ala
Asn Ile GluACCCCACGGCATC
GTG CCA ATCAAT CGT CT
G ACCThr His Gly MET
Val Pro MET Aan Arg L
eu ThrCAT CAT CCG CGCT
GG CTA CCG GCG ATG AGCG
AAAsp Asp Pro Arg Trp
T,eu Pro Ala MET Ser G
luCGCGTA ACG CGA ATG G
TG CAG CGCGAT CGT AAT
Arg Val Thr, Arg MET Val
Gln Arg Asp Arg AsnCA
CCCG AGT GTG ATCATCTGG
TCG CTG GGG AATHis Pr
o 5er Val Ile Ile Tr
p Ser Leu Gly AsnGAA
TCA GGCCACGGCGCT AAT
CACGACGCCG CTGGlu Ser G
ly His Gly Ala Asn His
Asp Ala LeuTAT CGCTGG
ATCAAA TCT CTCCAT 'CCT
TCCCGCTyr Arg Trp Ile
Lys Ser Val Asp Pro
Ser ArgPro Val Gln Tyr
Glu Gly Gly Gly Aha Asp T
hrThr Ala Thr Asp Ile
Ile Cys Pro MET Tyr AlaCG
CCTG CAT GAA GACCAG CCC
TTCCCG GCT GTGArg Val A
sp Glu Asp Gln Pro Phe
Pro Ala Val Pro Lys' Trp
Ser Ile Lys Lys Trp
Leu 6er LeuCCT GGA GAG
ACG CGCCCG CTG ATCCT
T TGCGAAPro Gly Glu Th
r Arg Pro Leu rle Le
u Cys GluTyr Ala His A
la MET Gly Asn Ser Leu G
ly GlyPro Arg Leu Gln Gl
y Gly Phe Val Trp Asp Tr
pGGCAAACCCG TGG TCG GCT
TACGGCGGT GAT TTTGly As
n Pro Trp Ser Ala Tyr G
ly Gly Asp PheGGCGAT ACG
CCG AACGAT CGCCAG TT
CTGT ATGGly Asp Thr Pro
Asn Asp Arg Gin Phe Cy
s MetAACGOT CTG GTCTTT
GCCGACCGCACCG CCG CATAs
n Gly Leu Val Phe Ala
Asp Arg Thr Pro HisCCA G
CG CTCACG GAA GCA AAA
CACCAG CAG CAGPro Ala
Leu Thr Glu Ala Lys
His Gin Gl'n GinTTT
TTCCAG TTCCGT TTA TCCG
GG CAA ACCATCPhe Phe
Gin Phe Arg Leu Ser
Gly, Gln Thr l1eGAA GTG
ACC' AGCGAA TACCTG, TTC
CGT CAT AGCGlu Val Th
r Ser Glu Tyr Leu Ph
e Arg I(is SerGAT AAC
GAG CTCCTG CACTGG ATG
GTG GCG CTGAsp Asn Glu
Leu Leu His Trp Met Val
Ala LeuGAT GGT AAG C
CG CTG GCA AGCGGT GAA
GTG CCTAsp Gly Lys P
ro Leu Ala Ser Gly Glu
Val Pr.

CTCGAT GTCGCT CCA CAA
GGT AAA CAG TTG ATTLeu
Asp Val Ala Pro Gln
Gly Lys Gin Leu IIe
GAA CTG CC'l' GAA CTA
CCG CAG CCG GAG AGCG
CCGlu Leu Pro Glu Leu
Pro Gin Pro Glu Ser
AlaGGG CAA CTCTGG CT
CACA GTA CGCGTA GTG C
AAGly Gin Leu Trp Leu
Thr Val Xrg Val Val Gl
nATCAGCGCCTGG CAG CAG
TGG CGT CTG GCG GAAlle
Ser Ala Trp Gin Gin Trp
Arg Leu Ala GluAACCTCAGT
GTG ACG CTCCCCGCCGCG
TCCCACAsn Leu Ser Val
Thr Leu Pro Ala Ala
Ser HisGCCATCCCG CAT
CTG ACCACCAGCGAA ATG G
ATala Ile Pro His Leu
Thr Thr Ser Glu Met A
spTTT TGCATCGAG CTG GG
T AAT AAG CGT TGG CA
APhe Cys Ile Glu Leu
Gly Asn Lys Arg Trp Gjn
TTT AACCGCCAG TCA GGCTT
T CTT TCA CAG ATGPhe
Asn Arg Gin Ser Gly
Phe Leu Ser Gln Met;
TGG ATT GGCGAT AAA AAA
CAA CTG CTG ACG CCGTr
p Ile Gly Asp Lys Lys
Gln Leu Thr Pr.

CTG CGCGAT CAG TTCACCC
GT GCA CCG CTG GATLeu A
rg Asp Gin Phe Thr Arg Al
a Pro Leu AspGACCCT AAC
GCCTGG GTCGAA CGCTGG A
AG GCGAsp Pro Asn Ala Trp
Val Glu Arg Trp Lys Ala
Ala Ala Cys Phe Asp Arg
Trp Asp Leu Pro LeuTCA G
ACATG TAT ACCCCG TACGTC
TTCCCG AGC8er Asp Met Ty
r Thr Pro Tyr Val Phe
Pr,o 5erAsn Tyr Gly Pro
His Gin Trp Arg Gly Asp
PheAla Glu Glu Gly Thr T
rp Leu Asn lie Asp GlyP
he His Met Gly Ile Gly G
ly Asp Asp Ser TrpGin Ly
s (Note) Nucleotides 3563:3567 are the first EcoRI sites present in the sequence from which this portion is derived, as described by Casadaban, Chou and Cohen.
.

Chou and Cohen), Noei. Bacteriolono (J, Bacteriology) 14
It has been changed as described in Vol. 3, p. 971 (1980), so it is expressed as zzz zzz. remaining 38
The nucleotide sequence from position 75 to position 6809 is derived from pBR322. The entire sequence can be written as follows2: AATTCCCGGGGATC552
: 5653, pSRBS(126:3435)
566:38744, PBR322(1
425:4359) 3875:6809-
Pharmaceutical Composition The γ-IFN species of the present invention can be combined with pharmaceutically approved carriers, excipients, and diluents by known methods to create compositions useful as pharmaceuticals. .

For suitable carriers and formulations, please refer to Remington (Remi
ngton) Pharmaceutical Thug'Rue 7
- described by E.W. Martin and is included here for reference. These pharmaceutical compositions will contain an effective amount of the interferon polyheptide of the present invention in combination with an appropriate amount of carrier for proper administration to the patient.

A. Parenteral Administration The human immune interferon of the present invention can be administered to people in need of antitumor or antiviral therapy or to patients exhibiting immunosuppressive symptoms. Doses and rates of administration will vary depending on the individual case, but have traditionally been used in other human interferon clinical trials, such as approximately
-10) XIO6 single V days, or if it has a purity of 1% or more, ≠ 50 x 106 units/
You may follow the rule that it is until the end of the day. The dosage of 1γ-IFH can be very high to obtain great effects. This provides one of the important advantageous effects obtained with the use of gamma-interferon 6 produced from seeds in the present invention.

The following microorganisms and Grasmid have been deposited with the American Type Culture Collection (ATCC) and have been given the following deposit accession number: Escherichia coli MC1,061 (
pGI-101) ATCC No. 39 Rock E. coli MC
1061 (pGZ 72) ATCC No. 39669 Escherichia coli MC1061 (psRBs) ATCC No. 3
It should be clear that 9th 39th can go home. Such changes are not to be considered as a departure from the spirit and scope of the invention, and all such modifications that are obvious to those skilled in the art are intended to be included within the scope of the following claims.

Patent applicant: Asahi Kasei Industries, Ltd.

Claims (1)

[Scope of Claims] 1. A polypeptide having human immune interferon activity, in which the amino acid at the 144th position from the N-terminus, which is usually the first amino acid of human immune interferon, is glycine. 2. A DNA sequence consisting of a sequence encoding the polypeptide according to claim 1. 3. A replicable expression vehicle capable of expressing the polypeptide according to claim 1 in transformed microorganisms or cell cultures. 4. A microorganism or cell culture transformed with the vehicle according to claim 3. 5. A pharmaceutical composition comprising a therapeutically effective amount of a human immune interferon species according to claim 1 and a pharmaceutically acceptable carrier or diluent or excipient. 6. Human immunization according to claim 1, comprising growing a culture of the transformed microorganism with a replicable expression vehicle to produce a human immunization interferon polypeptide and recovering said polypeptide. Method for producing interferon polypeptide. 7. A polypeptide having human immune interferon activity, in which the second amino acid from the C-terminal amino acid is glycine. 8. A DNA sequence consisting of a sequence encoding the polypeptide according to claim 7. 9. A replicable expression vehicle capable of expressing a polypeptide according to claim 7 in a transformed microorganism or cell culture. 10. A vehicle-transformed microorganism or cell culture according to claim 9. 11. A pharmaceutical composition comprising a therapeutically effective amount of a human immune interferon species according to claim 7 and a pharmaceutically acceptable carrier or diluent or excipient. 12. Human immunization according to claim 7, comprising growing a culture of the transformed microorganism with a replicable expression vehicle to produce a human immunization interferon polypeptide and recovering said polypeptide. Method for producing interferon polypeptide. 13. A polypeptide having human immune interferon activity having an amino acid sequence substantially represented by the following formula (I) [Amino acid sequence exists]. 14. A DNA sequence consisting of a sequence encoding the polypeptide according to claim 13. 15. A replicable expression vehicle capable of expressing a polypeptide according to claim 13 in a transformed microorganism or cell culture. 16. A microorganism or cell culture transformed with the vehicle according to claim 15. 17. A pharmaceutical composition comprising a therapeutically effective amount of human immune interferon according to claim 13 and a pharmaceutically acceptable carrier or diluent or excipient. 18. Propagating cells of the transformed microorganism with a replicable expression vehicle to produce a human immunointerferon polypeptide and recovering the polypeptide. Method for producing peptides. 20. A polypeptide having human immune interferon activity having an amino acid sequence substantially represented by the following formula (II) [Amino acid sequence exists]. 21. A DNA sequence consisting of a sequence encoding the polypeptide according to claim 20. 22. A replicable expression vehicle capable of expressing a polypeptide according to claim 20 in a transformed microorganism or cell culture. 23. A microorganism or cell culture transformed with the vehicle according to claim 22. 24. A pharmaceutical composition comprising a therapeutically effective amount of the human immune interferon of claim 22 and a pharmaceutically acceptable carrier or diluent or excipient. 25. The human immune interferon of claim 20, comprising growing a culture of the transformed microorganism with a replicable expression vehicle to produce a human immune interferon polypeptide and recovering said polypeptide. Method for producing polypeptide. 26. A plasmid capable of synthesizing the polypeptide of claim 1 in a suitable microorganism or cell culture. 27, pGI-101 and pGSII-102. 28, one of the plasmids according to claim 27
Microorganisms or cell cultures transformed using 29. The microorganism according to claim 28, which is obtained by transforming an E. coli strain with pGSII-102. 30, the microorganism is Escherichia coli MC1061 (pGSII-10
2) Claim 2 which is ATCC No. 39668
Microorganism according to item 9. 31. The microorganism is Escherichia coli MC1061 (pGI-101)
The microorganism according to claim 28, which is ATCC No. 39690. 32, a microorganism or cell culture transformed with a plasmid selected from the group consisting of pGZ72 and pSRBS. 33. The microorganism is Escherichia coli MC1061 (pGZ72)AT
The microorganism according to claim 32, which is CC No. 39669. 34. The microorganism is Escherichia coli MC1061 (pSRBS) AT
The microorganism according to claim 32, which is CC No. 39514.