JPS60172995A - Improvement of gamma-human interferon - Google Patents

Abstract

(57) [Summary] This bulletin contains application data before electronic filing, so abstract data is not recorded.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention is in the field of recombinant DNA engineering and relates to means and methods for using such engineering to discover preferred DNA sequences encoding the synthesis of human gamma-interferon.

More specifically, the present invention provides the design and construction of DNA sequences that identify the amino acid sequences of human gamma-interferon and improved human gamma-interferon, and the use of cilasmids containing these gamma-interferon DNA sequences. This invention relates to the transformation of microorganisms and the use of these transformed plasmids for the production of γ-interferon.

BACKGROUND OF THE INVENTION Interferons (FNs) are a group of proteins that are synthesized within mammalian cells in response to stimulation by viruses, drugs, and various other substances. Early research on interferons focused on their ability to induce an antiviral state in target cells;
Recently, it has been revealed that interferon has cell proliferation inhibiting and immune response regulating effects (1).

Interferons have the potential to be used to treat viral infections and malignant tumors, but research has not progressed due to the limited amount available and the presence of impurities.

The natural amino acid and DNA sequence of human immune (gamma) interferon is disclosed in British patent application GB2 107 71
8A and European Patent Application No. 008854OA2.

Human interferons are classified into six types depending on their antigenicity and biological and chemical properties.

IFN-α (white blood cells), IFN-β (fibroblasts) and IFN-γ (immune). Virus-induced acid-stable interferons (FN-α and FN-β)
has been studied in the most detail.

The use of recombinant DNA manipulation has made it possible to predict the amino acid sequences of FN-β and several FN-α species, and it has also been shown that FN-α and FN-β can be synthesized in E. coli. As a result, it has become possible to purify large quantities of interferon for biological activity.

On the other hand, little is known about the properties of FN-γ. FN-γ is acid-labile and does not cross-react with antisera against FN-α and FN-γ. IF
N-γ has a wide range of biological activities (2), (
3), (4). Engineering FN-γ is Engineering FN-α and IF
It enhances the antiviral activity of N-β, but differs from fluid-stable interferons in its virus and target cell specificity (2). However, impure IF
In vitro studies with N-γ showed that IFN, -
One of the important activities of γ has been shown to be the regulation of immune responses (3). Engineering FIJ for transformed cells
The antiproliferative effect of -γ is similar to that of FN-α and FN-β.
Furthermore, FN-γ has the potential to be applied to the treatment of malignant tumors (4). The mouse protein FN-γ exhibits significant antitumor activity against murine sarcomas (5).

Recently, cDIIA encoding human FN-γ was cloned into E. coli f6)i7). The amino acid sequence of engineered FN-γ was deduced and the mature polybezotide was found to have a length of 146 amino acids. FN-γ is expressed in E. coli and in eukaryotic systems (6
). Its expression level is 1F within the same expression system.
It is much lower than that of N-α and FN-β. The relatively low expression level of FN-γ in E. coli is due to
This may be due to the fact that codon usage within prokaryotic systems differs considerably from that of human genes (8). It is believed that if a synthetic γ-interferon gene constructed to allow optimal expression in prokaryotes is obtained, a larger amount of interferon will be synthesized and recovered. , in the field of recombinant DNA manipulation, and relates to means and methods for using such manipulation to discover favorable DNA sequences encoding the synthesis of human γ-interferon.

The present invention provides improved nucleotide sequences encoding the naturally occurring γ-interferon amino acid sequence. This novel nucleotide sequence has excellent properties and stability and can be used in the fermentation process to produce human interferon boripezotide. The present invention also discloses amino construction sequences, DNA nucleotide sequences and plasmids for improved γ-interferons.

More specifically, the present invention relates to the design and construction of a DNA sequence encoding information specifying the amino acid sequence of human γ-interferon, the design and construction of a plasmid containing the γ-interferon DNA sequence, and the design and construction of a plasmid containing the γ-interferon gene. The present invention relates to the transformation of microorganisms by microorganisms and the use of transformed microorganisms for producing γ-interferon.

Improved human γ-interferon has L7ell as G1n
111, and its amino acid sequence is as shown in FIG.

In order to clarify the background of the present invention and to provide further detailed reference in the Examples, literatures, patents, etc. are cited in the specification, and the references are indicated by numbers in the order of citation. The title of the document, etc. is indicated at the end of the specification.

Preferred Embodiments of the Invention The direction for improving the synthesis of IFN-γ was to construct synthetic genes that were optimized for expression in a particular host. Recent advances in the synthesis of deoxyoligonucleotides (9) have shown that engineered FN-α can be reduced to low Pτ
This made it possible to synthesize large double-stranded DNA such as genes. In addition to optimizing codon usage, chemical synthesis of genes offers several powerful advantages. In other words, the gene is designed so that it can be inserted into various expression vectors, and by introducing appropriate restriction sites, the DNA can be modified or hybrid interferon formed.
insertion or deletion has become easier.

A synthetic gene for human FN-r has been designed,
It was constructed. 446 bp duplex first pAT156
and then into an expression vector.

The gene encoding the amino acid sequence of human γ-interferon is shown in FIG. 1 along with the 46 oligonucleotides used to construct the complete gene. During the genetic engineering process to form the expression vector, two nucleotide changes occurred.

This is shown in FIG. These two nucleotide P changes result in 1) no effect or conservative nucleotide change in H1822 (OAT −+ OAO), and 2) (A
G1n11 of L7S111 by AA-+ OAA)
1 caused a phenotypic change.

Example 1 describes the construction of a gene that encodes the γ-interferon gene. Example 2 includes FN −γ(Gln
□Cilasmid encoding FN-γ (Ly
We describe the construction of a corrective plasmid encoding B1I). Example 6 includes engineering FN-γ (Lysll) and engineering FN-γ (Lysll).
The biological properties of N-γ (Gln 0,) are described.

The plasmid list below summarizes the nature of the cilasmids used and their origin or construction methods. INF r
(LYElxx,) and 工FNr (Gln, □)
Both Shirasumi P used in the production of both H1θ2
2 has an OAO codon. All cilasmids produced are ampicillin resistant and tetracycline sensitive. The pXY2 plasmid inserted an f promoter and a liposome binding site to improve transcription and expression. The term "mature" was used to indicate the absence of a fusion polypeptide and the presence of an N-terminal methionine.

List of cilasmids Plasmid Nature Origin or construction pXY 1 FN r (Gln Ill) pWT23+1 expression vector containing Figure 2 ((+B 2052513 A) p, TB 1 Low copy Trp Fitt fusion FNγ (G1n1□,) pWT 11100p Expression vector Fig. 4 Hycobi mutant (GB2052513 A) PJB 2 High copy Trp Htt fusion engineering FN r (Glnx, x) y-β from p124c Fig. 5 Expression vector (GB8102051) pXY 2 Low copy N-terminal Met 'tt engineering FN
r(Gln
L3”l1l) pac103 High copy N-terminal Mθt Figure 9 IFN
γ (I, ys engineering 11) poo 2 high copy N-terminal IFNr Figure 10 ("
n111) Highly expressed pac2[]3 High copy N-terminal engineering rNi〃(L7
θ111) Highly expressed Fi, coli strain used is Fi, C,0115to
ck center (Yale University
5chool of Meaicine, 5Q[
133Bedar 5treet, New Have
NII Connecticut, USA).

Cilasmid I) Co 2 and poo 203 were also added to E+coli K-12 strain HB10.1.
ericanType Culture Co11ec
tion (12601ParlclawnDrive
, Rockville% Marylana 2
Q852, USA). Those containing pac2 are ATea/Ig3 (L493, pac
Those containing 203 ゛ is ATOOyt639A'9
It is 4.

One synthetic gene encoded the synthesis of a modified human γ-interferon polypeptide in which L7'llll was replaced with G1n111. This gene was later identified as L3'811
1-containing human γ-interferon.

What is the sequence of the synthetic NA double strand? Shown in A1M. Several criteria were set for the design of this gene. Codon usage was optimized for expression in coli (8).
. The computer used a computer to examine sequences that might anneal to each other or cause problems with subsequent gougeons. These sequences were removed unless essential to the genetic code. Initially, the synthetic gene was
It was designed to clone into the d ll and Sa'l I sites. Several other restriction sites not included in pAT 153 were also inserted into the synthetic gene sequence. These unique restriction sites facilitate the analysis of cloned genes and the formation of hybrid P or modified sequences.

Insertion of the gene into pWT 231 results in trpffi
Enables expression of engineered FN-γ as a fusion polypeptide containing 12 amino acids from the polypeptide. Mature engineering FN
For expression of the -γ polypeptide, this synthetic gene was cleaved at the BamH1 site, attached to a synthetic linker with Ola and BamH ends, and then inserted into xx2 for expression (Fig. 6, Figure 5).

In summary, interferon genes and cilasmids were designed, in part, with the following goals.

1) As long as the amino acid sequence of human γ-interferon is provided, the nucleotide sequence was designed to allow the following optimization.

a) Provide a large number of restriction sites for ease of manipulation b) Introduce regularly spaced overlapping restriction sites into the synthetic gene to facilitate the formation of hybrid interferon genes c) Maximize expression in L. coli d) There is no unfavorable palindromulnizo formation. 2) Synthetic interferons are made with cilasmid and adjusted DN to optimize the synthesis of interferon protein.
Combined with A.

3), cilasmids were constructed to facilitate high copy number to increase γ-interferon gene dosage.

4) An intermediate gene was constructed that inhibited the expression of modified human γ-interferon in which Lys 0 was replaced with G1n 0.

The synthetic sequence was divided into 46 oligonucleotides with an average length of 19 nucleotides. There was an overlap of at least 6 bases on either side of all IJ r-john points.

Example 1 A chemically synthesized oligonucleotide of an interferon gene was synthesized by the phosphotriester method on polystyrene resin using a dimer block (9) (M'1yoshi et al.).

Methods for constructing this polynucleotide are well known.

See the method of Miyoθhl et al.

The polynucleotide product was stored as a solid and aliquoted and dissolved in pyridine for use in oligonucleotide synthesis.

Nucleotide resin (100~) was sequentially (11 2% in dichloromethane/butanol (7:5 V/V) (5 ml)
Benzene sulfonic acid, 5 minutes, (2) dichloromethane/
Butanol (7:3V/v) 5 X 5 +I+7! (3) Anhydrous bilicin 5 X 5 rnl (4) Dinucleotide iQQ+y and mesitylenesulfonylnitrotriazole in anhydrous bilicin (0,5g) for 45 minutes (5) Anhydrous pyricin 2 x 5 ml (6) Bilicin (5#u) 10 min in acetic anhydride (7) anhydrous bilicin 2 x 5 nt (8) dichloromethane/butanol (7:3 v/v) 5 x 5 IrL
treated with l.

The above cycle was repeated using the appropriate dinucleotide until the oligonucleotide was completed.

The coupling efficiency of each step was monitored spectrophotometrically by the absorption of free dimethoxytrityl alcohol at 504 nm.

After the synthesis is complete, the oligonucleotide polymer is separated from the polymer support and sequentially treated with dioxane/water (1: IV
/V) Medium Q, 5MN1. BTL, N2. N2-
Tetramethylguanidimiram 4-nitrobenzalrximate for 60 hours at room temperature (7 hours with aqueous ammonia)
All protecting groups were removed by treatment with 80% acetic acid for 4 hours at 0° C. and 60 minutes at room temperature. The oligonucleotide from which the protecting group was removed was placed on a Partisi 119 SAX column.
Purified by HPLO using a 1M to 4M triethylammonium acetate salt gradient at 50°C, pH 4,9.

Block paste r-ion on oligonucleotide (Figure 1) A sample (5 oopmole) of oligonucleotide r was added to 2Q #M-spermidine, 2Q mM-DT
T.

1Q mM -MgC42, 50mM -Tris -
Hci (pH 9,0) and 0.1 mM −11+
Phosphorylation was performed with 6 units of T4-induced polynucleotide kinase (EO2,7,1,78) for 60 minutes at 67°C in DTA. Each oligonucleotide P was precipitated with ethanol and denatured with 15% polyacrylamide (pi(8,3
) analyzed above.

After elution from the sample, the recovered amount was measured by OerθnkOV counting. There were a total of 46 oligonucleotides used to form the complete gene shown in FIG.

The procedure is to combine the oligonucleotides that form the wood-like blocks ('7'') into four double-stranded blocks, each with γ - Assemble to form a quarter of the interferon gene.

Finally, the double-stranded block is resorted to form a complete gene.

Four duplex blocks (each [], 4 pmole) are
The synthetic gene was assembled by annealing at 45° C. for 5 minutes in 50 μm water and then slowly cooling to room temperature. The mixture was lyophilized and coated with T4 DNA IJ gauze (106
, 5, 1, 1) gauze mixture containing 50 units 20
It was taken in μm. After reacting for 18 hours at room temperature, the reaction was terminated and the product was purified on a 5-layer natural polyacrylamide gel. The products were identified by size markers obtained from Haθ■ digestion of φX174 DNA. D K8. It was recovered by electroelution on l filter paper and phosphorylated with T4-induced polynucleotide kinase.

Example 2 Construction of cilasmid (Figure 2) cilasmid pAT 156 (BRL, Gaither
sburg.

MD) was digested with HlndIll and sal IL 7 donuclease, and the resulting 3036 bp fragment was isolated by Rosdal electrophoresis. Synthetic engineered FN-γ gene (20 μg) and vector DNA (1
600μg) onto the DK8i disk? then 1Q mM Tris-HCL, 1mM-E
Washed in DTA (pH 7,5). Next, add the filtrate to 1.
5N-NaC1,IQ mM-Tris-H(J
, 1 mM-FiDTAp) (7,5 (200 μm)
The medium was incubated at 67°C. The filtrate was removed by centrifugation and the DNA was recovered by ethanol precipitation. The DNA was washed twice with 170% ethanol and dried. Sur-John buffer (IQ μm, 66mM T
riθ-H(J PI-17,5゜6.6mM-Mg
(J2, I QmM DTT, Q, 4mM ATP.

1mM spermidine, 1 unit of T4 reagent) was added and the mixture was incubated at 15°C for 18 hours.

K. coli using standard transformation procedures
A portion (2.5 μm) of the resolution mixture was used for the transformation of K12 strain DH1. -Ampicillin 1 at night
After culturing on L-Agar plates containing 00 μg/ml, colonies were treated with ampicillin and tetracycline (2
0 μg/mJ) onto a plate. One colony showed insertion of the complete γ-interferon sequence into the Hjnd[1 and Elal I sites of pA'r t 53 by tetracycline sensitivity, colony hybridization with 32p-labeled oligonucleotides, and restriction enzyme mapping. I was able to identify. This operation is summarized in FIG. pXY1 was constructed in this way.

The engineered FN-γ gene in pXYi contains a converted nucleotide sequence (AAA 4 CAA ), which is I+711
11, encodes the synthesis of γ-interferon with G1n111 in place of (AAA). By inserting a new nucleotide 1 sequence into the υF+- John using the R-joon after restriction enzyme cleavage, the nucleotide tridelet-R
(CAA) was changed to AAA (Figures 5 to 10).
(see figure).

The gene encoding r-interferon (""zzz)'l was used to generate several cilasmids capable of expressing engineered FN-gamma mRNA and protein.

Construction of cilasmid pXY 103 Correction of GAA (Gln) to AAA (I, ys) was performed by insertion of nucleotides into pXM 1 (Figure 6).

Synthetic genetic CheFN7” in pXY I (G1n111
) no conversion nyo'), engineering IFM -r (Lysll
l) (y) Synthesis'? Shirasumi t'pXY105 containing the synthetic gene for cocoon was generated.

a) Vector preparation Plasmid pxy I DMA (5ttg) at 6mM
Tris-HCLPH7,5,6mM Mg (J2.
6mM 2-mercazotoethanol and 100μg,
/mA! It was digested with endonuclease Sac I (40 units) at 67° C. for 2 hours in a 1100 μm dilute buffer containing BSA.

5 M NaCL (1/41, final Na CL rij
After adding t5QmM), F! at 18N (
13 units) and the mixture was further incubated at 37 °C for 2 hours. 5'OOff1M-KDTA 6μm
After adding phenol-chloroform (
1:1.100μm) once, then chloroform (100μm) once.
After extracting twice with m) and adjusting the NaCL concentration to 0.3 M, precipitation was performed with 2.5 times the volume of ethanol. The DNA was collected by centrifugation, and the precipitate was diluted with 80% ethanol (0'0).
and dried in vacuo. Sa of pXY 1
The complete decomposition by C,l and BstEJI is )f/I/
Confirmed by electrophoresis (1 thiagarose, TBIC buffer). DNA7.5.0μmノ2 QmMNa(
J, I QmMTris-H (W PH8,0, Q,
Digested with bacterial alkaline phosphatase (7.5 units, 6 times 2.5 units each) for 1 hour at 65°C in 1 mM EDTA. Enzyme (2,5 units) for 10 minutes, 2
Additions were made after 0 and 60 minutes. Digest solution with phenol
It was extracted twice with chloroform (1:1.50 μm) and twice with chloroform (50 μm), and then precipitated with ethanol. After leaving at -20'C for 18 hours, DNA r1 was recovered by centrifugation, washed twice with 70'l ethanol, and dried in vacuo. 10mMT of KonoDNA
rle −H(J P+(7,5, Qj mM BDT
It was dissolved in A at a concentration of 0.5 μg/μm.

Double chain consisting of oligomer γ32-γ37 (Fig. 1) (
150 ng), mild KM solution (150 ng), 150 ng)
50 μm) at 67° C. for 2 hours with 5acI: (80 units). After adding Ha (J Y to 50 mM), Bst E II (8 units) was added and digestion was continued at 67'G for 2 hours.
After extracting twice with 0 μm) and adding NaC1k O-3M lc and tRNA Y, the mixture was precipitated with ethanol. The resolved duplexes were collected by centrifugation, washed twice with 70% ethanol, and dried in vacuo. The duplex was dissolved in 10 μm of water and 2 μm of 5× loading buffer (5XTBE, 2-part Ficol, o, i wide bromophenol blue and 0.1% xylene cyanazole) was added, followed by 2 min at 65°C. Heat and TBF! Loaded onto 7.5 polyacrylamide cells in buffer. After electrophoresis, the pills were autoradiographed on pre-exposed KOdaX X-ray film at -70"O for 18 hours. The fragments contained traces of (32p) label. The main band f visualized was cut out from the pill and electroeluted on DE8'1 filter paper.

After recovery from DE81 filter paper and precipitation with ethanol, the fragment was dissolved in 4' TE 25 μm. A portion (5 μm) was added to an equal volume of 2× loading buffer (9 μm).
0 part formamide, 2 part FiCO1,0,1% bromophenol del-10,14 xylene shear tool)
, heated to 60" for 2 minutes and applied to 15-striped polyacrylamide in 7M urea. After electrophoresis, pill autoradiography revealed that each strand of the resolved duplex had the expected size of 60". and a length of 41 nucleotides).

pXY1 (Figure 6) fragment (
100 ng) and vector (1 μg) in 66 mM Tr.
is-HCJ pH7,5,6-6mM-MgCLz
, I Q mM DTT , Q, 4 mM ATP, 1
mM, c-vermisoy containing slow io, l D tn , 1
5 'OK oiic, T4DNA Riga-ze (NEN
) Incubated with 1Q unit for 18 hours.

A portion (5 μm) of the resolution mixture was used to transform Conbiten) K, coli K12 strain H under standard transformation conditions.
Transformation of B101 was performed. When cultured for two nights on an L-Agar plate containing 100 μg/goambicillin, 4
, 500 colonies were obtained, 200 of which were picked onto a nitrocellulose funnel. Colony hybridization
John showed that the majority of colonons strongly hybridized to oligonucleotides γ11 and γ35 (contained in the Bst F;II-SaCI fragment). The cilasmid DNA from eight of these recombinants possessed the endonuclease restriction site theoretically contained within the synthetic 1FN-γ gene. Max the sequence of the bottom strand of the correction gene in f Lasmi FpXY1 [15
It was determined according to the method of Am and Gi'1bert. According to this, codon 111 was AAA. Conservative changes at the 5' end of the gene were still present in pXY103.

pWT 2611FN of the IFN-1 gene (Glz1411) - Hind II containing the gene
Insertion of the -Sad l fragment into the pw'r 251 vector creates a TrpE-fusion IFN-γ protein*
: Met-Gln-Thr, -G4n-Lys-Pr
o-Thr-8er-8er-8er-Lys-Leu
- Gln-Ala - produces expression of mature IFN-γ.

Plasmid DNA from pWT 261 was incubated at 67°C in 250 μM restriction endonuclease buffer.
Digested by Sal (4 units) and Hindi (10 units) for 16 hours. After extraction with phenol and recovery by ethanol precipitation, vector
The Hindl of pXY1 containing the synthetic IFN-γ gene ("n111) was treated with alkaline phosphatase.
The -Sal I fragment was separated by electrophoresis and eluted on a DE81 filter as described above. vector(
4μg11) WT 261Sal I-Hindll
) and IFN-γ gene (40 ng) were lysed in slow-resolution solution (20 μm) and treated with T4 DNA ligase (
51#) was added, and then incubated at 15°C for 18 hours. Half of the retention mixture (10,5 μm)
Competent E, coli K 12 strains HB101
It was used in the form of Negative Enkyo. 100! ! Twenty-two colonies obtained by culturing on L-Agar plates containing M/- of ampicillin were picked on ampicillin-enriched plates. By colony hybridization, these 20 colonies were infected with oligonucleotides γ11 and γ
46 was shown to hybridize with both.

Restriction digestion of these colonies revealed that they contained the human FN-γ gene fused to the 5' end of the TrpE gene.

As a detailed illustration of the genetic pre-testing method, pWT 11 is shown below.
1 cop and cilasmid pJB from pJBi 2
(Figure 4) shows the formation.

pWT 111 copDNA (10,4/jg)
, 20mM Tris-HCJ PI17.5.10
mM Mg(J2.5 Qu+M(NH4)2304
+ 6 in 100μg/-BSA (250μm)
At 7°C, endonuclease Pst engineering (11-2
The sample was disassembled for 10 hours in the sheep position. Min Is Temporary, 500[I
7.5 μl of M EDTA and 5 M Na(J
5 μm of Q was added, and the reaction solution was extracted with naphenol-chloroform.

DNA was recovered by ygethanol precipitation. The product of this reaction] am was reduced to 150 mM Na (J, 6 mM
Tris-HCJp) (7,5,6mM MgCl,
2,6mM 2-mercazotoethanol, 100μg/
16 in mBsA (250 μm) at 67°C and further with endonuclease 5alI (10 units).
Time resolved. After adding 50 μm of 5× TBE loading buffer, the sample was heated to 65°C for 2 min and TBE
One sip of 0.8% agarose in buffer was added to the electrolyte. Sal I-Pst I fragment (2227
bp) was recovered by air elution on DECI filter paper and precipitated with ethanol.

pJB I DNA (10 μg) was digested with the endonuclease Pst I (11,2 units) and Sal[(1) in the same manner as Pst.
The I-Sall fragment (1649 bp) was recovered by acidic elution over DE81 and ethanol precipitation.

pWTllicop, 5alI-PstI fragment (6 μg) and pJB i, Pst-Sal
l fragment (2 μg) was dissolved in ligase buffer (20 μg).
m) at 15°C in T 4 DNA ligase (1
0 units, NEW) and were allowed to resort to each other for 18 hours. A portion (5 μm) of this mixture was added to competent E
, was used for transformation of Co11 K12 strain aB101. When cultured overnight on L-Agar plates containing 100 μg/- of ampicillin, 219 colonies were obtained. Colony hybridization revealed that the majority hybridized strongly to oligomers γ11 and γ46. 1 colony 1) JB2
I chose this as a way of expressing myself. The restriction enzyme map of pJHB2 was consistent with expectations.

This operation is summarized in FIG.

戟) Preparation of vector 1) JB 2 DNA (10 μg) to 100 mM
NaCJ, 5 []mM Trig-HCJ,
PH7-5, 10mM Mg (J2, imMDT
T.

Digested with endonuclease Bau+HI (20 units) in 100 μg/−BsA (500 μl) at 67° C. for 16 hours. Equal volume of 5 Q mM NaCL,
1 C1mM Tris-H (JpH7,5,1Q'
After adding mM Mg (J2, lmM DTT,
The DNA was filtered at 7°C and treated with endonuclease Sal.
I (20 units) for a further 5 hours. Vector fragment (6474 bp) in TBE buffer
．． Separated by ridge aerophoresis on 8% aga p-spill, D
The air was eluted on No. 81 filter paper and recovered by ethanol precipitation. This DNA fragment was digested with 5QmM T
ris-H(Jpl(9,0, i mM MgCj2,
Q, 1mM ZnSO4, imM spermidine (50
μl) for 15 minutes at 67°C. Enzyme 2
After the third addition (10 units), the reaction was allowed to run for an additional 15 minutes. Heat the enzyme to 70°C for 10 minutes to inactivate it,
DNA fragments were extracted several times with phenol-chloroform and recovered by ethanol precipitation.

(bLrFN-γ gene-containing fragment 13am H
Preparation of I-8al I pCC106 DNA (20 pg) was digested with endonucleases Bann II (40 units) and 5al I (20 units) and recovered in the same manner as in (a).

BamHI-8al I fragment (460bp)
were separated by Dell electrophoresis, electrobathed on DBS'l filter paper, and precipitated with ethanol. Above, I dealt with the case of (c)) and my colleagues. This fragment was not treated with bovine primary phosphatase.

(C) ') Dageon and transformed Ban l (I
-Sal I corrective gene fragment (6 μg) and Sal I -Bam,H engineering pJB 2 vector DN
A (7 μg) was digested with T4 DNA ligase (about 10 years old, NEN) at 15° C. for 18 hours in salmon 9-shihun slow-strength solution. ) Add a portion (10 μl) of the 1%-John mixture to competent E, Co11 K12.
It was used to transform strain HB101. - When cultured at night, about 600 colonies were found at 100μg/-
It was obtained on a single agar plate. One colony, pJB2D5, was used for subsequent expression experiments.

Plasmid I) l 24 CDNA, 1Q Q mM
Na(J.

6mM Tris-HCfpE (8,6mM MgC
L,...BSA i Q [1 μg/- containing 11 il
Endonuclease C1a (25 units) and Sal (25 units) in 100 μl of solution J at 67°C.
I disassembled it for 4 hours and got 1 piece.

After adding 20 μm of 5× TBE loading buffer (same as above), heating to 60° C. for 5 minutes, and cooling at 25° C., the tube was directly passed through a 1% agarose del. After separation by electrophoresis, the gel was stained with 0.5 μg/ld ethidium bromide, and the bands were visualized with long wavelength U irradiation. Solid. Tar DNA (5114 bp) was electroporated onto DE81 filter paper.

Filter paper was treated with 1.5M NaCJ, 10mM Tris-
DNA was collected by incubation in H (J pH 7, 5, 1mM EDTA (200 pl)) at 67°C for 12 hours, centrifuged to remove the filter paper, and precipitated with ethanol. Washed twice with ethanol, dried in vacuo. Vector DNA
(6.7 μg) was aliquoted in 10 μm water.

(b) wsm plasmid pXY,,,I DNA of Bhm HI-8al I fragment of pXY1 (
40μg), 150mM NaCj! ,, 6 m
MTris-HCJ pH8, t5 mM 2-mercaptoethanol, 6 mM MgCj, 2 and 100
fig,/ml in Aya buffer solution containing BSA (500 μl),
At 67°C, the endonuclease Bam1HI (
cBarn HI-8al I IFN-γ gene fragment (42,5 bp) digested with 5alI (25 units) and 5alI (25 units) for 16 hours was separated by electrophoresis on 1% agarose del in TBE buffer and DE8
No. 1 was recovered by kick elution onto filter paper and ethanol precipitation. The fragment (4 μg) was then dissolved in 50 μm of water.

(C) Oligonucleotide linker (lower part of Figure 5) Two oligonucleotides were chemically synthesized by the method described above.

I A CGATCGAATGTGTTATTGTCA
G 22 base 1B TAGCTTACAC:AATAA
CAGTCCTAG 24 base oligomer rlA (4,2
5 μl, 66 ng) and rIB (2,5 μm, 6
6 ng) to 66111M Tris-HCLp)1
7.5.1Q InMMgCf2.10 mM 2-mercaptoe: 20 p Ci (r-3″) in fJ-h
p ) ATP (5,000Ci/+n n+01+
10 mCi/Amersham) and 6
Treated with polynucleotide kinase (o, 8 units, NEW) for 10 minutes at 7°C. 10 to the reaction mixture
mM ATP (5 μm), 10X buffer (1 μm)
, water (6 μm) and polynucleotide kinase (1 μm)
m, 8 units) and incubated for an additional 60 minutes at 67°C. After the reaction was completed, the mixture was heated to 70°C for 5 minutes to inactivate the enzyme and cooled to 25°C.

This reaction is illustrated in the upper left corner of FIG.

The kinase reaction mixture (25 μl) was purified by Ban HI-'
5alI DNA 7 fragments (1 μm, 5.0 mg
) and farted at 0°C. T4 DNA ligase (1μ
m, 10 units, NEN), the reaction solution was heated at 15°C to
Incubated for 6 hours. Bring this mixture to 70°0
Heat for 1 minute to denature the DNA ligase. NaC
After adding the endonuclease C1aI (10 units) and SalI (10 units), the reaction mixture was further incubated at 67°C for 90 minutes. After adding 10 μm of 5 x TBlii shellfish, the reaction was heated to 70°C for 2 minutes, cooled to 25°C and applied directly to a 7.5% polyacrylamide gel in TBE. . 'After electrophoresis, the gel was autoradiographed for 18 hours using pre-exposed Kodak X-ray film.

C1a I-Sal I fragment (446 bps
), and DNA was recovered by electroelution onto DE81 filter paper. After extraction, ethanol precipitation, recovery by centrifugation, and washing with 70% ethanol, the DNA pellet was dried in a Makabe dish.

The C1a I-Sal I fragment (50 μgz recovery was estimated to be 60%) was dissolved in 12 μm water.

This reaction is illustrated in the center of FIG. C1a', 1-
The Sal If fragment (50 μg, 12 μm) and vector DNA (2agz 5 μm) were incubated at 15′C in T4 solution (20 μm).
Religated with DNA ligase (10 units, NBN) for 18 hours. A portion of this resolution mixture (10 μm)
Competent E, Co11 K 12 strain H
B101 was transformed. Ungisu 7100a
When cultured on a L-Agar plate containing g/-,
250 colonies were obtained. Colony hybridization showed that one colony 'IJ) strongly hybridized to oligomers γ11 and γ46. This colony, pxy2, showed the desired quaternary structure by restriction enzyme mapping. This is IFN-r
(G1n4col) Contains M gene and is named pxy2.

Construction of cilasmid pCCi (Gln]1x) For the construction of cilasmid pCCi, cilasmids pXY 2 and pWT were used as illustrated in FIG. 8 and referred to below.
111 cop was used.

La) Vector and gene fragment preparation pW
Ti Icop DNA (10ag) and px
y2 DNA (10 μg) in 20 mM Tris
-HCJpH7,5, iQmMMgCJ-z, 50
mM (NH4), SO2, 100pg/ml BSA
(100 μl) at 57° C. for 4 hours with endonuclease Pstr ('i Q units).

iQmM Tris-H(lJpH7,5,10m
After adding y KDTA (200μm), decomposition D
NA was extracted once with phenol-chloroform and twice with chloroform. After recovery by ethanol precipitation, D
The NA digest was resuspended in 50 μl of water and added with buffer (100 μl).
11 (same as above) at 67° C. for a further 4 hours with endonuclease Sal r (20 units). DNA fragments were phenol-chloroform extracted and ethanol precipitated. DNA fragments were resuspended in 40 µm water, 5x TBE loading buffer, 10 µm
After adding m, the mixture was heated to 65'0 for 2 minutes and separated by 1 thiagarose gel electrophoresis in TBE'.

Fragment Pst I-Sal IIFN 7.
g gene (1614 bp) and Sal I, -P
The stI vector (2962 bp) was electroeluted onto DE81 filter paper, extracted and recovered by ethanol precipitation.

Pst I-8alI IFN-g gene fragment (1 μg) and Sal I-Pst I pWT 1
1 cop fragment (1 μg) was resuspended in 25 μm of resolution buffer. T4 DNA ligase (20
After addition of NEW, ), the reaction was incubated at 15"C! for 18 hours. 5 μl of the redundancy mixture was added to competent E.

12 strains of E.coli K. HB101 were used in the experiment.

Overnight culture on L-Agar containing 100 μg/− of ampicillin yielded 1.46 (Nml) colonies. By restriction enzyme mapping and colony hybridization, one colony was used for subsequent expression experiments.

As illustrated in Figure 2 and detailed below, plasmid p
ea 1 and pXY i 06 into plasmid pc10
Used in Article 5.4.

pCCI DNA (5 μg) was diluted with 150 mM Na (
J, 10 m1viTris-HCfpHa, o, i
QuxM Mg(42,1Q mM DTT.

Endonuclease Bam HI (25 units) in 100 μg/mBsA (100 μm) at 67°C.
and Sal I (position 7,5'I') for 2 hours.

After addition of 5 μl of 500 mM EDTA, the decomposition solution was extracted with phenol-chloroform and chloroform and collected by eta/-k precipitation. After treatment with bovine phosphatase, phenol-chloroform extraction and ethanol precipitation were performed, and the recovered DNA fragments were subjected to TBE.
Separation was performed by electrophoresis on 1% agarose gel in buffer solution.

Sal ■-Bam HI fragment of pea i (
6544 bp) was electroeluted onto DE81 filter paper, extracted from the filter paper, and recovered by precipitation with ethanol.

(b) 1AI-A pXY105 DNA (10 μg) of the M-positive gene BamHI-8al fragment was digested with endonucleases Bam E, I (50 units) and 5alI (2tJj = t1 position) in the same manner as in (a). did. This Baa+ jiI-8al+I gene fragment (
a 50 bp) was treated with phosphatase, and the 7 strands were released in the same manner as described above.

(C) Sal I-Bam HI fragment (0,4 μg) from ligation/pCC1 and I) P,a from XY 105
mI (I-Sal I fragment +1) = turbid in 20 μl of solution y*&. After adding T4 DNA ligase (90 units, NEW), add 1
Incubate for 18 hours at 5°C. Competent E, 'co
The li K 1.2 strain HB101 was exchanged into a type. Approximately 2-D when cultured overnight on L-Agar containing ampicillin 1001/ml.

A colony of individuals was observed. After creating a restriction enzyme map, one colony, pcc1o6, or the correct 44 wells, was identified and used for this elaborate expression.

1) CC2 and υ: pCC206 sofusmid pC
C105 (pCC1 to pCC4) and pWT 111 were used for reconnaissance of plasmids pCC2, pCC206, as illustrated in FIG. 10 and detailed below.

pWTillcopDNA (25 μg) was added to 100 m
M NaCj.

5 Q mM Tris-H (Jp) 17.5, i
Digested with endonuclease Bam HI (25 units) for 6 hours at 67° C. in Q mM Mg (J2 (200 μm)).

DNA fragments were recovered by phenol extraction and ethanol precipitation.

(b) Preparation of fragments Pea i DNA (20 μg) was added to the above TiK solution (
200 μm) at 67° C. for 4 hours with endonuclease 5alI (40 units). DNA fragments were recovered by phenol extraction and ethanol precipitation. pCC105 DNA (29 μg) was also processed in a colleague's reaction.

(c) Reaction pWT with T4 DNA polymerase
111 C01) A Bam HI llr piece of DNA (25 μg) was dissolved in 66 mM Tris-H (JpH7,
9,66mA KOAC, 1 [] rnMMg0AC,
0.5mM DTT, 100, l+g/-BSA, o
, ituMtiATP, 0.1 mMt, 1cTP, 0
．． 1 mMtiGTP, 0.1 mMdTTP (200
μm) Instep, T4 DNA poly/9--1 at 67°C!
'c40 units, ERL) for 1 hour. In addition, the mixture was extracted with phenol, and DN and A were extracted with ethanol If
Recovered with hN. pea i by Sal I
20 pg) and pcc 105 m'' pieces (20 μg) with Sal I were treated similarly.

pWTI 11 cap DNA (25
μg) 'i plate m1 (200 μm, same as above),
At 67°C, the endonuclease Pstll (40
It was decomposed for 4 hours according to the following criteria. Phenol extraction Shun, DN
The A fragment was recovered by ethanol precipitation. Sal
T4 DNA polymerase light JJ4pCC1 and pCCi 05 (18 μg) were treated in the same manner.
HI-Pst engineering (5267bp) and p'cc1
and pCC106Pstney Sal Engineering (1614b
p) Fragments were isolated by agitation on a 1% agarose gel in TBE slow splitting. DR8 DNA
The residue was placed in an electric bath on 16-liter soil, extracted using 4 filters, and precipitated with ethanol to be recovered.

pWT 111. c, op, Bam HI-Pst
I fragment (1.6 μg) was added to pCCl or p
ea 103 Pstney Sal I fragment in resolution grade IA solution (25 μm).
T4 DNA ligase (20°C, NEW
) for 18 hours.

A part of it (5 μ, 1) was transformed into competent E, coli
It was used for transformation of K12 strain HB101. 100μg/
m! When cultured overnight on ampicillin Miyaari L-Agar, 400 (pCC2) and 2000 cells (pcc
205) colonies were obtained. Each Tanigumi chiae body characterized by restriction enzyme mapping was used in the following second son experiment.

The starting cilasmids listed in the plasmid-transformation table have a cylasmid lineage map as exemplified in FIG. Shirasi PpCC2 and pea 205 each have AT
Deposited as CCA and ATCCA (.

Example 6 Interferon gene expression and biological activity An overnight culture of plasmid
) and grown in M9 medium containing ampicillin (100 μg/mj). Inoculum (0.5 ml)
is M9 medium (2511A', ampicillin 100μ
g/ml. Growth continued until the optical density at 670 nm reached 0.4. At this point, the β-indoleacrylic acid (10m?/mA!
x in tanol) was 20 μg/+++J. Growth continued at 67°C with vigorous stirring, and samples for quantitative and electrophoretic analysis were taken at 1 hour intervals.

Method A for quantifying interferon activity of a sample: 67
A culture solution with an optical density of 1.5 to 2.0 at 0 nm (10 to 20
TL1) (mid to late logarithmic phase) was removed and centrifuged to collect the cells. 50mTris-1101pH at 0°C
7.5. After suspending in filtration Q mM Na0Al (1 ml), lysozyme (57 W/d) was added to a final concentration of 50
ng/mA! Add this suspension to 000
and incubated for 1 hour. After adding RPM Engineering 1640 medium (IIA') and 10% fetal bovine serum, the suspension was sonicated for 24 seconds. The cell layer was removed by centrifugation and the supernatant was quantified.

Method B: In this method, cells are collected by centrifugation and immediately treated with 0.15 M Na0J, 1% Fe2.1Q mM
Same as method A except resuspension in Tris-Hea pH 8.0 (1 ml) and sonication for 3×20 seconds. The upper groove was recovered in the same manner.

A volume of cells corresponding to 0.5 densities units at 670 nm was removed from the culture medium, and the cells were collected by centrifugation. Cells were immediately treated with 6 QmM Tris-Hc
zpH6,8,0,05 Thipromophenol Delu, 5
Tiglycerol, 1% sodium dodecyl sulfate, 0.5
Resuspend in 2-mercaptoethanol and incubate at 100 °C for 6 min.
℃ and quickly frozen on dry ice. This boiling-freezing cycle was repeated 2 to 3 times to reduce the viscosity of the sample, and then the sample was boiled for 5 minutes and the Laem
5 μm was loaded onto a 15-th polyacrylamide gel prepared by the method of mli. Protein evaluation was performed by comparing the size of the engineered FN-γ band on these gels with that of total protein.

Quantitated cell extracts were monitored for their protective effects against the cytopathic effects of EMO (myocarditis) virus infection in 'VenO (African Greenosa/I/) cells in an in vitro microplate quantification system. Antiviral activity was quantified.

Results Tables 1 and 2 summarize the expression results when using the expression cilasmid containing the synthetically engineered FN-γ gene. Engineering F
The international unit of H is 2.62 based on the IFli-β standard used.
It was expressed in units of /d. All cultures were performed in 25 shake flasks, 10-20 of which were used for quantitative purposes. The antiviral activities in Tables 1 and 2 are as follows:
The figure shows the IFN virus activity in international units at an optical density of 1 at 670 nm for Atashi cells.

As a control for comparing the properties of the synthetic 1FN-γ gene,
The native engineered FN-γ gene was used in pEo 252.

The pharmaceutical composition, i.e., the human immune interferon product of the present invention, is mixed with a pharmaceutically acceptable carrier vehicle. .

Reming for suitable vehicles and their formulations.
tOn's Pharmaceutical 5cienc
es (LW.

Martin). This composition combines an effective amount of the interferon protein of the present invention with an appropriate amount of vehicle to form a pharmaceutical composition suitable for effective administration to a host.

Parenteral Administration The human immune interferons of the present invention can be administered parenterally to subjects in need of anti-tumor or anti-viral treatment and to subjects who are immunosuppressed. Doses and frequency of administration are currently the same as those used in other human interferon clinical trials. For example, approximately (
1 to 10) x 106 units, 5 if the purity is 1 or more
0x10 units/day are administered.

Dissolved in the following: If this solution is passed through a bacterial filter and the filtrate is aseptically packaged into 100 vials,
A formulation containing <5X10' units of interferon in a vial suitable for parenteral administration is obtained. This vial is preferably stored in a cool place (-20°C) until use.

Reference 1) Steward (Stewart, W, L) (
1979): The Int, erferon Sys
tem (Springer, New York)
2) Fleishmann (Fleishmann, W, R
,) IGeorgiades, ,T,A, ,0
eborne, L. L., & John80n, H.

M. (1979): Infect.
, 26:248-253゜6) Rubin (B, Y.) and Gupta (
Gupt a+S,L,) (198Q): P
roc, Natl, Acad, Sci.

U, S, A, 77:5928-5932゜4) Blulock (J, E,), Georgi
ades.

J.A., Langford, M.P. & ,To.
hnson, H.M. (1980) 2ce1. ．． I
mmun, 49: 390-394゜5) Crane, , r, L, Glasgow, L,
A.

Kern, ]lc, R. & Youngner, J.S.
(1978): J.

Natn, Cancer Engineering, 61:871
-874°6) Gray (P, W, ) and Leug.

D., W.), Penn1ca, D., Yelve.
rton, E., Najarian.

Simonsen, C., Deryck, R.
, Sherwood, P.

J., Wallac, D.M., Berger, S.L.
, , Levinson, A.

D. & Goeddel, D. V. (1982):
Nature, 295:503-508゜7)Devos(R,'),'0her
outre, H.

Taya, Y., Degrave, W., Van He
uberswyn, H., & Fiers, W. (198
2): Nucleic Ac1ds Res.

8:2487-2501゜8) Grantham (R,) + Ga
utier-, c.

& Guoy, N. (198Q): 1 Jucl
eic Ac1ds Res, .

8: 1 893-1 91 2゜9) Miyoshi (N1yoshi, K.), Arent
zen, R,.

Huarlg, T. & Takura, N. (19
8Q): Nucleic^ 'as Res,, 8
: 5507 55 1 7゜10) KJge
, N, D, ) + Greene, A, R,.

Heathcliffe, G. R., Meacock.
P, A,, 5chuch.

W., 5canlon, D.B., Atkinson,
T., O., Newton.

0, R., & Markham, A. F. (1981).
: Nature.

292 Near”56-762゜11) Mitchell (A, R,)
, 5tephen.

B., H., Er1ckeon, B., W., & Merri.
field, B, B.

(1976): Tetrahedron Lett
, , 3795-3798゜12) Reese (c, B,), 'Tit
maS, R, O, & Yau, L, (1978): T
etrahedron Lett.

2727-2730゜13) Tanaka, S. et al.: Nucl
eic Ac1dsRed, 1 1 (1983):
6° nucleotide sequence and restriction sites are shown. Numbers 1-46 above and below the gene are the oligonucleotide segments that were synthesized and finally used to assemble the complete gene.

Figure 2 shows the cloning of the synthetic γ-interferon gene duplex into plasmids 1) AT 153 and p
Construction of the expression cilasmid pJB 1 from XY i is shown.

Figure 6 shows the synthetic γ-interferon gene.
The nucleotide sequence of the gene encoding the natural amino acid sequence and the nucleotide sequence with an amino acid change (T-+G) of Lys Yu, l is G1n4□ are shown.

Figure 4 shows the construction of plasmid 1), TB203.

FN-γ encoding human FN-γ of Lye□, 1
A high copy Trp 15 vector containing the gene.

Figure 5 shows the construction of cilasmid pXY 2 from pXY i and pl, 24c.

Figure 6 shows plasmid pXY containing the IFN-γ gene encoding human γ-interferon of G1n111.
103 construction is shown.

Figure 7 shows the construction of a high copy Trp E vector, cilasmid pJB 203.

Figure 8 shows a high-copy mature engineered FN-γ vector harboring G1n11□ human FN-γ, plasmid pc.
! The construction of 01 is shown.

Figure 9 shows a high-copy engineered FN-γ vector encoding human FN-γ in L7Slll, plasmid po0.
103 construction is shown.

Figure 10 shows a high copy vector, cilasmid pac 203, substituted with the BamH-Sal fragment.
shows the construction of

FIG. 11 is a restriction enzyme map of the expression plasmid used as the starting DNA.

FIG. 12 shows the amino acid sequence of γ-interferon in which position 111 is Gln.

Agent Asamura Hajime Engraving of drawings (no *changes to the content) kuhi<t<do<H (p.

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-<s ■o <l-(ri o (J(ri <to0(ri OL) l-([l-< o([)-○■ 00 ■○ Q○ to<to< 〆] kuhi cqo Dimension Q (!:l Q (ri (!:l OUO<)-+< SJ TokkuH dimension 00 ku t< (ri O(ri Q 6 0(ri)-([<)--41 -' U (D RI0 緘 <)-<)-)-([■○<)-o (!:1 〇Q　〇〇〇〇〇〇〇〇〇〇〇〇〇〇〇〇〇〇〇〇〇〇〇〇〇〇〇〇〇
~<rq)-size"0's ■lContinued from page 1 Qlnt, CI,' Identification symbol Seiichi Osaka @ Inventor Paul Gavin Baugh United Kingdom Suriy Country Pax, High Wycombe, Sands, Heathed Road, Touchwood (No address) Procedural amendment (method) March 9, 1932 Director-General of the Japan Patent Office 1, Indication of the case Showa Patent Application No. 2 xrb<t, i No. 2, Name of the invention ← Inderful Qn 3, Relationship with the case of the person making the amendment Patent applicant 4, agent 5, date of amendment order A] First year of 1985 816th 6, Number of inventions increased by amendment 7, amendment Target drawing of

Claims (1)

Scope of Claims: (1) A composition consisting of the DNA nucleotide sequence shown in FIG. 1
DNA nucleotide sequence according to claim 1 (3) A DNA nucleotide sequence according to claim 1 operably linked with a DNA sequence capable of regulating the synthesis of human γ-interferon (4) A DNA nucleotide sequence according to claim 1 capable of regulating the synthesis of human γ-interferon DNA nucleotide sequence (5) according to claim 2, in which the DNA sequences are operably linked, consisting of the amino acid sequence shown in FIG. 12, in which L7s111 of the amino acid sequence of human γ-interferon is replaced with G1n111 Improved γ-interferon i61 pcc 2 (ATCO@No. 69,496)
and pco 203 (ATOO No. 39,494)
(7) an i-savior transformed with the plasmid according to claim 6; (8) a therapeutically effective amount of the polypeptide according to claim 5; A composition suitable for administration as a medicine (9) A method for producing human γ-interferon (11) characterized by fermenting a microorganism containing the DNA nucleotide sequence according to Claim 1. Claim 6 A method for producing human γ-interferon, which comprises fermenting a microorganism transformed with Shirasumi P as described in 2.