JPS61108397A - Production of interferon-gamma protein - Google Patents

Abstract

PURPOSE:A transformant containing DNA having a specific base sequence is cultured and the resultant solution containing IFN-gamma protein is purified using antiIFN-gamma monoclonal antibody whereby the yield of the IFN-gamma produced is improved. CONSTITUTION:A transformant containing DNA having a base sequence represented by formula I (X is TAA, TAG) is cultured and the resultant culture mixture containing interferon-gamma protein is purified using antiinterferon- gamma monoclonal antibody. the collection yield is almost 100% and the specific activity of the interferon-gamma is 1X10<7>-1X10<9>IU/mg protein.

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial Field of Application] The present invention relates to a method for producing inter7eron-r protein by genetic engineering techniques.

[Conventional technology]

Interferon (hereinafter abbreviated as IFN)-r has remarkable immunosuppressive, antiviral, and anticellular effects, and also has a synergistic effect on the activities of IFN-α and IFN-β, and also has a synergistic effect on the activities of K IFN-α, It has a higher antiproliferative effect on tumor cells than IFN-β.

Therefore, there are great expectations for the clinical effects of IFN-r as a medicine.

On the other hand, since IFNK has high species specificity, human-derived IFN must be used for human applications. However, since interferon-r is difficult to mass-produce, there is a desire for the development of a technology that can easily mass-produce highly purified IFN-r at a lower cost.

Conventionally, xFN-r&'z obtained by genetic manipulation, 14
It was composed of six amino acids (Unexamined Japanese Patent Application Publication No. 1983-1983).
IFN-r, which has 143 amino acids, which is 3 fewer amino acids than γ-γ, is superior in yield.
The present invention provides a method for producing the protein, which involves purifying a solution containing FN-γ protein using an anti-IFN-r monoclonal antibody.

(5ri cAc GAG (1: cA TAT GT
A AAA GAA GCAGAA AAG G
TT AAG AAA TAT TTT AAT GC
AGGT CAT TCA GAT GTA GCG
GAT AAT GGAA (7I' CTT TTG
TTA GGCATT TT(r AAG AATTG
GAAA GAG GAG AGT GAG AGA
AAA ATAATC, CA (1, AGOCAA A
TT (, TOTOOTTT TACTTCAAAG
TT TTT AAA AACTTT AAA GAT
GACGAG AGOATCCAA AAG A(,T
GTG GAGAce ATCAAG GAA GA
G ATCAAT (, TOGCG (I) TTT T
TCAAT AGCAACAAA AAG AAA C
GAGAT GAG 'rTG GAA leg α1m, AC
T AAT TATTCG GTA ACT GACT
TG AAT GTCCAA CGCAA GOA
ATA CAT GAA CTCATOGAA GTG
ATG GOT GAA GTG TCG CGA G
CA GCT AAAAAA GGG AAG CGA
AAA AGG AGT GAG ATGCTG T
TT CGA GGT CGA AGA GCA TO
The DNA represented by formula (I) has a signal sequence and/or ATG at its 5' end. You can also.

The DNA represented by formula (1) VC is preferably linked downstream of a promoter. As a promoter, tr
Examples include p promoter, 1&c promoter, amylase promoter, 5V4Q promoter, λ7age promoter, tBB promoter, and hybrid promoters thereof.

In the present invention, IF cells such as human peripheral blood lymphocytes, for example,
Cells that secrete N-r are cultured, and IFN-
The Sumi messenger mRNA encoding r is isolated, and single-stranded Sho4 cDNA is synthesized using, for example, reverse transcriptase, and double-stranded DNA is synthesized.
A and introduce it into a plasmid.

For example, double-stranded DNA encoding pIFN-r can be produced by transforming E. coli and isolating a cDNA-containing plasmid therefrom.

The base □ base sequence of this double-stranded DNA is
-Gilbert method (Maxam, A, and G
11bart, W, 1979゜Method8in
Knz7ilog765.499-560) to confirm the presence of the IFN-r gene.

Next, all or part of the IFN-r gene was excised from the obtained clone, and ligated downstream of an appropriate promoter and SD (Shine and Dalgarno) sequence.
This can also be introduced into a suitable host.

Examples of the promoter include those mentioned above, and examples of the host include E. coli, #mother, and Bacillus subtilis.

Examples include animal cells, and preferably Escherichia coli.

The host thus obtained is cultured in a known medium.

After culturing, the cells are collected by a known method, suspended in a buffer solution, for example, and then disrupted and centrifuged to obtain a supernatant.

IFN-r in the above supernatant can be purified using an anti-IFN-r monoclonal antibody.

Monoclonal antibodies are produced by cell fusion methods.

The cell fusion method is carried out by known means, such as by reacting proliferative cells and rhinocytes producing the desired antibody in the presence of polyethylene glycol. This method produces cells that have both sex and antibody-producing ability at the same time, and the antibody produced by these cells is a single antibody that reacts only with one antigenic determinant.

For example, myeloma cells are proliferative cells.
Antibody-producing 72 cells include 1, for example, white blood cells.

Monoclonal antibodies to IFN-r are produced using reticuloendothelial cells such as lymphocytes, peritoneal, lung, spleen, etc. cells.

The present inventors have discovered a fusion cell line that produces antibodies against IFN-γ, but the monoclonal antibodies produced by this fusion cell line are common to IFN-α, IFN-β, Escherichia coli, Bacillus subtilis, or animal serum. No reaction, IFN-
It reacts specifically to r.

A water-insoluble immobilized anti-IFN-γ antibody is obtained from the anti-IFN-r thus obtained. Examples of this method include the method of binding anti-IFN-γ to a water-insoluble carrier as described below.

]1) Copolymers of amino acids (J, Biol, ,
Chem.

236, 1970 (1961)) (2) Cellulose (Nature, 189.576
(1961)) (3) Agarose or Sephadex (Nature, 215.1491 (1967),
Nature, 245.3059 (1970) (4) Polyacrylamide (Bioahem,...
8.4074 (i966)) In the present invention, adsorption of IFN-r is carried out by combining the supernatant containing IFN-r and a water-insoluble immobilized anti-IFN-γ antibody, preferably under gentle stirring, for example, at about room temperature. A method of mixing (stirring) for about 0.5 to 2 hours, and a method of filling a column with a water-insoluble immobilized anti-IFN-r antibody and flowing the supernatant solution (preferred conditions: room temperature) are used.

The supernatant liquid may be subjected to the adsorption treatment as described above, but preferably, prior to adsorption, the supernatant liquid is desalted,
It is preferable to perform treatments such as pH adjustment (preferably PH5, 6 to 9, more preferably pfj 6 to 8), dialysis, and freeze drying.

Wash out the residue by washing with a water-insoluble immobilized anti-IFN-r antibody with an IFN solution, such as physiological saline. Elution of IFN-r is carried out with a buffer of pH 5,6-9, especially a high ionic strength buffer of pH 6-8.

The eluted (developed) IFN,r is recovered by following its activity in the eluate and is recovered in high yield as a single peak without impurities.

The IFN-r protein thus obtained preferably has the formula (
It is composed of polypeptide having the amino acid sequence shown in [1], and can be used as it is or with the addition of a suitable stabilizer, preferably by desalting and dialysis.

(N)H-Y Gln Asp Pro Tyr Va
l Lys Glu A: LaGlu Asn Leu
L7B L78 Tyr Phe Asn Ala
Gay Hls Ser Asp 'Van Aha A
sp Asn Gxy Thr LeuPhe Leu
Gay IIs Leu Lye Asn Trp
Lya G'1uIce Val Ser Phs T
yr Phe Lys Lau Pha LysAgn
Phe Lye Asp Asp Gln Ser
Ile G], n LysSer Van GLu T
hr 11e Lys Glu Asp M@t As
nMal Lye Phe Phi Aan Ser
Aan Lye Lye Lye Arg Asp As
p Phe Glu Lye Leu Thr Asn
TyrSsr Val Thr Asp Lau,
l1kJn VaIGln Arg LyaAlt 1
1e Hls Glu Leu 11e G1. nV
aIMet AlaGxu Leu Ser Pro
Ala A'la Lye Thr G17 LyaA
rg Lye Arg Ssr Gin Met Le
u Phi Arg G]7Arg Arg Aha
Ser Gxn-OH(C) [wherein, Y represents Mat j or a bond. [Furthermore, according to the present invention) the vehicle containing the IFN-r protein may contain 1 to 99 elb of other active ingredients such as IFN-α or IFN-β or lymphokines such as interleukin-2 to the substance of the present invention. You can leave it there.

〔Effect of the invention〕

The method for producing FN-r according to the present invention has a recovery rate of about 1
009! i, the specific activity of the obtained IFN-r is 1×
107~1x10! 6 things about the engineering U/ln9 protein,
This method is suitable for producing IFN-r on an industrial scale.

〔Example〕

Hereinafter, the present invention will be explained in more detail with reference to Examples, but the present invention is not limited to these Examples. In addition, the activity of IFN-r in the Examples is FL-8ind
It was measured by a vis virus CPE inhibition method.

Human peripheral blood] After culturing the prepared leukocytes at 37°C in RPMI-1640 medium containing human AB type serum for 1 to 10 hours,
PHA (Phytohemagglutinin)
was added and the culture was continued to induce IFtJ-. After 20 to 60 hours, the culture solution was centrifuged to collect the cells.
4M Gua-Schiff f-ocyanate (Gu-H8CN), 0
．． 1M2-mercaptoe fi/-#, 0. I M
Homogenized into Tris-HGIC pH 7,5). 5.7M suspension.

0, I M EDTA IICX layer City Tsukuman 60
Centrifugation was performed at Tiop- (35#OOrpm% 12°C, 17 hours). The obtained precipitate was dissolved in 6M guanidine hydrochloride CG.
a-HCI), l, QmM dithio x lytol, 10m
M EDTA (pH 7,0) suspended in K, 65°C, 3
After heating for a minute, 0.04 parts by volume of IN acetic acid and 0.5 parts by volume of ethanol were added, and the mixture was cooled with Tri-ice-ethanol. After centrifugation (10,000 rpm, 0°C, 3
0 minutes), the precipitate was washed with 70% ethanol, dried and
．． Dissolved in 01M TriB-H (l (voice 7.5)).After heating and quenching at 65°C for 3 minutes, 1l of 0.5M Na was added.
Ori: I" (CLT) - Cellulose 'Scarum Poly Prisoner R
NA was concentrated. The obtained po17(A) RNA fraction was precipitated with ethanol and then centrifuged on a 5-201 sucrose density gradient.
It was fractionated and 8 fractions were obtained (Baakman 5W28
rotar, 22. OOOrpm, 5°C, 17 hours). The RNA of each fraction was dissolved in sterile water to a volume of approximately 250 μL, heated and rapidly cooled at 65°C for 3 minutes, then injected into African frog oocytes, incubated at 25°C for 48 hours, and then homogenized. , the supernatant was extracted.

The activity of IFN-r in the obtained extract was determined by F'L.
When examined using the cell-8indbis vl and 'rata system, fraction 4 (17-20s) [IFN activity 26
5IU/- was detected.

Moreover, this IFN activity was abolished in more than 5 cases by adding anti-IFN-r antibody.

2. Preparation of cDNA The outline is shown in Figure 1.

(4) Creation of ciT-tilp2imer Pharmac
Plasmid psV71 purchased from ia-P.L.
86. was digested with Kpnl. 37 liters of the following reaction solution
After incubation for 5 minutes at °C, 200 U of terminal deoxynucleotidyl transferase (TdT) was added, and when the average length of the dT chain reached about 60±10 bases, the reaction was stopped and the dT-tisib 2 imer was recovered.

Reaction solution 1: 100mM K-force:x dilate 2mM GaCl2 (FH7,5) 0.2mM
DTT 0.25mM (L'1TP 0.5μC1α-PaTTP reaction solution 300μ!) After collecting and digesting the dT-til primer with Hpa, electrophoresis was performed on 1 thiagarose, and a fragment corresponding to about 2.7 kb was extracted with DKAE. -?- The product was collected using an electro-element filter using an oligomer (L(A) cellulose) and further purified using an oligo(L(A) cellulose column).

(2) Synthesis of cDNA First, IFN-r mRNA and dT-tail primer were mixed in reaction solution 2 below, heated at 65°C for 3 minutes, and then rapidly cooled. The remaining components were added and reacted at 37°C for 90 minutes.

Reaction solution 2: *10XRT buffer 7 2μ! 30mM DTT 2 pi (3mM)20
<6-*14d~? 2μ! (2-component) [α-”P)
acTP ~10μCIIFN-r mRNA
(12-168) 111. #Jf! (4μ9)aT-
Tail Brimer 1.55μiL, 4μl) Reverse transcriptase 2μ, g (28.8 units)◎ d
Addition of Ctil and Hlnd J [digestion] The obtained primer-cDNA-mRNA:l conjugate was reacted in the following reaction solution 3 at 37°C for 25 minutes, and dCtil (5 or 6 pieces) was added.

The resulting DNA was digested with HinaI.

Reaction solution 3 Nibry W--cDNA-mRNA Konishugate 11μ1sxTaT buffer
4μJ 1mM ac'rp
L32ttl (α-32P) acTP
~10,1iBSA (201
1f/7) 1tt
lpolycA) (0,1r!I9!/ml)
2AIPharmacia-P
psV1932 obtained from Co., Ltd., L., was digested with Patl. After keeping the following reaction solution 4 at 37°C for 5 minutes,
60U oTaT omm L, aG chain power 10bas
e The reaction is stopped when K is reached, and aG till is added to D.
NA was collected.

100mM K-force codylate 2mM CoG12 (pH 7,5) 0.2mM
DTT O, 1mM aGTP 1μC1α-32p modified GTP Pstl digested psV 1932 (~1004F) reaction solution 50μl Next, after digesting the aGtil-added DNA with Hlndl, t
, S* agarose electrophoresis was performed to recover the desired aG till-added linker DNA (approximately 0.28 kl:I).

■ Circularization and replacement of mRNA with DNA After heating the following reaction solution 5 at 65°C for 5 minutes, further VC at 42°C.
After incubating for 30 minutes and rapidly cooling on ice, reaction solution 6 was reacted at 12°C overnight, and the primer-cDN
A-mRNA consulate and linker were ligated and circularized.

10XT] 1.3” 1u7
tiG-label linker-1, mJ (7 μ1) H2O, 9
μJ Reaction solution 5 10μ110×ligation buffer” 10ttlBSA (
20mp, 4) 1μJ5
mM β-NAD 2plE
, Co11 ligase (1tryrnl)
0.6 ttlH2076, 4μJ Next, by reacting reaction solution 7 at 12°C for 1 hour!
J mRNA was replaced with (!DNAK.

Reaction solution 7: Reaction solution 5 10100AJ15
0β-NAD 0.3ttlE,
Co11 +) Curse (11119/m)
0.4 ttl DNA polymerase I
lμ! RNase HO, 5111 (
■ Reaction solution 7 after the completion of the transformation reaction, E. coli RRI
was transformed. Selection was made on an ampicillin-containing LI plate, and 5.559 transformants were obtained.

Human) IFN-γ cDNA reported by Gray et al.
Base sequence (P, W, Gray, D, W, Leung
, D., Penn1ca, E.

Yelverton, R., Najarian, C.
CoSimoC05i, R, Deryck.

Sherwood, P.J., Wallace, D.M.
, S.L., Berger, A.D.

Levinaon, and D.V., Goeddel.
(1982) Nature, 295°503-5
08), the nucleotide sequence (5'-TGCAGGTCATTCA) near the IFN-rON terminal
GA-3 was chemically synthesized.

Reaction solution 8 below was reacted at 37°C for 1 hour, and elution buffer (t, oMNaCl, 10mM Tria-HC
J (PH7,2).

1mM EDTA) 3.5 pi and 1rn9,4
' 10.5 μJ of tRNA was added. After heating and quenching at 65° C. for 3 minutes, it was applied to a NAC3-52 column (manufactured by BRL) and eluted with an elution buffer to obtain 32p-labeled oligonucleotide 9.

Reaction solution 8: T4 polynucleotide kinase 1 μl (4 units) Each of the obtained ampicillin-resistant transformants was immobilized on an S&S membrane filter.

This was hybridized with the above oligonucleotide probe. Eighteen hybridized strains were isolated by autoradiography.

Blasmi) DNA was isolated from these strains using the minipress method. As reported by Gray et al.
Various restriction enzyme cleavage sites were investigated from the base sequence of FN-r. The obtained plasmid DNA was digested with BstN, and plasmids having the expected cioobp oM fragment were selected, and two plasmids, pYslo and pYs130, were obtained.

A restriction enzyme map of the cDNA insert of pYslo is shown in FIG.

Next, the base sequence of a part of the cDNA insertion part was extracted using Maxam-
It was determined by Gilbert's method. The base sequence is shown in Figure 3.

The outline is shown in Figure 4.

IQmar oligonucleotide 5'-GTCCTGC
ATGr=e' was chemically synthesized, and the following reaction solution 9 was heated at 37°C.
The 5' end was phosphorylated by reacting for 30 minutes. Reaction liquid 9 was extracted with water-saturated phenol and chloroform to obtain an aqueous phase.

Next, the fragment of 2.1 kl) obtained by cleaving this synthetic NA and pYslo with Avail was added to the following reaction solution 1.
Connected in 0. Furthermore, the obtained DNA was combined with Pvu ■ and BamH reaction solution 9: Synthesis 10mer 1ti9C1pno) = 150pmo
11 mhd ATP 1.5 μ = 1500
pmo! 10X Kinase Buffer 8 3 μl Polynucleotide r Kinase 2 μl (8 units)
H2O22, 5μ no pYslOAval fragment (2,xkb) 5plc
~5t4) Phosphorylation synthesis IQmar 8μ
l synthesis 11mar 1μl (1μg) 1
0X11gation buffer 2plT4 ligase 1μl (3,2 units) plasmid rpYN6 containing part of the tryptophan promoter (
Patent Application No. 59-18133)
It was digested with H-engine to obtain a fragment of about 2 kb. Next, this DNA
was ligated with the above 1180 bp fragment to prepare a plasmid for expressing IFN-r.

(2) Transformation When this plasmid was used to transform E, col engineered HBIOI, 83 transformed strains were obtained.

Plasmid DNA was extracted from 60 of these using the minipretubation method, and a fragment of approximately 2.1 kb was found to be present after Aval digestion.
As a result of screening for plasmids that yielded fragments of approximately 250 bp after digestion, two clones were found to have the desired plasmid (named pYs 51) (named clones 424 and 453, respectively). ) ■ Figure 5 shows an outline of the introduction of the terminator.

) to Bgl l[linker, PL Bioc
1ac Z of pUC9 obtained from Chemicals
(beginning of the gene) was inserted into the BamH site immediately after P. The obtained plasmid) pTA2 was incubated with EcoRI.
and SmaI to obtain the larger fragment. Next, this fragment was ligated with a 900-1000 bp fragment obtained by digesting pYs51 with Hasl and EcoRI, and E. coli HBIOI was transformed with this plasmid.

Since 168 transformants were obtained, EcoRI and Hi
Four strains were selected that had a plasmid (named pYs61) that produced a fragment of approximately B o o bp upon digestion of ndlll.

5. Expression of 1FN-γ in E. coli HBIOI (
E. coliHB 101/pYS 51. (same below) was cultured in M9 medium containing glucose and casamino acids, and indole acrylic acid (IAA) was added to continue the culture. When IFN-r was extracted from the obtained bacterial cells, 100-200 IU of IFN-r was extracted per 1 d of culture solution.
γ activity was detected. At the same time, E. coli HBIO
When similar measurements were performed for I/pYs24, 5-10 IU of IFN-r activity was detected per 1 portion of the culture solution. pYS24 is a plasmid almost identical to pys51, but in the amino acid sequence of IFN-r it encodes, Cys-Tyr-Cys is present at the N-terminal Gln.
oj with three extra amino acids added, namely -Cy
By removing the N-terminal 3 amino acid sequence of s-Tyr-Cys, the production amount (yield) of IFN-γ was reduced to 10-20%.
It was thought to have doubled.

Similar measurements were performed on E. coli HB 101/pYS 61. As a control, pY
pYs31 prepared from pYs24 in the same manner as s61
E. coli HB 101/pYs 31 was used. As a result, E. coli HB101/pYs3
In E. 1, 000 to 2 (LOOOIU) of IFN-γ was produced per 1 culture solution, whereas in E.

co1iHB101/pYs61 produced 10Q000 to 50QOOIU of IFN-γ per 1M culture solution. In addition, in jar culture, LOO per culture Qld
More than QOOIU of IFN-γ was produced.

BALB/C-type Mac, 'I, K was immunized with IFN-r for 10 weeks, and after confirming that the blood antibody titer had increased, the tile cells (B cells) were collected. This cell and mouse myeloma cell X63-Ag3653 (USA, FLO
(obtained from W Company) in the presence of polyethylene glycol Luna 1000 to fuse them. Among these fused cells, IFN
IFN-r antibody-producing strains were obtained by examining cells producing antibodies against -γ using methods such as hemagglutination, enzyme immunoreaction, and neutralizing antibody reaction.

This cell line was cultured intraperitoneally in a mouse, and mouse ascites was isolated on the 7th to 10th day. This mouse ascites strongly inhibited the activity of IFN-r. This monoclonal antibody in mouse ascites was bound to GNBr-activated Sepharose (manufactured by Pharmacia) according to a conventional method to prepare a water-insoluble immobilized monoclonal antibody.

This water-insoluble immobilized monoclonal antibody 10- was packed into a column, and the supernatant of the lysate was diluted at room temperature for 50 hr. Ll/vP
The solution was allowed to flow down at a flow rate of 100 mL to adsorb IFN. Total 2
After adsorbing X108IU of FN, 0.1~1.
The column was washed with OM NaCl solution to remove impurities.

Next, 3.5M KS (J solution) was injected into the column, and IFN
-r was eluted.

rose to 300 times that of the starting material.

[Brief explanation of drawings]

Figure 1 shows the outline of the cDNA preparation method, and Figure 2 shows the pY
This is a restriction enzyme map of the cDNA inserted part of slo. The figure shows the outline of the preparation of the expression plasmid, and Figure 5 shows Termine (and 3 others), $3 figure leg ei Ava[411i AV@IL AsnTrpLysGluGluScrAspArgL
ys Ile〜IetG1nScrMTTG G AA
AG AG G AGAG TG A CAG AAA
AAT AATG CAG AG C No. 5111 Procedural Amendment December 72, 1980 Patent Application No. 229864 of 1988 2, Title of invention Method for producing interferon-r protein 3, Person making the amendment Relationship to the case: Patent application Name Midori Juji Co., Ltd. Kasumigaseki Building Post Office PO Box No. 49 Eiko Patent Office Telephone (581)-9601 (Representative) 7
, Target of amendment 1) Ministry of Specification, page 8, line 20, insert: ``pH5,9J is corrected to ``pH5,6J.'' 2) Same page, page 18, line 6, ``prob'' is corrected to ``probe.'' do. 6) In the 20th line of the 18th member, correct “oligonnucleotide” to “oligonnucleotide P”. 4) On page 19, line 6 from the bottom of the same page, amend "no-ibudaizu" to "hybridize." 5) Same page 19, line 5 from the bottom, “...Ibridice J
't-r-・Ibridize'' is corrected. 6) Same page 22, line 19, [900-1000J]
Corrected to "approximately 800". that's all

Claims (1)

[Claims] A transformant containing DNA having the base sequence represented by formula (I) is cultured, and interferon-
A method for producing interferon-gamma protein, which comprises producing and accumulating gamma protein and purifying the resulting interferon-gamma protein-containing solution using an anti-interferon-gamma monoclonal antibody. [There is a gene sequence] [In the formula, X represents TAA, TGA or TAG. ]