JPS6234760B2 - - Google Patents

Description

[Detailed description of the invention]

Technical Field The present invention relates to a method for purifying human gamma interferon. BACKGROUND ART Interferon (hereinafter sometimes abbreviated as IFN) is generally defined as a substance produced by living organisms that has antiviral effects, but it has been proven that it has many other biological activities. It has attracted particular attention because of its antitumor effects [Brad, 55 , 711 (1980); Same Journal, 55 , 875 (1980)].
Possible methods for suppressing tumor growth include direct suppression of tumor cell proliferation and indirect tumor suppression via the host's immune response.In the latter case, for example, natural killer cells (NK )
Possible causes include activation of macrophages, activation of killer T cells, etc. In fact, in addition to its direct effects, IFN has been proven to have various immune-enhancing activities.
Biophysica Acta, 516 , 231 (1978)]. γ
type interferon (hereinafter sometimes abbreviated as IFN-γ) is immune interferon (hereinafter I-
(sometimes abbreviated as IFN), the various activities that lead to these antitumor activities in vitro and the antitumor activities in vivo are similar to IFN-α and
Its importance has been strongly pointed out because it is much higher than IFN-β [Cellular Immunology,
49 , 390 (1980)]. However, in vitro induced IFN-γ
The large-scale production and purification of IFN-γ is difficult because the titer of IFN-γ is generally low, there are few suitable IFN-γ-producing cell lines, and purification is difficult due to poor stability against heat and acids. α and
It is significantly delayed compared to IFN-β. Very recently, there has been a report that a single form of natural IFN-γ has been produced [Proceedings of the National Academy of Sciences, 79 , 1820]
(1982)], recovery of the activity is very poor, and a more effective purification method is awaited. On the other hand, recently, the cloning of the human IFN-γ gene has been reported, and a molecular species of approximately 17 kilodaltons consisting of 146 amino acids, at least as a type of IFN-γ, has been produced in Escherichia coli. , 295 , 503 (1982); Nuclei Tsukuba Research, 10, 2487 (1982)], but since the purification method has not been established, there have been no reports that it has been obtained as a highly pure protein. Therefore, it has been desired to establish a purification method that can obtain IFN-γ as a substantially pure protein. DISCLOSURE OF THE INVENTION The present invention provides a polypeptide represented by the formula H-Lys-Arg-Lys-Arg-Ser-Gln-Met-Leu-Phe-Arg-Gly-Arg-Arg-Ala-Ser-Gln-OH () Human IFN-γ using a novel monoclonal antibody against human IFN-γ, especially human IFN-γ obtained by genetic recombination technology (hereinafter referred to as
(sometimes abbreviated as rIFN-γ). The present inventors have made a report on the amino acid sequence of human IFN-γ deduced from the nucleotide sequence of the structural gene [Nucletics Research
10 , 2487 (1982)], a novel polypeptide () having a partial structure on the C-terminal side of the amino acid sequence was chemically synthesized and further chemically bonded to a carrier protein to form a protein complex. A mammal was immunized with the polypeptide () or protein complex obtained here, and the removed spleen cells were fused with homologous or heterologous lymphoid cells to form a hybridoma, which was then cloned. The hybridoma thus obtained was inoculated into mammals to produce and accumulate monoclonal antibodies, which were collected to produce monoclonal antibodies against the polypeptide. Then, using the monoclonal antibody obtained here, human IFN-γ was extracted from the crude human IFN-γ-containing material.
The present invention was completed by establishing a method for purifying . That is, the present invention provides human IFN-γ, which is characterized in that a crude human IFN-γ-containing product is purified using a monoclonal antibody against a polypeptide ().
The present invention provides a purification method for According to the purification method of the present invention, by purifying a crude human rIFN-γ-containing material using a monoclonal antibody against the novel polypeptide (),
Substantially pure human rIFN-γ protein can be produced. To purify IFN-γ by the method of the present invention, the purified monoclonal antibody is coupled to a suitable carrier such as activated agarose gel beads in accordance with a conventional method, and then loaded onto a column. Culture supernatant or crushed bacterial cells, etc.
Materials containing IFN-γ are applied to a column, adsorbed, washed, and then eluted using a chaotropic reagent such as KSCN or under weakly acidic conditions that do not deactivate IFN-γ. Can be purified. An antibody column can be produced by the following method, for example, by coupling the monoclonal antibody of the present invention purified from ascites inoculated with a hybridoma to an appropriate carrier. Any carrier may be used as long as it allows IFN-γ to be specifically and efficiently adsorbed after coupling and is then eluted appropriately. Activated agarose gel beads, such as Afhuigel-10 (Bio-Rad), are conveniently used in the manner described below. The reaction between Afuigel-10 and the antibody is carried out in a buffer of 0.001 to 1M, preferably 0.1M, such as bicarbonate. The reaction conditions are 0° to 20°C, 10 minutes to 24 hours, various pH values are possible, but preferably 4°C, 4 hours, pH 3.
~10 conditions are used. Afui gel to mix
The amount ratio of 10 to antibody can be adjusted within this range, up to approximately 50 mg of antibody per 1 ml of Afui gel, as the more antibodies are attached, but it may vary depending on binding efficiency and affinity column chromatography. 10-30 mg of antibody is conveniently used considering purification efficiency. After thoroughly washing the antibody-carrier conjugate thus prepared with the buffer used in the reaction,
After blocking the remaining unreacted active groups by leaving it for several days or by adding ethanolamine/hydrochloric acid at a final concentration of 0.05M and reacting for 1 hour at 4°C, it is packed into an appropriate column. Can be used as an antibody column. When performing purification using the antibody column described above, for example, a human IFN-γ protein-containing material is dissolved in a near-neutral buffer, such as a phosphate buffer or a Tris/HCl buffer, and then adsorbed onto the antibody column. Next, after washing the column with the same buffer, IFN-γ is eluted. Examples of elution solutions used include weakly acidic solutions such as acetic acid solutions, solutions containing polyethylene glycol, solutions containing peptides that bind more easily to the antibody than the material, highly concentrated salt solutions, and solutions that combine these. It is preferable to use a substance that does not significantly promote the degradation of IFN-γ. The column eluate is neutralized with a buffer solution using a conventional method. If necessary, the purification operation using the antibody column described above can be performed again. The human IFN-γ protein solution obtained here is subjected to dialysis, and if necessary, it can be lyophilized to form a powder. Stabilizers such as sorbitol, mannitol, dextrose, maltose, and glycerol can be added during freeze-drying. Human rIFN-γ obtained by the method of the present invention
The protein exhibits a specific activity of 10 ⁷ U/mg or more in viral activity measurement using a cytopathic effect inhibition test of vesicular stomatitis virus (VSV) on human amnion-derived WISH cells. Here, U/ml (units/ml) as the IFN activity was calculated as follows. The unit's established international standard IFN-α and white blood cell-derived crude
VSV of IFN-γ against human amnion-derived FL cell line
The titer of leukocyte-derived crude IFN-γ was determined from the comparison of the titers and used as a standard product of IFN-γ. in the target material
Always use this standard for titration of IFN-γ.
IFN-γ was lined up and assayed using the WISH-VSV system described above, and the titer was calculated from the ratio. The human rIFN-γ protein to be purified in the present invention has, for example, the general formula (N)H-A-Z ₁ Tyr Z ₂ Gln Asp Pro Tyr Val Lys Glu Ala Glu Asn Leu Lys Lys Tyr Phe Asn Ala Gly His Ser Asp Val Ala Asp Asn Gly Thr Leu Phe Leu Gly Ile Leu Lys Asn Trp Lys Glu Glu Ser Asp Arg Lys Ile Met Gln Ser Gln Ile Val Ser Phe Tyr Phe Lys Leu Phe Lys Asn Phe Lys Asp Asp Gln Ser Ile Gln Lys Ser Val Glu Thr Ile Lys Glu Asp Met Asn Val Lys Phe Phe Asn Ser Asn Lys Lys Lys Arg Asp Asp Phe Glu Lys Leu Thr Asn Tyr Ser Val Thr Asp Leu Asn Val Gln Arg Lys Ala Ile His Glu Leu Ile Gln Val Met Ala Glu Leu Ser Pro Ala Ala Lys Thr Gly Lys Arg Lys Arg Ser Gln Met Leu Phe Arg Gly Arg Arg Ala Ser Gln−OH(C) () [In the formula, A represents Met or a bond, and Z ₁ and Z ₂ each represent Cys or 1/2 Cys]. According to the purification process of the present invention, polypeptides containing 90% or more, particularly 95% or more of the polypeptide on a dry basis can be obtained. By the purification method of the present invention, for example, the substantially pure human rIFN-γ protein shown below can be obtained. (1) Molecular weight measured by SDS-polyacrylamide gel (17.5%) electrophoresis shows 17000±1000. (2) Has cysteine or half-cystine or methionine as the amino terminal amino acid. (3) H-Lys-Arg-Lys-Arg-Ser-Gln-Met
−Leu−Phe−Arg−Gly−Arg−Arg−Ala−
Binds with monoclonal antibody against Ser-Gln-OH. The human IFN-γ protein purified by the present invention can be used for the same purposes and in the same way as IFN-γ obtained by conventional methods, but it contains fewer contaminant proteins and pyrogens than conventional products, so it can be injected. It is safer to use as a drug substance, etc. The human IFN-γ protein produced according to the present invention exhibits antiviral, antitumor, cell proliferation suppressive, and immunoenhancing effects. Human IFN produced according to the present invention
-γ protein can be mixed with sterile water, human serum albumin (HSA), physiological saline, or other known physiologically acceptable carriers, and can be administered parenterally or topically. For example, 100,000 to 100 million units, preferably 50 million to 60 million units, can be administered by intravenous or intramuscular injection per adult. Formulations containing the human IFN-γ protein of the present invention may also contain other physiologically acceptable active ingredients such as salts, diluents, adjuvants, other carriers, buffers, binders, surfactants, and preservatives. May contain. Preparations for parenteral administration may be prepared in ampoules of sterile aqueous solution or suspension in physiologically acceptable solvents, or as sterile powders (usually IFN-γ solutions) which can be diluted for each day with physiologically acceptable diluents. (obtained by freeze-drying) is provided as an ampoule. Furthermore, the above-mentioned preparations containing human IFN-γ protein may contain 1 to 99% of other active ingredients such as IFN-α or IFN-β or lymphokines such as interleukin-2, based on the substance of the present invention. Good too. A monoclonal antibody against the above-mentioned novel polypeptide () can be produced by, for example, immunizing a mammal with the polypeptide () or its protein complex, making a hybridoma by cell fusion of the removed spleen cells, cloning this, and then injecting it into a mammal. The monoclonal antibody can be produced by inoculation, producing and accumulating monoclonal antibodies, and collecting the monoclonal antibodies. The polypeptide () can be produced by conventional methods of peptide synthesis. Either a solid phase synthesis method or a liquid phase synthesis method may be used, but the liquid phase synthesis method is often advantageous. The means of synthesis of such peptides is
For example, “The Peptides” Volume 1 (1966), Schleder and Lubke, Academic Press, New York, USA or “Peptide Synthesis”;
Izumiya et al., Maruzen Co., Ltd. (1975), or "Biochemistry Experiment Course, Vol. 1, pp. 207-400" by Haruaki Yajima, Tokyo Kagaku Dojin Co., Ltd. (1977), for example: Azide method, chloride method, acid anhydride method, mixed acid anhydride method, DCC method, active ester method, method using Woodward's reagent K, carbodiimidazole method, redox method, DCC/additive (e.g. HONB, HOBt, HOSu) law, etc. The polypeptide () consists of (a) a raw material having a reactive carboxyl group corresponding to a part of the polypeptide, both of which may be protected, and (b) a reactive amino acid corresponding to the remainder of the polypeptide (). When raw materials having a group are condensed using a conventional method for peptide synthesis, and the resulting condensate has a protecting group, it can be produced by removing the protective group using a conventional method. Protection of functional groups that should not participate in the reaction of raw materials, protective groups, removal of the protective groups, activation of functional groups that participate in the reaction, etc. can also be appropriately selected from known methods or methods. Examples of the protecting group for the amino group of the raw material include carbobenzoxy, t-butyloxycarbonyl,
t-amyloxycarbonyl, isobornyloxycarbonyl, p-methoxybenzyloxycarbonyl, 2-chlorobenzyloxycarbonyl, adamantyloxycarbonyl, trifluoroacetyl, phthalyl, formin, o-nitrophenylsulfenyl, diphenylphosph Examples include inothioyl and 4-methoxy-2,3,6-trimethylbenzenesulfonyl. Examples of carboxyl protecting groups include alkyl esters (e.g. methyl, ethyl, propyl, butyl, t
-butyl etc.), benzyl ester group, p-nitrobenzyl ester group, p-methoxybenzyl ester group, p-chlorobenzyl ester group, benzhydryl ester group, carbobenzoxyhydrazide group, t-butyloxycarbonylhydrazide group group, trityl hydrazide group, etc. As a protecting group for the guanidino group of arginine,
Examples include nitro group, tosyl group, p-methoxybenzenesulfonyl group, carbobenzoxy, isobornyloxycarbonyl, adamantyloxycarbonyl, 4-methoxy-2,6-dimethylbenzenesulfonyl group, pentamethylbenzenesulfonyl group, etc. Ru. The guanidino group may also be protected in the form of an acid (eg, benzenesulfonic acid, toluenesulfonic acid, hydrochloric acid, sulfuric acid, etc.) salt. The hydroxyl groups of threonine and serine can be protected, for example, by esterification or etherification. Examples of groups suitable for this esterification include lower alkanoyl groups such as acetyl groups,
Examples include aroyl groups such as benzoyl groups, groups derived from carbonic acid such as benzyloxycarbonyl groups, and ethyloxycarbonyl groups. Further, examples of groups suitable for etherification include benzyl group, tetrahydropyranyl group, and t-butyl group. However, the hydroxyl group of threonine does not necessarily need to be protected. Methionine may be protected in the form of sulfoxide. Examples of raw materials with activated carboxyl groups include corresponding acid anhydrides, azides, active esters (bentachlorophenol, p-nitrophenol,
N-hydroxysuccinimide, N-hydroxybenztriazole, N-hydroxy-5
-esters with norbornene-2,3-dicarboximide, etc.). The peptide bond-forming reaction may be carried out in the presence of a dehydrating agent (eg, a carbodiimide reagent such as dicyclohexylcarbodiimide, carbodiimidazole, etc.). This peptide condensation reaction can be performed in the presence of a solvent. The solvent can be appropriately selected from those known to be usable in peptide condensation reactions. For example, anhydrous or hydrous dimethylformamide, dimethyl sulfoxide, pyridine,
Examples include chloroform, dioxane, dichloromethane, tetrahydrofuran, ethyl acetate, N-methylpyrrolidone, and appropriate mixtures thereof. The reaction temperature is appropriately selected from the range known to be usable for peptide bond forming reactions, usually about -40°C to about 60°C, preferably about -20°C to about 0°C.
It is appropriately selected from the range of °C. After the completion of the condensation reaction, if the product has a protecting group, it can be removed by a conventional method. Such conventional methods include, for example, reductive methods (e.g. hydrogenation using catalysts such as palladium black, reduction with metallic sodium in liquid ammonia), acidolysis (e.g. trifluoroacetic acid, hydrogen fluoride, methanesulfonic acid or Examples include acidolysis using the above acids or a mixture thereof in the presence of a sulfur-containing compound such as anisole. Peptide produced as above ()
can be collected from the reaction mixture by conventional peptide separation means, extraction, partitioning, column chromatography, etc. Regarding the protein complex of polypeptide () and carrier protein used to immunize mammals, the type of carrier protein and the mixing ratio of the carrier and hapten (in this case, peptide) are determined by the hapten immunized by coupling with the carrier. Any substance may be coupled in any ratio as long as an antibody can be efficiently produced against the hapten. ~
A method of coupling at a ratio of 5 is used. Further, various condensing agents can be used for coupling the hapten and the carrier, and glutaraldehyde, carbodiimide, etc. are conveniently used. When immunizing with a polypeptide () or protein complex, the mammals to be immunized are experimental animals such as sheep, goats, rabbits, guinea pigs, rats, and mice. , a mouse is preferred. The immunization method is
For example, when immunizing mice, it is possible to immunize mice by any route such as subcutaneous, intraperitoneal, intravenous, intramuscular, or intradermal, but it is preferable to inject subcutaneously, intraperitoneally, or intravenously (especially subcutaneously). . Also,
The immunization interval, immunization dose, etc. are also highly variable, and various methods are possible.
A method using post-spleen cells is often used.
The immunization dose is the amount of peptide at one time, per mouse.
It is desirable to use 0.1 μg or more, preferably 10 μg to 300 μg. Furthermore, before removing the spleen, it is desirable to perform a partial blood sampling, confirm an increase in antibody titer in the blood, and then perform a fusion experiment using spleen cells. The above-mentioned cell fusion between spleen cells and lymphoid cells can be carried out, for example, by converting isolated mouse spleen cells into cells with hypoxanthine-guanine-phosphoribosyltransferase deficiency (HGPRT ^- ) or thymidine kinase deficiency (TK ^- ). The cell line is fused with an appropriate allogeneic or xenogeneic (preferably allogeneic) myeloma or other lymphoid cell line containing a marker. For fusion, a fusion agent such as Sendai virus or polyethylene glycol (PEG) is used. Of course, dimethyl sulfoxide (DMSO) and other fusion promoters can also be added. The polymerization degree of PEG is usually 1000 to 6000, the time is 0.5 to 30 minutes, and the concentration is 10% to 80%. As an example of preferable conditions, PEG6000 is treated at 35 to 55% for 4 to 10 minutes. This allows efficient fusion. The fused cells were grown in hypoxanthine-aminopterin-thymidine medium [HAT medium; Nature;
256 , 495 (1975)]. The culture supernatant of the proliferated cells can be screened to determine whether the desired antibody is produced or not, and the antibody titer can be screened as follows. That is, in this case,
First, as a first step, the presence or absence of antibody production against the immunized peptide can be examined using a method such as RIA or EIA, but various modifications of these methods are also possible. As an example of a preferred measurement method, one method using EIA will be described. For example, a rabbit anti-mouse immunoglobulin antibody is coupled to a carrier such as cellulose beads according to a conventional method, and the culture supernatant to be measured or mouse serum is added to this and kept at a constant temperature (hereinafter referred to as 4-40℃) for a certain period of time. ). After this, the reaction product is thoroughly washed, and an enzyme-labeled peptide (purified after coupling the enzyme and peptide according to a conventional method) is added, and the reaction is allowed to occur for a certain period of time at a constant temperature. After washing the reaction mixture thoroughly, the enzyme substrate was added and incubated for a certain period of time.
After the reaction is carried out at a constant temperature, the colored product produced can be measured by absorbance or fluorescence. It is desirable to clone cells from wells that have grown in the selective medium and have antibody activity against the peptide used for immunization using a limiting dilution method or the like. By similarly screening the supernatant of the cloned cells and increasing the number of cells in wells with high antibody titers, monoclonal antibody-producing hybridoma clones that exhibit reactivity with the immunized peptide can be obtained. Next, it is necessary to examine whether the antibodies produced by these clones show reactivity not only with the peptide used for immunization but also with the IFN-γ molecule itself. A method of examining reactivity using the RIA method or EIA method, a method of determining whether biological activity (IFN-γ activity) is absorbed by the antibody, etc. are used. The latter case can be advantageously used since it is not necessary to purify IFN-γ. An advantageously used example will be described below, but of course other methods are also possible, such as measuring residual IFN-γ activity in the supernatant after removing the immunoprecipitate using protein A. For example, a rabbit anti-mouse immunoglobulin antibody is coupled to a carrier such as cellulose beads according to a conventional method,
The hybridoma supernatant or mouse serum to be measured is added to this, and the mixture is allowed to react at a constant temperature for a certain period of time.
After this, the reaction mixture is thoroughly washed and a certain amount of IFN-γ is added. For example, IFN-γ was induced from human peripheral blood lymphocytes with lectin and phorbol ester.
A culture supernatant containing IFN-γ or an extract containing recombinant IFN-γ produced using Escherichia coli or the like can be used. After adding IFN-γ and reacting at constant temperature for a certain period of time, the IFN-γ contained in the reaction supernatant
Measure γ activity. In this manner, the ability of the antibody of interest to absorb IFN-γ activity can be measured. Hybridomas thus cloned are grown in liquid culture or intraperitoneally in mammals. Specifically, for example, a liquid medium such as
The monoclonal antibody can be obtained from the culture solution by culturing it for 2 to 10 days, preferably 3 to 5 days, in a medium containing RPMI-1640 and 0.1 to 40% bovine serum. By inoculating the cells intraperitoneally into an appropriate mammal such as the following, proliferating the cells, and collecting ascites fluid, it is possible to efficiently obtain a large amount of antibodies with a much higher titer than that obtained from cell culture supernatants. For this, for example, in the case of a mouse,
BALB/c pre-inoculated with mineral oil etc.
1×10 ⁴ to 1×10 ⁷ , preferably 5
×10 ⁵ to 2 × 10 ⁶ hybridomas are inoculated intraperitoneally, and ascites fluid etc. are collected 7 to 20 days later, preferably 10 to 14 days later. Antibodies produced and accumulated in ascites are
For example, monoclonal antibodies can be easily isolated as pure immunoglobulin by ammonium sulfate fractionation, DEAE-cellulose column chromatography, etc. The above monoclonal antibody has the following properties. (1) Binds to the polypeptide () used for immunization. (2) Binds to IFN-γ molecules, producing IFN-α and IFN-β
It does not combine with (3) The antibody belongs to the IgG2b or IgG1 subclass as determined by Ouchterlony's method. (4) SDS-polyacrylamide gel electrophoresis shows only two bands that completely match the H and L chains of standard immunoglobulin. This monoclonal antibody can be safely produced, used, and stored. In addition, the human of the present invention
It can be advantageously used for producing antibody columns for rIFN-γ purification. In this specification, when amino acids, peptides, protective groups, active groups, etc. are indicated by abbreviations,
They are IUPAC-IUB (Commission on
Biological Nomenclature) or common abbreviations in the field,
Here are some examples: Furthermore, when an amino acid or the like can have optical isomers, the L-isomer is indicated unless otherwise specified. DNA: Deoxyribonucleic acid A: Adenine T: Thymine G: Guanine C: Cytosine RNA: Ribonucleic acid dATP: Deoxyadenosine triphosphate dTTP: Deoxythymidine triphosphate dGTP: Deoxyguanosine triphosphate dCTP: Deoxycytidine triphosphate ATP: Adenosine triphosphate EDTA: Ethylenediaminetetraacetic acid SDS: Sodium dodecyl sulfate Gly: Glycine Ala: Alanine Val: Valine Leu: Leucine Ile: Isoleucine Ser: Serine Thr: Threonine Cys: Cysteine Met: Methionine Glu: Glutamate Asp: Aspartate Lys: Lysine Arg: Arginine His: Histidine Phe: Phenylalanine Tyr: Tyrosine Trp: Tryptophan Pro: Proline, Asn: Asparagine Gln: Glutamine Z: Carbobenzoxy Boc: t-Butoxycarbonyl Mtr: 4-Methoxy-2, 3, 6 -trimethylbenzenesulfonyl Pme: pentamethylbenzenesulfonyl OBu: t-butyl ester ONB: N-hydroxy-5-norbornene-
2,3-dicarboximide ester DCC: N・N′-dicyclohexycarbodiimide DCU: N・N′-dicyclohexylurea HONB: N-hydroxy-5-norbornene-
2,3-Dicarboximide HOBt: N-Hydroxybenzotriazole CHA: Cyclohexylamine DCHA: Dicyclohexylamine TEA: Triethylamine TFA: Trifluoroacetic acid MSA: Methanesulfonic acid THF: Tetrahydrofuran DMF: Dimethylformamide MeOH: Methanol AcOEt: Ethyl acetate Below The present invention will be explained in more detail with reference to Examples and Reference Examples, but the present invention is not limited thereto. In addition, mouse B hybridoma γ2-11.1 and mouse B disclosed in Reference Examples 2 and 3 below
Hybridoma γ3-11.1 has been deposited with the CNCM of the Institut Pasteur [Paris, France] under deposit numbers No. I-242 and No. I-243, respectively. In addition, the strains disclosed in Reference Example 3 (2)-(iv) below
E.coli 294/PHITtrp2101 was given the accession number to the Microbial Research Institute (FRI), Agency of Industrial Science and Technology, Ministry of International Trade and Industry.
It has been deposited as FERM P-7550. Example Purification of rIFN-γ A Production of antibody column 25 ml (65.3 mg) of the monoclonal antibody (γ2-11.1 monoclonal antibody) of the flow-through fraction purified by the method described in Reference Example 1B (6) was dissolved in 0.1 M NaH.
Dialysis was performed overnight against a CO ₃ , PH 8.3 solution.
On the other hand, Afuigel-10 (Bio-Rad) 25ml
After thorough washing with water using a glass filter, the suspension was suspended in a 0.1M NaHCO ₃ PH8.3 solution, mixed with the antibody, and reacted at 4°C for 4 hours with gentle stirring. After that, after leaving it at 4℃ overnight, 0.1M
Washed thoroughly with NaHCO ₃ PH8.3 solution. Add 0.1M ethanolamine and 0.15M NaCl to the reacted gel.
25 ml of a solution containing (PH8.0) was added and shaken at 4°C for 1 hour to block any unreacted active groups that may remain. The gel was then thoroughly washed with PBS and suspended in 25 ml of PBS containing 0.1% _NaN3 .
Stored at 4°C. From the amount of antibody added and the amount of antibody in the recovered solution, it was found that 2.35 mg of antibody was bound per ml of gel. The reaction product thus obtained was filled into a column and used as an antibody column. B Purification of rIFN-γ using an antibody column 60 ml of crude rIFN-γ-containing supernatant obtained in Reference Example 2 (4)
1 m MEDTA, 20mM containing 0.15M NaCl
After diluting to 150 ml with Tris.HCl, PH7.6 (TEN), it was applied to the anti-IFN-γ antibody column (9 ml) prepared by method A above. After thorough washing with TEN, the plate was further washed with TEN containing 0.01% Nonidet P-40 (manufactured by Ciel) and 0.5M NaCl. Then, IFN-
Elute γ, and the eluate is immediately diluted with 1M Tris-HCl.
Neutralized at PH7.6. The resulting solution was dialyzed against distilled water at 4°C for 16 hours, frozen with acetone-dry ice, and then freeze-dried to form a powder. The results were as follows.

[Table] The specific activity of the final human rIFN-γ protein obtained here (as determined by virus activity measurement by the cytopathic effect inhibition test of VSV on WISH cells (described above)) was 7.5 × 10 ⁷ U/mg. It was hot. The properties of this protein were as follows. Properties of human rIFN-γ protein (i) Molecular weight The protein obtained in step B above was treated with 2-mercaptoethanol and subjected to SDS-polyacrylamide gel (17.5%) electrophoresis (15 mA, 6 hours), and Coomassie blue. When stained, the protein was confirmed as a single band (first
figure). Furthermore, the molecular weight of the protein was estimated to be 17,000 ± 1,000 from the relationship between the migration distance of the molecular weight marker and the migration distance of the protein, which were electrophoresed at the same time.
figure). On the other hand, in the protein that was not treated with 2-mercaptoethanol, another band was detected at a molecular weight of 33,000±2,000. This value is
Since this corresponds to about twice the molecular weight of IFN-γ, 17,000±1,000, this band is considered to be derived from an IFN-γ dimer. (ii) Amino acid analysis Place the protein obtained in B above in a glass hydrolysis test tube, add 200 times the volume (V/W) of constant boiling point hydrochloric acid containing 4% thioglycolic acid, and seal the tube under reduced pressure. After that, it was hydrolyzed at 110°C for 24, 48, and 72 hours. After hydrolysis, the tube was opened, hydrochloric acid was removed under reduced pressure, the residue was dissolved in 0.02N hydrochloric acid, and amino acid analysis was performed using a Hitachi model 835 high-speed amino acid analyzer. Cystine and cysteine were determined using the Haas method [Methods of Enzymology 11 , 197
(1967)], the protein was oxidized with performic acid, then hydrolyzed for 24 hours in the same manner as above, and quantified as cysteic acid using an amino acid analyzer. Amino acid analysis values were determined by averaging the values obtained after 24, 48, and 72 hours of hydrolysis. However, the values of serine, threonine, tyrosine, and tryptophan were determined by extrapolating the hydrolysis time to 0 hours. The results are shown in Table 1.

【table】

[Table] (iii) Amino-terminal amino acid analysis The protein obtained in step B above was analyzed using the Haas method [Methods of Enzymology 11 , 197]
(1967)], followed by performic acid oxidation according to a modified method of the Edman decomposition method by Iwanaga et al.
Journal of Biochemistry, 8 ,
189 (1969)], its amino-terminal amino acid analysis was performed. The generated phenylthiohydantoin-amino acid (PTH-amino acid) was purified using an Ultrasphere ODS column [manufactured by Artex (USA), 4.6 x 250 mm, particle size 5 μm] using the method of Alchier [Artex Chromatogram, 3 , 8 ( 1980)], Varian high performance liquid chromatograph model 5040 (USA)
It was identified and quantified. As a result, PTH-methionine sulfone and PTH-cysteic acid were detected. As is clear from the above examples, according to the purification method of the present invention, a substantially pure human γ-interferon protein having a specific activity of 7.5×10 ⁷ U/mg or more can be produced. Reference Example 1 Monoclonal Antibody Production A Immunogen Production and Immunization In the following Reference Example 1A(1), thin layer chromatography was performed using a Merck silica gel plate.
60F ₂₅₄ or cellulose plate manufactured by Funakoshi Pharmaceutical Co., Ltd., Avicel SF was used, and the following developing solvent was used. Rf ¹ : Chloroform: Methanol: Acetic acid = 9:
1:0.5 Rf ² : Ethyl acetate: Pyridine: Acetic acid: Water = 30:
10:3:5 Rf ³ :Chloroform:methanol:water=7:
3:0.5 Rf ⁴ :n-butanol:pyridine:acetic acid:water=
30:20:6:24 Rf ⁵ : Ethyl acetate: n-butanol: acetic acid: water = 1:1:1:1 (1) Production of polypeptide (i) Production of Z-Ser-Gln-OBu ^t Z-Gln-OBu ^t 10.1g methanol 500
ml, and the palladium black was reduced to the catalyst in a hydrogen stream. After filtering off the catalyst and distilling off the solvent, 7.5 g of Z-Ser-OH was added to the residue.
It was dissolved in 250 ml of DMF along with 6.8 g of HONB and cooled on ice. 7.15 g of DCC was added under ice-cooling, and the mixture was stirred at 0° C. for 4 hours and at room temperature for 12 hours.
The precipitated DCU was filtered off, the solvent was distilled off, and the residue was extracted into 300 ml of AcOEt.
Washed with _NaHCO3 water, 0.2N hydrochloric acid, water, and dried over anhydrous sodium sulfate. Distill the solvent,
The precipitated crystals were filtered with ether, dried, and then recrystallized from CH ₃ CN. Yield 6.5g,
Yield 51.2%, mp.97-100℃, [α] ^25D _- 24.1
° (c=0.40, methanol), Rf ¹ 0.64 Elemental analysis As C ₂₀ H ₂₉ O ₇ N ₃ Calculated value: C, 56.72; H, 6.90; N, 9.92 Experimental value: C, 56.21; H, 6.72; N, 9.76 (ii) Production of Z-Ala-Ser-Gln-OBu ^t 4.23 g of Z-Ser-Gln-OBu ^t was dissolved in 300 ml of methanol and reduced in a hydrogen stream using palladium black as a catalyst. The catalyst was filtered off and the solvent was distilled off, and the residue was Z-Ala-OH2.34
g, HONB2.27g, AcOEt200ml
was dissolved in a mixed solvent of 200 ml of dioxane and 100 ml of DMF, and cooled on ice. DCC2.38 under cooling
After stirring at 0° C. for 4 hours and at room temperature for 12 hours, DCU was filtered off and the solvent was distilled off. CH ₃ CN and ether were added to the residue to obtain crystals, which were collected by filtration, dried, and then recrystallized from CH ₃ CN. Yield 3.72g, yield 75.3%, mp.165
-170℃, [α] ²⁵ _D -41.2゜ (c=0.50, methanol), Rf ¹ 0.60 Elemental analysis Calculated value as C ₂₃ H ₃₄ O ₈ N ₄ :
C, 55.86; H, 6.93; N, 11.33 Experimental value:
C, 55.58; H, 6.74; N, 11.12 (iii) Z-Arg(Pme)-Ala-Ser-Gln-
Production of ^OBut 3.46 g of Z-Ala-Ser-Gln-OBu ^t was dissolved in 300 ml of methanol and reduced in a hydrogen stream using palladium black as a catalyst. The catalyst was filtered off and the solvent was distilled off, and the residue was purified by Z-Arg.
Z prepared from 4.54 g of (Pme)-OH・CHA
-A-g(Pme)-OH and 1.9 g of HOBt were dissolved in 150 ml of DMF and cooled on ice. under cold ice
Add 1.9g of DCC and store at 0℃ for 4 hours, then at room temperature.
Stirred for 15 hours. DCU was removed by filtration, the solvent was distilled off, and AcOEt was added to the residue to obtain a gel-like precipitate, which was collected by filtration. After drying, methanol and
It was reprecipitated from AcOEt. Yield 4.55g, yield
75.5%, mp.130-134℃, [α] ²⁵ _D -24.1゜ (c=0.26, methanol), Rf ¹ 0.57 Elemental analysis As C ₄₀ H ₆₀ O ₁₁ N ₈ S・1/2H ₂ O Calculated value: C, 55.22; H, 7.07;
N, 12.88; S, 3.69 Experimental value: C, 55.23; H, 6.93;
N, 12.54; S, 3.48 (iv) Z−Arg(Pme)−Arg(Pme)−Ala−
Production of Ser−Gln−OBu ^t Z−Arg(Pme)−Ala−Ser−Gln−
Dissolve 4.3g of OBu ^t in 400ml of methanol,
Palladium black was reduced to a catalyst in a hydrogen stream. The catalyst was filtered off and the solvent was distilled off, and the residue was used as Z-Arg(Pme)-OH prepared from 3.24 g of Z-Arg(Pme)-OH.
It was dissolved in 200 ml of DMF along with 1.42 g of HOBt and cooled on ice. Add 1.41g of DCC while cooling to 0°C.
The mixture was stirred at room temperature for 4 hours and at room temperature for 20 hours. DCU
was removed by filtration and the solvent was distilled off.
AcOEt was added to obtain a precipitate, which was collected by filtration. After drying, it was reprecipitated from methanol and AcOEt. Yield 4.4g, yield 71.7%, mp.125-130
°C, [α] ²⁵ _D -18.0° (c = 0.40, methanol), Rf ¹ 0.63 Elemental analysis C ₅₇ H ₈₆ O ₁₄ N ₁₂ S ₂ · H ₂ O Calculated value: C, 54.96; H, 7.12;
N, 13.50; S, 5.15 Experimental value: C, 54.66; H, 6.92;
N, 13.32; S, 5.34 (v) Production of Z-Arg(Pme)-Gly-OBu ^t 13.0 g of Z-Gly-OBu ^t was dissolved in 500 ml of MeOH and catalytically reduced in a hydrogen stream using Pd black as a catalyst. The catalyst was separated, the liquid was concentrated under reduced pressure, and the residue was dissolved in 200 ml of DMF. To this, Z−Arg(Pme)−OH・
Z-Arg (Pme) prepared from 20.0g of CHA
- Add 5.4 g of OH and HOBt and cool on ice.
8.2 g of DCC was added and stirred for 48 hours. After separating and concentrating the precipitated DCU, AcOEt500
It was dissolved into ml. AcOEt layer with 4% _NaHCO3
After washing with water and 10% citric acid water, it was dried over Na ₂ SO ₄ . After concentration, petroleum benzine was added to obtain a precipitate. Yield 19.8g (96.8
%). mp.71-73℃, [α] ^23D _+0.2゜(c=
0.9, DMF), Rf ¹ 0.62 Elemental analysis as C ₃₁ H ₄₅ O ₇ N ₅ S Calculated value: C, 58.93; H, 7.18;
N.11.09; S, 5.08 Experimental value: C, 59.42; H, 7.58;
N, 10.95; S, 4.84 (vi) Production of Z-Phe-Arg(Pme)-Gly-OBu ^t 10.0g of Z-Arg(Pme)-Gly-OBu ^t
After catalytic reduction in 500ml of MeOH,
Transferred to 300ml of DMF. Z-Phe- to this
Add 4.72g of OH and 2.35g of HOBt and cool on ice.
3.59 g of DCC was added and stirred for 15 hours.
After separating the precipitated DCU and concentrating the liquid,
The residue was dissolved in 400 ml of AcOEt. AcOEt
The layer was washed with 4% _{aqueous NaHCO3} , 10% aqueous citric acid, and dried over _Na2SO4 . After concentration, add ether to collect crystals, MeOH
- Recrystallized from ether. Yield 10.5g
(85.3%). mp.101-103℃, [α] ²⁶ _D -8.3゜ (c=0.9, DMF), Rf ¹ 0.64 Elemental analysis As C ₄₀ H ₅₄ O ₈ N ₆ S Calculated value: C, 61.67; H, 6.99;
N, 10.79; S, 4.12 Experimental value: C, 61.66; H, 6.56;
N, 10.93; S, 4.14 (vii) Z−Leu−Phe−Arg(Pme)−Gly−
Production of OBu ^t Z−Phe−Arg(Pme)−Gly−OBu ^t 5.5
After catalytic reduction of g in 300 ml of MeOH,
Transferred to 300 ml of DMF. To this, Z-Leu
-OH Z-Leu prepared from 3.31g DCHA
- Add 1.47g of OH and HONB and cool on ice.
1.68g of DCC was added and stirred for 48 hours. After separating the precipitated DCU and concentrating it, AcOEt300
It was dissolved into ml. AcOEt layer is 4% _NaHCO3
Washed _with water, 10% citric acid, and dried over _Na2SO4 . After concentration, ether was added to obtain a powder. Yield 6.2g (98.3%)
mp.171−173℃, [α] ²⁶ _D −15.5゜(c=
0.9, DMF), Rf ¹ 0.64 Elemental analysis C ₄₆ H ₆₅ O ₉ N ₇ S Calculated value: C, 61.93; H, 7.34;
N, 10.99; S, 3.59 Experimental value: C, 62.02; H, 7.37;
N, 11.08; S, 3.59 (viii) Boc−Met−Leu−Phe−Arg(Pme)−
Production of Gly−OBu ^t Z−Leu−Phe−Arg(Pme)−Gly−
After catalytic reduction of 6.0 g of ^OBut in 200 ml of MeOH, the mixture was transferred to 150 ml of DMF. to this,
Boc-Met-OH prepared from 3.0 g of Boc-Met-OH/DCHA and 1.39 g of HONB were added and cooled on ice, and 1.59 g of DCC was added and stirred for 15 hours. Take the precipitated DCU and concentrate it,
It was dissolved in n-BuOH-AcOEt, washed with 10% citric acid water, and dried over Na ₂ SO ₄ . After concentration, ether was added to obtain crystals. Yield: 6.2g (93.1%). mp.192−195
°C, [α] ²⁶ _D -19.7° (c=1.0, DMF),
Rf ¹ 0.64 Elemental analysis C ₄₈ H ₇₆ O ₁₀ N ₈ S ₂ Calculated value: C, 58.27; H, 7.74;
N, 11.33; S, 6.48 Experimental value: C, 58.48; H, 7.79;
N, 11.34; S, 5.98 (ix) Boc−Gln−Met−Leu−Phe−Arg
Production of (Pme)-Gly-OH Boc-Met-Leu-Phe-Arg(Pme)-
50 ml of TFA was added to 5.5 g of Gly-OBu ^t and the mixture was shaken at room temperature for 10 minutes, concentrated, ether was added, and the mixture was dried. this,
The mixture was dissolved in 50 ml of DMF, cooled on ice, and 1.8 ml of TEA was added. To this, 1.85g of Boc-Gln-OH,
Prepared from 1.49g of HONB and 1.90g of DCC
Boc-Gln-ONB was added and stirred for 15 hours. After concentration, AcOH was added and AcOEt was added to obtain a precipitate. Yield 5.3g (89.8
%). mp.181-183℃ (decomposition), [α] ²⁶ _D −
19.6゜ (c=1.0, DMF), Rf ¹ 0.30 Elemental analysis C ₄₉ H ₇₆ O ₁₂ N ₁₀ S ₂ Calculated value: C, 55.45; H, 7.22;
N, 13.20; S, 6.04 Experimental value: C, 55.39; H, 7.17;
N, 13.34; S, 6.20 (x) Production of Z-Ser-NHNH-Boc Dissolve 10.0 g of Z-Ser-OH and 6.2 g of t-butylcarbazate in 150 ml of DMF, cool on ice, and dissolve 8.0 g of HONB and 9.0 g of DCC. Added 3g and stirred for 15 hours. Take the precipitated DCU and
After concentration, it was transferred to AcOEt. The AcOEt layer was washed with 4% _NaHCO3 water, 10% citric acid water, and dried _{with Na2SO4} . After concentration, ether was added to obtain crystals. yield
7.3g (50.4%). mp.95-98℃, [α] ²⁶ _D −
6.2゜ (c=0.8, DMF), Rf ¹ 0.62 Elemental analysis Calculated value as C ₁₆ H ₂₃ O ₆ N ₃ :
C, 54.38; H, 6.56; N, 11.89 Experimental value:
C, 54.77; H, 6.88; N, 12.29 () Z-Arg(Pme)-Ser-NHNH-
Production of Boc 3.9g of Z-Ser-NHNH-Boc in MeOH300
After catalytic reduction in 50 ml of DMF, the mixture was dissolved in 50 ml of DMF. To this, Z−Arg(Pme)−OH・
Z-Arg(Pme)- prepared from 6.2 g of CHA
Add OH, HOBt1.5g, cool on ice, DCC2.3
g was added and stirred for 15 hours. precipitated
DCU was taken, concentrated, and dissolved in AcOEt. The AcOEt layer was washed with 4% _NaHCO3 water, 10% citric acid water, and dried _{with Na2SO4} .
After concentration, ether was added to obtain a precipitate. Yield 7.45g (93.7%). mp.100−101
°C, [α] ²⁶ _D -0.9° (c=1.2, DMF),
Rf ¹ 0.51 Elemental analysis As C ₃₃ H ₄₉ O ₉ N ₇ S Calculated value: C, 55.06; H, 6.86;
N, 13.62; S, 4.46 Experimental value: C, 55.49; H, 6.94;
N, 13.14; S, 3.86 () Z−Lys(Mtr)−Arg(Pme)−Ser
-Production of NHNH-Boc Z-Arg(Pme)-Ser-NHNH-Boc3.9
g was dissolved in 300 ml of MeOH for catalytic reduction, and then transferred to 50 ml of DMF. To this, Z-
Z-Lys(Mtr)-OH prepared from 3.4 g of Lys(Mtr)-OH/DCHA and 0.88 g of HOBt were added and cooled on ice, and 1.34 g of DCC was added and stirred for 20 hours. The precipitated DCU was separated, concentrated, and AcOEt was added to take it as a powder.
Recrystallized from MeOH-AcOEt. Yield 5.1
g (93.4%). mp.103−105℃, [α] ²⁶ _D −
7.2゜ (c=0.8, DMF), Rf ¹ 0.57 Elemental analysis C ₄₉ H ₇₃ O ₁₃ N ₉ S ₃ Calculated value: C, 55.50; H, 6.94;
N, 11.89; S, 6.05 Experimental value: C, 55.70; H, 7.15;
N, 11.60; S, 5.63 () Z−Arg(Pme)−Lys(Mtr)−Arg
Production of (Pme)-Ser-NHNH-Boc Z-Lys(Mtr)-Arg(Pme)-Ser-
4.8 g of NHNH-Boc was dissolved in 300 ml of MeOH, subjected to catalytic reduction, and then transferred to 70 ml of DMF. To this, Z−Arg(Pme)−OH・
Z-Arg(Pme)- prepared from 2.8g of CHA
Add 0.74g of OH and HOBt and cool on ice.
1.12g of DCC was added and stirred for 15 hours. The precipitated DCU was separated and concentrated, then dissolved in AcOEt, and the AcOEt layer was washed with 4% NaHCO ₃ water and 10% citric acid water, and dried over Na ₂ SO ₄ .
After concentration, ether was added to obtain a precipitate, which was reprecipitated from MeOH-ether. Yield 5.8g (89.8%). mp.136−137℃, [α]
²⁶ _D -6.6゜ (c=1.0, DMF), Rf ¹ 0.58 Elemental analysis C ₆₆ H ₉₉ O ₁₆ N ₁₃ S ₃ Calculated value: C, 55.56; H, 6.99;
N, 12.76; S, 6.74 Experimental value: C, 55.34; H, 7.07;
N, 12.50; S, 6.59 () Z−Lys(Mtr)−Arg(Pme)−Lys
(Mtr)−Arg(Pme)−Ser−NHNH−Boc
Production of Z−Arg(Pme)−Lys(Mtr)−Arg
(Pme)-Ser-NHNH-Boc3.0g
The mixture was dissolved in 150 ml of MeOH, subjected to catalytic reduction, and then transferred to 60 ml of DMF. To this, Z-
Z-Lys(Mtr)-OH prepared from 1.54 g of Lys(Mtr)-OH/DCHA and 0.31 g of HOBt were added and cooled on ice, and 0.57 g of DCC was added and stirred for 15 hours. The precipitated DCU was separated, concentrated, and dissolved in AcOEt, and the AcOEt layer was reduced to 4%
Wash with NaHCO ₃ water, 10% citric acid water,
Dried _{with Na2SO4} . After concentration, ether was added to obtain crystals, which were recrystallized from AcOEt. Yield 2.70g (72.8%) mp.123−
125℃, [α] ²⁶ _D -5.9゜ (c=1.1, DMF),
Rf ¹ 0.57 Elemental analysis C ₈₂ H ₁₂₃ O ₂₀ N ₁₅ S As ₄ Calculated value: C, 55.73; H, 7.02;
N, 11.89; S, 7.26 Experimental value: C, 55.89; H, 7.30;
N, 11.72; S, 7.08 () Boc−Gln−Met−Leu−Phe−Arg
(Pme)−Gly−Arg(Pme)−Arg(Pme)−
Production of Ala−Ser−Gln−OBu ^t Z−Arg(Pme)−Arg(Pme)−Ala−
1.0 g of Ser-Gln-OBu ^t was dissolved in 100 ml of MeOH, subjected to catalytic reduction, and then transferred to 20 ml of DMF. In addition, Boc−Gln−Met−Leu−Phe
−Arg(Pme)−Gly−OH0.85g,
135 mg of HOBt was added and cooled with water, and 210 mg of DCC was added and stirred for 20 hours. Deposited DCU
After separating and concentrating, MeOH was added to obtain a precipitate. Yield 1.50g (87.4%).
mp.216-217℃ (decomposition), [α] ²⁶ _D -11.8゜ (c=1.0, DMF), Rf ¹ 0.43 Elemental analysis C ₉₈ H ₁₅₄ O ₂₃ N ₂₂ S As ₄ Calculated value: C, 55.09; H , 7.27;
N, 14.42; S, 6.00 Experimental value: C, 54.81; H, 7.33;
N, 14.23; S, 5.79 () H-Lys-Arg-Lys-Arg-Ser-
Gln−Met−Leu−Phe−Arg−Gly−Arg−
Production of Arg−Ala−Ser−Gln−OH Boc−Gln−Met−Leu−Phe−Arg
(Pme)−Gly−Arg(Pme)−Arg(Pme)−
Add 10 ml of TFA to 1.0 g of Ala-Ser-Gln-OBu ^t , shake at room temperature for 50 minutes, concentrate, add ether and collect as a precipitate.
Dry. On the other hand, Z−Lys(Mtr)−Arg(Pme)−
Lys(Mtr)−Arg(Pme)−Ser−NHNH−
10 ml of TFA was added to 0.83 g of Boc, and the mixture was shaken at room temperature for 10 minutes, concentrated, and ether was added to form a precipitate, which was then dried. this
Dissolve in 10ml of DMF, cool with dry ice-acetone, 6.2N HCl/AcOEt0.23
ml and isoamyl nitrite (0.075 ml) and kept at -25° to -20°C for 20 minutes (hydrazine test: negative). Cool this again with dry ice-acetone and add 0.25ml of TEA.
was added to neutralize it. Amine component in DMF40
ml, cooled on ice, added 0.16 ml of TEA, and then added the azide solution prepared earlier.
Stir at 4°C for 72 hours. The reaction solution was poured into dilute aqueous acetic acid, and the precipitated gel was collected and washed with hydrous CH ₃ CN. Yield 1.40g (81.5
%), Rf ¹ 0.09. 400mg of this
Dissolve in 60 ml of TFA-thioanisole-methyl sulfide (8:1:1) containing 0.15 MMSA, shake at room temperature for 2 hours, and then
400 mg of AcONH ₄ was added and concentrated, ether was added to precipitate, and the mixture was dried. This was dissolved in a small amount of 1N AcOH and applied to a Sephadex G-25 column (2.2 x 120 cm). It was eluted with 1N AcOH, and fractions of 160 ml to 260 ml were collected and lyophilized, and then passed through a column of Amberlite IRA-410 (acetic acid type) and lyophilized. Add this further to TSK−
LS-410 column (2.14 x 7.5 cm + 2.14 x 30
The desired product was obtained by purification by HPLC using CM). Yield 45 mg, [α] ²⁴ _D -45.9° (c = 0.7,
0.1N AcOH), Rf ⁴ (cellulose) 0.20 Amino acid analysis: Lys1.71, Arg4.71,
Ser1.69, Glu2.12, Gly1.00, Ala1.03,
Met0.32, Leu1.30, Phe1.08 (average recovery rate 77%) (2) Synthesis of carrier protein and polypeptide complex Synthesis of the polypeptide obtained in (1)-() above and thyroglobulin (hereinafter referred to as TG) The conjugation was carried out according to the method of Gutfriend et al. [Science, 144 , 1334 (1964)]. That is, 2.5 mg of the polypeptide was mixed with 3.75 mg of TG,
ml of 50mM phosphate buffer was added and stirred well in ice water. After slowly adding 30.4mg of carbodiimide hydrochloride dissolved in 200μ of distilled water drop by drop,
The reaction was allowed to take place while stirring in ice water for an hour. After the reaction, 4.7 mg of the protein complex was obtained by thorough dialysis with distilled water and freeze-drying. (3) Preparation of antigen for EIA for antibody detection Antigen for EIA was prepared by the method of Kitagawa et al. [Journal of Biochemistry, 79 , 233
(1976)]. (i) Introduction of maleimide group into polypeptide The polypeptide (350 nmoles) obtained in (1)-() above was dissolved in 1 ml of 100 mM phosphate buffer PH6.8, and N-(4-
585 μg (1.75 μmoles) of N-hydroxysuccinimide ester of carboxycyclohexylmethyl)maleimide and 70 μg of N.
Add to N-dimethylformamide solution and mix, stir at 30°C for 30 minutes to react, then fractionate with a Sephadex G-25 column to obtain a polypeptide fraction with a maleimide group introduced.
Obtained 185 nmoles. (ii) Maleimide group-introduced polypeptide and β-D
- Binding with galactosidase 16.5 nmoles of the maleimide group-introduced polypeptide obtained in (3)-(i) above and 3.3 nmoles of β-D-galactosidase were mixed, and after reacting at 4°C for 18 hours, 412.5 nmoles of β-mercaptoethanol was added. The reaction was stopped. β-D-
The polypeptide bound to galactosidase was fractionated using a Sepharose 6B column and used in the following experiment. (4) EIA method Antibody activity in the serum or hybridoma supernatant of mice immunized with the protein complex obtained in (2) above was detected using the EIA method [Immunopharmacology, 1 , 3 (1978). )]. That is, serum or hybridoma supernatant was mixed with buffer A (20mM Na ₂ HPO ₄ , 100mM
NaCl, 0.1% _NaN3 , 1mM _MgCl2 , PH7.0)
100μ of this was mixed thoroughly with 100μ of the polypeptide derivative obtained in (3) above, and reacted at 24°C for 24 hours. After reaction, 3% cellulose conjugated with rabbit anti-mouse IgG
100μ was added and reacted at 24°C for 4 hours.
After the reaction, the cellulose was thoroughly washed with Buffer A containing 0.5% Tween 20, and 4-methylumbelliferyl-β-D-galactoside 20 was added.
Add 500μg/ml and react at 37℃ for 2 hours, then add 100mM carbonate buffer (PH
10.5) was added to stop the reaction, and the fluorescence intensity in the supernatant was measured using a fluorometer (excitation 365 nm, emission 450 nm). (5) Immunization Six 7- to 8-week-old BALB/C female mice each were inoculated subcutaneously with 40 μg of the protein complex obtained in (2) above mixed well with Freund's Complete adjuvant as an antigen. (first immunization). Two weeks after the first immunization,
Freundin complete with the same amount of antigen
It was mixed well with an adjuvant and inoculated subcutaneously (secondary immunization). Furthermore, two weeks later, a tertiary immunization was performed in the same manner as the secondary immunization. Six days after the third immunization, a portion of blood was collected from the mouse.
The antibody titer in the serum was measured by the EIA method described in (4) above. Of these, γ-2 showed high antibody titer.
Final immunization was performed by intravenously inoculating mice with 120 μg of the antigen dissolved in 0.5 ml of saline. The antibody titer of each mouse is shown in Table 2.

[Table] B. Production of monoclonal antibody γ2-11.1 (1) Cell fusion Immunization was performed using the method described in A (5) above, and 3 days after the final immunization, the spleen was removed from the γ-2 mouse and compressed with a stainless steel mesh. The cells were filtered and suspended in Eagle's Minimum Essential Medium (MEM) to obtain a spleen cell suspension. As cells used for cell fusion,
BALB/C mouse-derived myeloma cells P3-×
63.Current using Ag8.U1 (P3U1)
Topics in Microbiology
and Immunology, 81 , 1 (1978)]. Cell fusion is performed using the original method [Nature, 256 , 495
(1975)]. i.e., spleen cells and P3U1, respectively, are serum-free.
Wash 3 times with MEM, mix spleen cells and P3U1 at a ratio of 5:1, and rotate at 800 rpm.
Cells were pelleted by centrifugation for 15 minutes. After thoroughly removing the supernatant, lightly loosen the precipitate,
45% polyethylene glycol (PEG) 6000
(manufactured by Kotsuholite) was added, and the mixture was allowed to stand for 7 minutes in a 37°C hot water bath to effect fusion. After fusion, add MEM to the cells at a rate of 2 ml per minute.
After adding a total of 12 ml of MEM, the mixture was centrifuged at 600 rpm for 15 minutes and the supernatant was removed. 10 of this cell precipitate
P3U1 was suspended in RPMI1640 medium (RPMI1640-10FCS) containing % fetal bovine serum at a concentration of 2 x 10 ⁵ cells per ml, and 1 ml per well was added to a 24-well multidish (manufactured by Linbro).
Each was seeded in 144 wells. After seeding, cells
The cells were cultured at 37°C in a 5% carbon dioxide gas furan vessel. twenty four
After hours HAT (hypoxanthine 1 × 10 ^-4 M,
Aminopterin 4×10 ^-7 M, thymidine 1.6×
HAT selective culture was started by adding 1 ml of RPMI1640-10FCS medium (HAT medium) containing 10 ^-5 M) per well.
HAT selective culture was continued by adding 1 ml of HAT medium after discarding 1 ml of the old solution 3, 5, and 7 days after the start of culture. Hybridoma proliferation is observed 10 to 14 days after cell fusion, and when the culture medium turns yellow (approximately 1 x 10 ⁶ /
ml), the supernatant was collected and searched for the presence of antibodies using EIA method. In this way, when we examined the supernatant of 141 wells in which hybridoma growth was observed, we found that 2 wells (γ2-11, γ2-11,
100), 2 wells (γ2-
62, γ2-70). (2) Cloning Three wells (γ2-
11, 62, and 100) were cloned by the limiting dilution method. In other words, the number of hybridomas should be 2/ml.
Float in RPMI1640-20FCS, per well in a 96-well microplate (manufactured by Nunc)
It was dispensed in 0.1 ml portions. During dispensing, BALB/C mouse thymocytes were added as feeder cells at a density of 5 x 10 ⁵ cells per well.
In this way, after about two weeks, cell proliferation was observed, and the supernatant was collected.
The presence or absence of antibodies was examined by the EIA method described in A(4) above. As a result, in γ2-11, 8 out of 19 clones
Antibody activity was observed in 3 out of 54 clones for γ2-62 and 5 out of 47 clones for γ2-100 (Table 3). (3) Monoclonal antibody against IFN-γ

[Table] Binding ability The binding ability of monoclonal antibodies to IFN-γ was examined using the following method. That is, a 3% cellulose solution bound with rabbit anti-mouse IgG antibody.
To 300μ, 300μ of culture supernatants of two to three types of clone cells derived from γ2-11, γ2-62, and γ2-100 were added, and the mixture was reacted at room temperature for 18 to 20 hours. After the reaction, the cellulose was thoroughly washed with physiological saline, and 550 U/ml of IFN-γ was obtained as follows.
was added and reacted for 3 to 4 hours. After the reaction, the supernatant was collected and the IFN-γ activity in the supernatant was measured by the cytopathic effect (CPE) reading method using a microplate [Applied Microbiology, 16 , 1706 (1968)]. In other words, in all wells of a 96-well microplate (manufactured by Nunc)
Serial 2-fold dilutions were performed by adding 50μ of MEM and adding 50μ of IFN sample to the first well. Add 50μ of a cell suspension containing WISH cells in MEM containing 20% FCS to 4 x ¹⁰ cells per ml to each well, and
The cells were cultured at 37°C for an hour in a carbon dioxide gas furan vessel. After culturing, vesicular stomatitis virus (New Jersey strain) was added to 2000TCID ₅₀
The cells were adjusted with MEM, 50μ of which was added to each well, and cultured at 37°C in a carbon dioxide gas furan vessel.
Approximately 35 hours later, when 100% CPE has occurred in the cells in the wells to which no IFN sample has been added, the CPE in each well is observed under a microscope, and the number of dilutions of the IFN-sample in the wells in which 50% CPE has occurred is determined. The reciprocal of the value was taken as the IFN titer. The IFN-γ sample used was human peripheral blood lymphocytes mixed with 40 μg of concanavalin A/
ml and 12-O-tetradecanoylphorbol-13
The supernatant was collected 72 hours after stimulation with 15 ng/ml of acetate, and 1 ml of this culture supernatant contained human
It contained 4400 units of IFN-γ (unstable to acid and PH treatments). If there is an antibody capable of binding to IFN-γ in the culture supernatant of the cloned cells, the IFN-γ activity added later will bind to the antibody on the cellulose, and the IFN-γ activity added later will bind to the antibody on cellulose and Activity should be reduced. The results showed that relatively strong IFN-γ binding ability was observed in the supernatant of the γ2-11 clone, and 50 to 75% of the added IFN-γ (550 U/ml)
% bound to the antibody (Table 4).

【table】

[Table] (4) Ascites formation of monoclonal antibody-producing hybridomas ¹⁰⁶ γ2-11.1 clone cells (mouse B hybridoma γ2-11.1) that produce antibodies capable of binding to IFN-γ were prepared in 0.5 ml in advance. Ascites was performed by intraperitoneally inoculating BALB/c mice to which mineral oil had been intraperitoneally administered. Ten days after intraperitoneal administration of the hybridoma, ascites was collected and the antibody titer was measured by EIA, and antibody activity was observed up to 10 ⁷ times dilution. Although the antibody activity in the culture supernatant of the cloned cells was observed up to 10 ⁴ -fold dilution, the antibody activity increased approximately 1000-fold by ascites formation. (5) Purification of monoclonal antibodies Using 4 ml of ascites obtained in (4) above as a starting material, Staelin et al. [Journal of Biological Chemistry, 256 , 9750]
(1981)] monoclonal antibodies were purified. First, in order to remove fibrin-like substances from ascites, centrifugation was performed at 10,000 rpm for 15 minutes, followed by phosphate buffer-saline (PBS: 8.1mM
_-NaH2PO4 , _{1.5mM KH2PO4 ,} 2.7mM
KCl, 137mM NaCl, PH7.2) to a concentration exhibiting a value of ultraviolet absorption at 280nm ( _A280 ) of 12 to 14. After dilution, add saturated ammonium sulfate solution to the sample to give a concentration of 47%.
Salting out was carried out for 60 minutes with stirring at 4°C, followed by centrifugation (10,000 rpm, 15 minutes) to obtain a precipitate. Precipitate containing 50mM NaCl
Dissolved in 20mM Tris buffer solution (PH7.9),
The same solution 2 was subjected to dialysis. After 2 hours, change to the same new dialysate in step 2, and then
Dialysis was performed for 15 hours. After dialysis, centrifugation was performed at 10,000 rpm for 15 minutes to remove the precipitate, and the supernatant was adjusted to have an _A280 value of 20 to 30. Add this sample to a sufficient amount of 50mM-
8 ml of water conditioned with Tris buffer containing NaCl.
DEAE cellulose column (Watmann
DE ₅₂ ) and fractionation was carried out using a Tris buffer containing 50 mM NaCl at a flow rate of 1.5 ml/min. Under these conditions, antibody activity was mainly observed in the flow-through fraction (Figure 3). The monoclonal antibody γ2-11.1 obtained here was confirmed using the method of Laemli et al.
227, 680 (1970)], the SDS-polyacrylamide gel electrophoresis (SDS-PAGE) method was used. That is, several fractions fractionated using an ammonium sulfate precipitation-DEAE cellulose column were each reduced with 2-mercaptoethanol and electrophoresed on a 17% SDS gel at 30 volts for 24 hours. As a result, in agreement with the antibody activity, the H chain, with a molecular weight of approximately 55 kilodaltons,
Two bands of L complex were observed around 28 kilodaltons (Figure 4). We tested whether the antibody fraction 17 purified in this way showed binding ability to IFN-γ by adding 2200 U/ml of IFN-γ according to method A (3) above. It was found that about 50% showed binding to the antibody (Table 5).

[Table] (6) Subclass to which monoclonal antibodies belong γ2-11.1 monoclonal antibody belongs to
For IgG subclasses, fraction 17 purified using method (5) above was diluted 10 times and goat anti-mouse IgG1, G2a, G2b, G3 antibodies (Miles) were used.
The agar precipitation reaction method (Ochterlony method: Immunological Methods Gel-Diffusion Techniques, Blackwell Oxford, 1964) was used. Result is,
One prominent band was observed between the monoclonal antibody and the goat anti-mouse IgG2b antibody,
No band formation was observed between it and other anti-IgG antibodies. Therefore, the monoclonal antibody was found to belong to IgG2 (Table 6).

[Table] C Production of monoclonal antibody γ3-11.1 The method for producing γ3-11.1 monoclonal antibody obtained from γ-3 mice is shown below. This monoclonal antibody can also be used for purification of IFN-γ in the same manner as the γ2-11.1 monoclonal antibody. (1) Immunization The γ-3 mice listed in Table 2 of A(5) above were given the fourth immunization in the same manner as the second and third immunizations, and two weeks after the fourth immunization, the γ-3 mice listed in Table 2 of A(5) above were given the fourth immunization. similarly
Final immunization was performed by intravenously inoculating 120 μg of antigen dissolved in 0.5 ml of saline. (2) Cell fusion γ-3 days after the final immunization described in (1) above
The spleen was removed from 3 mice, and cell fusion and HAT selection culture were performed by the method described in B(1) above. As a result, hybridoma growth was observed in 43 wells, and when the presence or absence of antibodies was searched using the EIA method described in A(4) above, 2 wells (γ
3-11, γ3-19), strong antibody activity was observed. (3) Cloning Two wells (γ3
-11 and γ3-19) were cloned by the method described in B(2) above. As a result, in γ3-11, 9 out of 21 clones
For clone γ3-19, antibody activity was observed in 15 out of 25 clones (Table 7). (4) Binding ability of monoclonal antibodies to IFN-γ Binding of monoclonal antibodies to IFN-γ

[Table] Performance was examined using the method described in B(3) above. However, the IFN-γ sample used in this reference example was obtained by incorporating the human IFN-γ gene into a plasmid and expressing it in E. coli (recombinant IFN-γ).
γ; see Reference Example 2 (4)) and was adjusted to 1100 U/ml. The results are γ3-11.1, γ3-
Strong IFN-γ binding ability was observed in the supernatant of clone 19.20, and about 90% of the added IFN-γ bound to the antibody (Table 8).

[Table] (5) Subclass to which the monoclonal antibody belongs γ3-11.1, γ3-19.20 clone cells were made into ascites by the method described in B(4) above,
The obtained ascites was purified by the method described in B(5) above. The subclass of the obtained monoclonal antibody was examined by the agar precipitation reaction method described in B(6) above. The result is γ3−11.1,
A distinct band was observed between each of the γ3-19.20 monoclonal antibodies and the goat anti-mouse IgG1 antibody, and no band was observed between the other anti-IgG antibodies. Therefore, the γ3-11.1 and γ3-19.20 monoclonal antibodies were found to belong to IgG1 (Table 9).

【table】

[Table] Reference Example 2 Production of transformants and production of IFN-γ-containing supernatant Crude IFN used in Example B and Reference Example 1C(4)
-γ-containing supernatant was obtained by the method shown in the following reference example. (1) Production of plasmid PHIT3709 containing the IFN-γ gene (i) Isolation of mRNA encoding human IFN-γ Lymphocytes prepared from human peripheral blood were treated with 12-0-tetradecanoylphorbol-13-acetate (TPA). (15ng/ml) and concanavalin A
(40 μg/ml) in RPMI-1640 medium (10
% of fetal bovine serum) at 37°C, and incubated with IFN-
γ was induced. After 24 hours, this induced 1
×10 ¹⁰ human lymphocytes were cultured in thioguanidine denaturing solution (5M guanidine thiocyanate, 5
% mercaptoethanol, 50mM Tris.
After denaturation with a Teflon homogenizer in HCl PH7.6, 10mM EDTA)
- Sodium lauroyl sarcosinate 4
% and homogenized mixture.
5.7M cesium chloride solution [5.7M cesium chloride, layered on 6 ml of 0.1M ethylenediaminetetraacetic acid (EDTA),
24000rpm30 at 15℃ using SW27 rotor
Centrifugation was performed for several hours to obtain RNA precipitate. This RNA precipitate was dissolved in a 0.25% sodium N-lauroyl sarcosinate solution and then precipitated with ethanol to obtain 8.3 mg of RNA.
This RNA was added to a hot solution [0.5M NaCl, 10mM
Tris・HCl PH7.6, 1mM EDTA, 0.3
%SDS] on an oligo(dT) cellulose column, and mRNA containing poly(A) was dissolved in a low salt solution (10mM Tris-HCl・PH7.6, 1mM
By elution with EDTA, 0.3% SDS), 700 μg of mRNA containing poly(A) was collected. This mRNA was further precipitated with ethanol, and 0.2 ml of solution (10mM Tris・HCl PH
7.6, 2mM EDTA, 0.3% SDS), treated at 65°C for 2 minutes, and centrifuged on a 10-35% sucrose density gradient (centrifuged at 20°C, 25000 rpm for 21 hours using a Bakeman SW27 rotor). After fractionation, 22 fractions were obtained. For each fraction, a portion of the RNA was
Injected into Xenopus oocytes,
Interferon activity in the synthesized protein [measured using a test for inhibiting the cytopathic effect of vesicular stomatitis virus (VSV) on human amnion-derived WISH cells. Antiviral activity [The Interferon System, Springer, New York, p. 11, 1979] was measured, and an activity of 195 units/μg RNA was detected in fraction 12 (sedimentation constant S value was 12-14S). did. m of fraction 12 thus obtained
RNA was approximately 20 μg. (ii) Synthesis of single-stranded DNA Using the mRNA and reverse transcriptase obtained above, 100 μg of reaction solution (5 μg of mRNA, 50 μg of
μg oligo (dT), 100 units of reverse transcriptase, 1mM each of dATP, dCTP, dGTP and dTTP, 8mM MgCl ₂ , 50mM
KCl, 10mM dithiothreitol, 50mM
After incubating in Tris-HCl (PH8.3) at 42°C for 1 hour, protein was removed with phenol and treated with 0.1N NaOH at 70°C for 20 minutes to remove RNA.
was decomposed and removed. (iii) Synthesis of double-stranded DNA 50μ of the single-stranded complementary DNA synthesized here
Double-stranded DNA was synthesized by reacting for 2 hours at 42°C in a reaction solution (the same reaction solution as above except that it did not contain mRNA and oligo-dT). (iv) Addition of dC tail Add 50μ of nuclease S1 to this double-stranded DNA.
Reaction solution (double-stranded DNA 0.1M sodium acetate PH4.5, 0.25M NaCl, 1.5mM ZnSO ₄ , 60
The DNA was reacted for 30 minutes at room temperature in a reaction mixture (double-stranded DNA, 0.14M potassium cacodylate) of terminal transferase (Unit's S1 nuclease), deproteinized with phenol, and precipitated with ethanol. ,0.3M
Tris (base) PH7.6, 2mM dithiothreitol, 1mM C ₀ Cl ₂ , 0.15mM dCTP, 30
Approximately 20 deoxycytidine chains were extended at both 3' ends of the double-stranded DNA by reacting the DNA in unit terminal transferase (unit terminal transferase) for 3 minutes at 37°C. Through these series of reactions, double-stranded DNA containing approximately 300 ng of deoxycytidine chains was obtained. (v) Cleavage of E. coli plasmid and addition of dG tail Meanwhile, 10 μg of E. coli plasmid
Add restriction enzyme PstI to pBR322 DNA in 50μ reaction solution [10μg DNA, 50mM NaCl, 6mM
Tris・HCl (PH7.4), 6mM MgCl ₂ , 6
mM2-Mercaptoethanol, 100μg/
ml, bovine serum albumin, 20 units of PstI]
After reacting for 3 hours at 37°C to cleave the PstI recognition site present in pBR322 DNA and deproteinizing it with phenol, add terminal transferase to 50μ of the reaction solution [10μ of DNA].
g, 0.14M potassium cacodylate, 0.3M Tris (base) PH7.6, 2mM dithiothreitol, 1
3 minutes in mM _C0Cl2 , 0.15mM dGTP, 30 units _of terminal terminal transferase.
The above plasmid pBR322DNA was incubated at 37°C.
Approximately eight deoxyguanine chains were extended at both 3' ends of the molecule. (vi) Association of cDNA and transformation of E. coli The synthetic duplex thus obtained
0.1μg of DNA and 0.5μ of the above plasmid pBR322
g to 0.1M NaCl, 50mM Tris・HCl PH
7.6, 65°C in a solution consisting of 1mM EDTA
The method of Enea et al.
Molecular Biology, 96 , 495
(1975)] was transformed into E. coli χ1776. (vii) Isolation of cDNA-containing plasmids Approximately 8500 tetracycline-resistant strains were thus isolated, and the DNA of each of these was immobilized on a nitrocellulose filter. [Proceedings of National Academy of Sciences USA 72 , 3961 (1975)] On the other hand, a report by Goeder et al.
295 , 503 (1982)], amino acids No. 1-5 (Cys,
Tyr・Cys・Gln・Asp) and amino acid No.77
−82 (Lys・Gln・Asp・Met・Asn・Val)
More inferred base sequences (respectively

【formula】 and

[Formula] ) was chemically synthesized using the triester method [Claire et al., Proceedings of the National Academy of Sciences USA, 75 , 5765 (1978)]. T4 for this oligonucleotide
Using polynucleotide kinase, 50μ reaction solution (0.2μg oligonucleotide, 50mM
Tris・HCl PH8.0, 10mM MgCl ₂・10mM
Mercaptoethanol, 50μCiγ-
³² PATP, 3 unit T4 polynucleotide kinase) for 1 hour at 37°C to remove the 5′ end.
Labeled with ^32P . Using this labeled oligonucleotide as a probe, the method of Lown et al.
(1981)], and four bacterial strains that reacted with the two types of oligonucleotide probes were isolated by autoradiography. Plasmid DNA was extracted from the cells of each of these strains using the alkaline method [Birnboim and Dolly, Nucleitsu Research, 7 ,
1513 (1979)]. Next, the inserted part of the plasmid DNA was cut out using the restriction enzyme PstI, and among the separated plasmids, the one containing the longest fragment of the inserted part was selected, and this plasmid was named PHIT3709. Then inserted into this PHIT3709 plasmid
The primary structure (base sequence) of the cDNA sequence was determined by the dinucleotide synthesis chain termination method and the Maxam-Gilbert method. Its primary structure was as shown in Figure 5. The base sequence of the IFN-γ coding region (codon No. S1 to No. 146) of PHIT3709 can be found in published literature [Nucleitsu Research, 10 ,
2487 (1982), Figure 3]. (2) Production of expression plasmids and expression transformants (i) Promoter region for tryptophan synthesis in Escherichia coli as an expression plasmid [DNA fragment containing promoter and operator, 276 base pairs, Bennett et al., Journal of Molecular Biology] , 121 113 (1978)] containing plasmid ptrp601 (vector is
pBR322) was constructed. Meanwhile, restriction enzyme plasmid pBR322
EcoRI containing the tetracycline resistance gene obtained by cutting with EcoRI and restriction enzyme AvaI
-The margin of the AvaI fragment was filled in with DNA polymerase I large fragment. This fragment was added to the Pvu of ptrp601 using T4DNA ligase.
ptrp701 was constructed by binding to the cleavage site of (Fig. 6). (ii) Next, restriction enzymes present in two locations in ptrp701
To erase one of the ClaI cleavage sites,
ptrp701 was partially digested with ClaI to obtain a product in which only one of the two ClaI cleavage sites was cleaved. After filling the resulting margin with DNA polymerase I large fragment,
Religation with T4 DNA ligase yielded ptrp771 (Figure 6). (iii) PHIT3709 was cut with the restriction enzyme PstI and IFN-
A PstI fragment containing the structural gene of γ was obtained. This fragment was partially digested with the restriction enzyme BstNI, and IFN-
A BstNI-PstI fragment was obtained by cutting the BstNI site within the γ structural gene. After filling in the margin of the BstNI cleavage site with DNA polymerase I large fragment, an oligonucleotide adapter CGATAATGTGTTACTGCC TATTACACAATGACGG containing the protein synthesis initiation codon ATG, chemically synthesized by the triester method described above, was ligated with T4 DNA ligase. On the other hand, IFN-
The fragment obtained by cleaving ptrp771 of the γ gene with restriction enzymes pstI and ClaI was inserted downstream of the tryptophan promoter and ligated using T4 DNA ligase to construct the IFN-γ expression plasmid PHITtrp1101 (No. 7 figure). (iv) Further improve PHITrp1101 and create the following PH
I built ITtrp2101. First, ptrp601 is treated with restriction enzyme ClaI and restriction enzyme Hpa to contain the trp promoter.
A ClaI-Hpa fragment of 0.33 Kb was obtained. This fragment was ligated to PHITtrp1101, which had been cut with ClaI and treated with alkaline phosphatase, using T4 DNA ligase to obtain PHITtrp2101, in which two trp promoters were inserted in series (Fig. 8). Using this plasmid PHITtrp2101, Escherichia coli 294 was transformed according to the method of Cohen et al. [supra] to obtain a strain E. coli 294/PHITtrp2101 containing this plasmid. (3) Cultivation of transformants for expression E.coli294/PHITtrp2101 was cultured at 37°C in a 1 Meyer medium containing 200 ml of M9 medium containing 8 μg/ml tetracycline, 0.4% casamino acids, and 1% glucose, and grown. When the amount reached KU220, 3β-indolylacrylic acid (IAA) was added.
It was added at a concentration of 30 μg/ml and cultured for an additional 4 hours. (4) Production of IFN-γ-containing supernatant Centrifuge the culture solution 1.2 obtained in (3) above to collect bacterial cells, and add this to 60 ml of 0.05 M Tris・HCl PH7.6 containing 10% sucrose. Suspended.
Add 0.3ml of 0.2M phenyl-methyl-sulfonyl fluoride (PMSF) to this bacterial suspension, and add 5M
NaCl 2.4ml, 0.2M ethylenediaminetetraacetate (EDTA) 2.4ml, 1M spermidine 2.4
ml, add 2.4 ml of 5 mg/ml lysozyme and incubate at 0°C.
After leaving it for a while, it was treated at 37℃ for 5 minutes, and then it was further processed with an ultrasonic crusher manufactured by Artek (USA).
It was treated at ℃ for 30 seconds. This lysate was centrifuged at 105,000 xg for 1 hour, and 66 ml of supernatant was collected.

[Brief explanation of the drawing]

FIG. 1 shows the results of electrophoresis of human immune interferon protein obtained by the purification method of the present invention shown in Examples, and FIG. 2 shows the results of molecular weight measurement of the same protein. Figure 3 shows the elution pattern of the monoclonal antibody of the present invention on a DEAE cellulose column in Reference Example 1C (5), and Figure 4 shows the elution pattern of the monoclonal antibody of the present invention in Reference Example 1C (5).
The results of monoclonal antibody electrophoresis in (5) are shown. Figure 5 shows the PH obtained in Reference Example 2 (1)-(vii)
The primary structure (base sequence) of IT3709 plasmid is
Figure 6 shows ptrp701 and ptrp771 of Reference Example 2(2)-(i) and -(ii), and Figure 7 shows PH of Reference Example 2(2)-(iii).
ITtrp1101, Figure 8 is the PH of reference example 2 (2)-(iv)
Each construction diagram of ITtrp2101 is shown.

Claims (1)

[Scope of Claims] 1 Crude human γ-interferon containing material has the formula H-Lys-Arg-Lys-Arg-Ser-Gln-Met -Leu-Phe-Arg-Gly-Arg-Arg-Ala -Ser-Gln A method for purifying human γ-type interferon, which comprises purifying using a monoclonal antibody against a polypeptide represented by -OH. 2. The purification method according to claim 1, wherein the crude human γ-interferon-containing material is subjected to affinity column chromatography using a monoclonal antibody coupled to a carrier.