WO1984004745A1 - Novel polypeptides and their use - Google Patents

Abstract

Novel polypeptides represented by the following formula:$(3,)$(wherein X represents a bond or a peptide or amino acid residue comprising 1 to 16 amino acids$(3,)$counted from the side of the C terminal, and Y represents a peptide or amino acid residue comprising 1 to 5 amino acids of$(3,)$counted from the side of the N terminal) and their protein conjugates, hybridoma and monoclonal antibody induced from said polypeptides or said protein conjugates, and method for detecting and purifying human gamma-interferon utilizing them.

Description

 Specification

, New e. Ribeptide and its uses

 Technical field

 The present invention relates to novel polypeptides and uses thereof.

 Background art

 Developed by Koehler and Misstein (Nature, Vol. 256, p. 495, 1975), the method of producing monoclonal antibodies using hybridomas, which has become popular in recent years, has been developed for each antigenic determinant. In addition, antibodies that exhibit monospecificity can be obtained, and antibodies obtained from crudely purified samples require an absorption procedure. In addition, from the aspect of antibody acquisition, many advantages such as the ability to obtain high-titer antibodies freely, in large quantities, and with high reproducibility of a homogeneous sample with good reproducibility by ascites of the hybridoma. There is. In this sense, the usefulness of the method for obtaining monoclonal antibodies by hybridomas has been highly evaluated in many fields. In addition, its usage was not limited to antigen detection, and was used to purify the micro-agitated components through the production of an antibody resistance arm.])

 '285, p. 4.46, 1980), and its application to diagnostics and therapeutics (Chapter 9, Bianjar Nanoreoimmunology, Vol. 9, p. 94, 1979).

It is known that human interferon (IFN) has at least three types of a, β, and antigenically different a, β, types (Netiya, Vol. 286, 110, 1980, 1980). Year). It has been found that r-type interferon (IFlflfr) is mainly released from τ-lymphocytes by mitogen and antigen stimulation] ?, also known as immune interferon (I-IFN). ) (The Interferon System, Springer, -Eu York, 1979). IFN-r is expected to be produced in vivo in response to various immune responses, and is considered to play an important role in immune regulation. In addition, the properties of IFN-r include different antigenicity from type a interferon (IFN-a) and type 3 interferon (IFN- / 9), and different types of inducers. It is also known that the stability of foams, acid, and heat is poor (The Interferon System, Sp !; Nanger, New York, 1979).

 —Generally, IFN is defined as having an anti-viral effect produced by living organisms, but it has also been demonstrated that it has many other biological activities i) (Blood, Vol. 55, p. 71, 1980; Ibid., Vol. 56, p. 875, 1980).

Methods for suppressing tumor growth include a method of directly suppressing the growth of tumor cells and a method of suppressing the tumor indirectly through the host's immune response. In the latter case, for example, Natura killer cells (NK ), Activation of macrophages, or activation of killer T cells. In fact, IFN has been proved to have various immunopotentiating activities in addition to its direct action (Biochemica et al., Vol. 516, p.231, 1978). FN- _r has much higher antitumor activity at INVIT and antitumor activity at in vivo than IF-a and IF! T- / 3 Its importance has been strongly pointed out (Cellular Immunology, Vol. 49, p. 390, 1980). However, the titer of IFN-r induced in the mouth is generally low or appropriate. ほ と ん ど Most IFN-r-producing cell lines are difficult to purify due to poor heat and acid stability. Mass production and purification of IFN-T lags far behind IFN-a and IFN-i9 I have.

 Very recently, it has been reported that a single natural IF IT-machine was created (Procedures of the National Academy of Sciences, Chapter 1).

Vol. 79, p. 1820, 1982), the recovery of the activity is very poor, and an effective purification method is expected.

 —How recently?]? Cloning of the human IFN-α gene has been reported, and at least as one type of IF IT-α, a molecular species of about 17 kDa, consisting of six amino acids, has been identified in bacteria. As it is possible, Necchia, Vol. 295, 503 Sada, 1982; Nucleic Acids Lisachi, Vol. 10, pp. 2487, 1982 Various molecular weights of the produced FN-α have been reported.], But the correspondence between the molecular species is still unknown.

From this point of view, obtaining monoclonal-nano antibodies against various IF IT-'r is not only important for establishing the correspondence between molecular species, but also using natural or genetically modified E. coli etc. IF iT - 7 · ₃ most recently Ru extremely powerful weapon and ¾ on the purification of the optimal '?, natural - monochrome against § - get monoclonal antibody has been reported (Ne - Chiya -, 296, pp. 2 P. 58, 1982), when IFN-r is composed of multiple molecular species, a genetic recombination method was used to obtain monoclonal antibodies against cloned molecular species. It is extremely important to do ₃

 Disclosure of the invention

 The present inventors performed cloning of the IFN-α gene based on the mBNA of IFiT-, which was derived from human peripheral blood lymphocytes, and clarified its nucleotide sequence.

• Although it was clarified (see PCT ZJ I> 82/0 CI 080 specification), it has a partial structure at the C-terminal side of the amino acid sequence deduced based on this. formula

 (N) (C),,

H-X-Lys Arg Ser Gin Met Le eu Phe Arg Gl -Y-OH (I)

_A (N)

 (In the formula, X Ji ^ joint hand or lie Gin Val Me t Ala Glu Leu

 (C) ―

 Ser Pro Ala Ala Lys Tiir Gly Lys rg ぺ チ 鎖 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1

 (N) (C) One or amino acid residue, Υ is Arg Arg Ala Ser Gin: Peptide or amino having 1 to 5 amino acids counted from its N-terminal in the peptide chain [The acid residue is shown.] Was chemically synthesized and further chemically bonded to a carrier protein to form a protein complex.

 A mammal was immunized with the polypeptide or the protein complex obtained here, and the extracted 陴 贜 cells were fused with allogeneic or heterologous lymphoid cells by cell fusion.] The hybridoma was cloned. .

 The obtained hybridoma was inoculated into a mammal to produce a monoclonal antibody, which was collected to produce a monoclonal antibody against the above-mentioned polypeptide.

Further, a method for purifying human IFN- _r from crude human IFN-I content using the nocloclonal antibody obtained here, a radioimmunoassay (RIA) method, and a radiobiology enzyme immunoassay (EIA) method By

]? A method for detecting human I N -r was established, and the present invention was completed.

 In other words, the present invention provides a novel polypeptide (I), a protein complex thereof, a novel cloned hybridoma, a novel monoclonal antibody, a method for producing the same, and a use of the monoclonal antibody. It is preferable that Y is Arg Arg Ala Ser Gin with respect to the polypeptide (I), and! In addition, X is preferably Lys Arg. In addition, polypeptide (I) is not a specific number of amino acid residues.

OMPI — B—

 It is preferably 15 to 25 pieces.

 The peptide can be produced by conventional means of peptide synthesis. Either the solid phase synthesis method or the liquid phase synthesis method may be used, but the liquid phase synthesis method is advantageous in many cases. The method for synthesizing such a peptide is described, for example, in “The Peptides-, Vol. 6 6), Schroder and Lbke, Academic Press, New York, USA, or "Peptide Synthesis", Izumiya et al., Maruzen Co., Ltd. (t975), al. , Pp. 207-400 "Haruaki Yajima, Tokyo Kagaku Dojin Co., Ltd. Π 977. J Examples include azide method, chloride method, acid anhydride method, mixed acid anhydride method, DCc method, and activity. Esthetic method, method using wood reagent E, carbodimidazo method, redox method, DC CZ active (eg, HOWS, HOBt, HOSu) method.

 The peptide has a reactive carboxy group corresponding to one of two types of fragments that are bisected at an arbitrary position in the peptide bond, and a reactive amino group corresponding to the other fragment. When the condensate formed is condensed by a conventional method for synthesizing a peptide and the resulting condensate has a protecting group, it can be produced by removing the protecting group by a conventional means.

Protection of functional groups that should not be involved in the reaction of the raw materials and protective groups, removal of the protective groups, activation of the functional groups involved in the reaction, etc. ₃ you can choose

 Examples of the protecting group for the amino group of the raw material include canolepobenzoxy, t-butynoleoxycanoleboninole, t-aminole; , 2 black

'Nore-Zinnore, Chicka Bo-Nore, Adamantinoreoxycanolebonil, Trifle, Loacetinole, Phthalinore, Honoreminore, ◦ -Nitrofenii Snorefe

O PI — A—

 Nyl, diphenylphosphinoinoiloyl 4-methoxy2,3,6-trimethylpentenesulfonate and the like. Examples of the protecting group for the carboxyl group include, for example, akiester (eg, meth, eth, propyl, buty, t-butyl ester group), pendi est group, pout benzene V est group, "0— Methoxy pent diester group, one mouth; 1 pentyl 1 / ester / 1 group, penzhydri ester group, force; bopenzoxy hydrazide group, 1-butoxy carbazyl hydrazide group, triti / hydrazide group Can be

 Protecting groups for the arginine guadino tomb include, for example, nitro, tol, p-methoxybenzenesulfonium, kabobenzoki ^, isoboni, and adaman. Tiloxycarb; = / t, 4-Methoxy 2,6-dimethylbenzenesulfonol group, pentamethynolensens / U-hole group and the like. In addition, the guadino group of ^: may be protected in the form of an acid (eg, venense; Hongjun, tonosulfonic acid, hydrochloric acid, sulfuric acid, etc.) salt.

The hydroxyl group of threonine can be protected, for example, by esterification or esterification. Suitable groups for this esterification include, for example, groups derived from carbonic acid such as acetyl group, lower alkano group, penzoi group, alkaro group, benzyloxycarbo-nore group, ethynoleoxycanorebo-group and the like. Examples of groups suitable for _{3- or} etherification, such as benzyl, include benzyl, tetrahydroxy-, and 7-butyl groups. However, the hydroxyl group of threonine does not have to be protected. Methione may be protected in the form of a sulfoxide. Examples of the activated carboxyl group in the raw material include, for example, the corresponding chrysanthemic anhydride, azide, and hot-pottenol (pentachlorophenol, p--trophenol) Nore, IT-Hydroxysuccinimide, N-Hydroxydzintriazole, N-zdoxydoxy 5 -Nonolevo ^ nene-2, 3-Zikaboxymidido, etc.) can give. The peptide bond formation reaction may be carried out in the presence of a dehydrating agent (eg, a carbodiimide reagent such as dicyclohexylcarbodimid or cabodimidazo).

 The present 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 coupling reactions. For example, anhydrous or water-containing dimethylformamide, dimethyl sulfoxide, pyridine, chloroform, dioxane, dichloromethane, tetrahydrofuran, acetic acid, U, —methylbilidone, or a mixture thereof. Can be

 The reaction temperature is appropriately selected from a range known to be applicable to the peptide bond formation reaction, and is usually about -4 Ot to about 60, preferably about

 -20 is appropriately selected from the range of about 0.

 If the product has a protecting group after completion of the condensation reaction, use a standard method! ) You can leave. Such conventional methods include, for example, reductive methods (eg, hydrogenation using a catalyst such as palladium black, reduction with metal sodium in a liquid ammonia), and acid lysis (eg, triflic acid, fluoride). In the presence of a sulfur-containing compound such as hydrogen, methanesulfonic acid, or thioazole, the above-mentioned acids or acid lysis by a mixture thereof are exemplified. The peptide of the present invention produced as described above can be collected from the mixture after completion of the reaction by ordinary means for separating peptides, extraction, distribution, column chromatography, and the like.

'In terms of polypeptide-protein complexes, the carrier' and the mixing ratio of carrier and hapten (in this case, peptide) depend on the carrier. If an antibody can be efficiently produced against the hapten immunized by priming, any hapten may be coupled at any ratio.For example, bovine serum albumin, bovine thyroglobine, etc. may be used in a weight ratio. In this case, a method is used in which a force is applied to the hub ten at a rate of 0.1 to 20, preferably 1 to 5.

 In addition, various condensing agents can be used for the hapten and carrier force pulling, and gnoletaldehyde dehydrobodimide and the like are conveniently used.

When immunizing with the polypeptide (I) or the protein complex, mammals to be immunized include experimental animals such as sheep, goats, rabbits, mo / remots, rats, and mice. In order to obtain a u 杬 body, a rat mouse is preferable. For example, when immunizing a mouse, it is possible to immunize a mouse subcutaneously, intraperitoneally, intravenously, intramuscularly, intradermally, etc. Prefer to inject. The immunity interval, immunity amount, etc. are also highly variable, and various methods are possible. For example, immunization is performed 2 to 6 times at 2 week intervals, and 1 to 5 days after the final immunization, preferably 2 to 4 times. A method using cells after days is often used. The amount of immunization should be the amount of peptide at a time, per mouse]? 0.1 g or more, preferably! It is preferable to use 0 g to 300 g. Also, before擠出the脬, partial blood collection line, after confirming the increase of antibody titer in blood, Shi desirable to perform fusion experiments with ^^ cells ₃

The above-mentioned saturating fusion between the ferocytic cells and the lymphoid cells can be performed, for example, by excising the extracted mouse cells from hypoxanthine-guanine-phosphoribosyltransferase deficiency (HGBBT ") or thymidine kinase-deficient (K-). _Three fusions to lymphoid-like cell lines, such as appropriate homologous or heterologous (preferably homologous) myeomas with the ability of -y—

 Inores, E. A fusion agent such as lietin glyco-nore (PEG) is used. Of course, it is also possible to add dimethisoxide (PMSO) or other fusion promoters. The polymerization degree of PEG is usually 100 to 6000, the time is 0.5 to 30 minutes, and the concentration is 10 ^ -80. The cells can be fused at a high impeachment rate by treating them for 4 to 10 minutes at ~ 55 to 6. The fused cells can be grown in a hypoxanthine-aminopterin-thymidine medium (HAT medium; Nayya, Vol. 256, (P. 495, p. 1975) can be used to selectively proliferate.

 The culture supernatant of the cultured cells can be screened for the production of the desired antibody, but the antibody titer can be screened as follows. I can do it. That is, in this case, the presence or absence of antibody production against the immunized peptide can be first examined as a first step by a method such as the BIA method or the EIA method, but various modifications of these methods are also possible. It is possible. As an example of a preferable measurement method, one method using EIA will be described. A carrier such as cellulos beads is coupled with, for example, a persimmon anti-Macsimiminoglobulin antibody according to a conventional method, and the measured culture supernatant or mouse serum is added thereto, and the mixture is incubated at a constant temperature for a certain period of time. Less than

4 to 40). After that, the reaction product is thoroughly washed, and an enzyme-labeled peptide (purified after force-purifying the enzyme and the peptide according to a conventional method) is added, and the reaction is carried out at a constant temperature for a certain period of time. After thoroughly washing the reaction product, the enzyme substrate is added, and the reaction is allowed to proceed at a constant temperature for a certain period of time. Then, the resulting color product can be measured by absorbance or fluorescence.

 -Cells that show growth in selective medium and have only antibody activity against the peptide used for immunization should be subjected to limiting dilution].

OMPI — L υ—

Is desirable. Similarly, screening of the cloned cell supernatant is performed to reduce the glaze of the antibody.], A monoclonal antibody that is reactive with the immunized butide A hybridoma clone is obtained.

 Next, it is necessary for the antibodies produced by these clones to show reactivity with IF IF-r molecules themselves as well as the peptide used for immunization, or to haze. Used IF If-r! ? A method of examining reactivity using the IA or EA method, a method of checking whether or not biological activity (Ι Ι ί-Γ activity) is detected by this antibody] are used. In the latter case, it is necessary to purify IFIT-r. One example that is advantageously used is described below. Of course, a method of measuring the remaining IF1ST-II activity in the supernatant from which the immunoprecipitate has been removed using protein A is also possible. For example, a heron anti-mouse immunoglobulin antibody

 -Capsule is added to a carrier such as beads according to a conventional method, and the hybridoma supernatant or mouse blood to be measured is added thereto and reacted at a constant temperature for a certain period of time. After that, the reaction mixture is added to the reaction solution in a constant amount.

For example, as an I i * -a, a recombinant containing IFK-r derived from human peripheral blood lymphocytes with lectin and phorbo este, etc. After adding _β- IFH-r, which can be used as a solution, react at constant temperature for a certain period of time, and then determine the activity of IFr contained in the reaction. In this way, the limb yield of IF-r activity of the target antibody can be measured.

 Hybridomas cloned as described above can be used, for example, in a greedy medium, such as BPMI-164, medium supplemented with 0.1 to 40% bovine serum for 0 days, preferably 3 to 40%. 5 3 times, the culture solution

OMPI The monoclonal antibody can be obtained from the above, but in addition to the above, by inoculating intraperitoneally into a suitable mammal such as a mouse, and culturing the cell, and collecting ascites, the cell culture can be performed much more. It is possible to efficiently obtain a large amount of crab high titer antibody. For this purpose, for example, in the case of ^{mouse, 1 X 1 0 4 ~ 1} X 1 0 7 or mice BALB ZC etc. Also connexion inoculated before Minera old I and the like, preferably 5's 1 0 ⁵ - 2 I 0 ⁶ Invite the hive 9-doma into the SC cavity, etc., and collect ascites narrow 7 to 20 days later, preferably 10 to 4 days later. Antibodies produced and accumulated in ascites fluid are collected, for example, by Bowan's fraction, DEAE-cellose column chromatography, etc.! ;), The antibody can easily be isolated as pure immunoglobulin.

 The Noclo-na antibody of the present invention has the following properties.

 (1) Binds to the polypeptide (I) used for immunization.

 (2) Binds to IFN-r molecule and does not bind to IFN or IFif-

(3) It belongs to the IgGnb or IgGi subclass of antibodies by the assay method.

 (4) In SDS-polyacrylamide gel electrophoresis, only two bands completely matching the H chain and the: L chain of the standard immunoglobulin are shown.

 This monoclonal antibody can be safely manufactured, used and stored O

 According to the present invention, the obtained monoclonal antibody can be obtained by utilizing the EIA method or the B.IA method], and the amount of FN in the living body or in a test tube can be increased.

-In addition to being able to be used for the detection of r, for example, the production of antibody chromosomes has made natural or genetic The resulting IFN-r can be used to purify very efficiently.

E PN - for detection of r, as an example, the measured I 5 ¹ 7 * previously including materials or standard IF N-r, e.g., radioactive ® -? By the de or enzyme etc. j labeled the monochrome -After reacting with antibody and then precipitating an immune complex with protein A, for example, and reacting for a certain period of time, measure the radioactivity or Xe activity of the immunoprecipitate to easily reduce the amount of DiFir-r. Can be measured. When the competition method using an antigen is used, for example, the polypeptide (I) is radioactively or enzymatically labeled, and when a certain amount of the unlabeled monoclonal antibody of the present invention is reacted, the measurement is performed. The co-existing material containing IFN-r was used to precipitate the immune complex.], Or by binding to an anti-mouse antibody! ?, IF5-r can be quantified. As the radioactive or enzyme complex used here, purified I Fir- _r can be used instead of polypeptide (I). When using the antibody competition method, for example, partially purified IP If

-Immobilize r or polypeptide (I) on a solid phase such as a micro tray. As a fixing method, for example, I i Ν Ν -r or polybutide (I) is added to a phosphoric acid solution containing 0.1〗 weight and nou to 0.,

Float it preferably in 10-20, put this 100 ^ in each channel of the microphone p tray and process for 24 hours! It is possible. The material containing iPN-r, which was determined to be the antimonochrome of the present invention, was added to this report at 37! After reacting for 20 hours at 4 ° C, add radioactive or enzyme-labeled mouse heron antibody, and so on! 2.) IF _r can be quantified. The enzyme-labeled anti-mouse (eg, heron-antibody, etc.) in these series of EIA methods — 丄 —

 (By the Enzyme Link Dom-So-Vent Atsay) method]. A quick Atsey method using Machiscan (Fuguchi-sha) is possible. In addition, when the monoclonal P-na antibody of two types of hybrid-macrones having different antigen recognition sites is used, the so-called Sandwich method is used.)) It is possible to measure. Of course, these

 Various modifications are possible as long as an antibody suitable for the quantification of IFr using BIA can be obtained.], * Not limited to the above method.

 In order to purify IFN-7 ", the purified antibody is coated on a suitable carrier such as activated agarose beads according to a conventional method, and then applied to a column. Material containing crude I-r such as culture supernatant or disrupted cells is applied to the column, washed after adsorption, and then washed with a pick-up reagent such as ∑sc ir or IFN-r Depending on the method of elution under weakly acidic conditions with a high degree of inactivation], purification can be performed efficiently.

 The antibody column can be prepared, for example, by force-purifying the monoclonal antibody of the present invention purified from ascites or the like inoculated with the hybridoma with an appropriate carrier], and the following methods. .

Any carrier can be used, as long as IFN-r is specifically and efficiently attached after cou- pling, and then can be appropriately eluted. Activated agarose beads such as Affige-10 are conveniently used in the manner described below. The reaction between Affige-10 and the antibody is carried out in a buffer solution of 0.001-1 M, preferably 0.1 M, such as biforce-bonate. Reaction conditions are 0 ^β ~ 20,! Various values of ρ 可能 are possible from 0 minutes to 24 hours, but preferably, the conditions of 4 * C, 4 hours, and pH 3 to 10 are used. The amount ratio of Abuigel-10 and antibody to be mixed is such that the amount of antibody

OMPI circulation — I 4—

 Antibodies of up to 50 W are more likely to be repelled as much as possible. Therefore, any antibody within this range may be used, but an antibody of 30 or less is preferably used in consideration of the r ratio. The antibody-carrier conjugate thus obtained is thoroughly washed with the buffer used for the reaction, and then left for a few days or at a final concentration of 0.05 M.

O PI " Use a method such as adding Tanoamine / hydrochloric acid and reacting with a rock for 1 hour. D. After blocking the remaining unreacted active groups, fill them with an appropriate solution], use as antibody column it can.

 When purifying with the antibody column described above, for example, a sample containing human immunogenic protein is dissolved in a buffer solution near neutral, for example, phosphate buffer solution, tris-hydrochloric acid buffer solution, and adsorbed to the antibody column. . After washing the camb with the same buffer, IFN-r is eluted. Examples of the solution include a weakly acidic solution such as an acetic acid solution, a solution containing polyethylene glycol, a solution containing ぺ butide, which is more easily D-bound to the antibody than the sample, a high-purity salt solution, and a combination thereof. It is preferably used to promote the degradation of human IFN-IFN]).

 The column eluate is neutralized with a buffer according to a conventional method. If necessary, the above-described purification operation using the antibody force column can be performed again.

 The human I-IFIT protein solution obtained here is subjected to dialysis, and if necessary] this can be made into a powder by freeze-drying]. For freeze-drying, stabilizers such as sonorebitose, man = 1 tonole, dextrose, ma / u tose, and glucose can be added.

Thus obtained human immune Intafe port down protein shows a 1 0 ⁷ UZ more specific activity in WE scan activity measurement by cytopathic effect deter test vesicular stomatitis Huy scan (VSV) on human amnion-derived WISH cells .

 Here, the method of producing U ZW (cut ^) as the activity of IFN was performed as follows. The international standard IFN- <∑ and the crude IFN-r derived from leukocytes were determined using the VSV cytopathic effect inhibition test on FL cell line derived from human amniotic membrane, and the titers were compared. Of leukocyte-derived crude IFN-r

OMPI The value was determined, and the standard product of IFN-r was used. IFii-r's power in the intended feed

In order to calculate 偭, the efficiency IFN-r was always arranged, and the results were calculated using the WISH-VSV system described above, and the titer was calculated from the ratio.

 The human immune interferon protein purified in this study is

(N) H-AZ _x Tyr Z ₂ Gin Asp Pro Tyr Val Lys Gl Al Gl Asn Leu Lys L73 Tyr Phe Asn Ala Gly His Ser As Yal Ala Asp Aan GI7 Thr Leu Phe Leu Gly lie Leu Lys Asn Trp Lys Glu Glu Ser Asp Arg Lys lie Met Gin Ser Gin lie Val Ser Phe Tyr Pile Lya Leu P e Lys Asn Phe Lys Asp Asp Gin Ser lie Gin Lys Ser Val Glu Thr lie Lys Glu Asp Met Asn Val Lys Phe Plie Asn Ser Asn Lys Lys Lys Arg Asp Asp Phe Glu Lys Leu Thr Asn Tyr Ser Val Thr Asp · Leu Asn Val Gin Arg Lys Ala lie His Glu Leu lie Gin Yal Me Ala Glu Leu Ser Pro Ala Al Lya Thr Gly Lys Arg L s Arg Ser Gin Met Leu Phe Arg Gly Arg Arg Ala Ser Gln-OH (C) (IE)

Those containing polypeptide that from wherein, A is Met or indicates a bond, Zi, Z ₂ represents a Cys or ½ Cys respectively] Amino acid sequence of. According to the purification treatment of the present invention, it is possible to obtain a polypeptide containing the polypeptide in an amount of 90 or more, especially 95 <1> on a dry matter basis.

According to the purification method of the present invention! ? Human ^ obtains a fin ^ "Feron protein bets can. 1) SDS one polyacrylic § Mi Doge _(17.5 ¾) IPO β indicating the molecular weight measured at 17,000 ± 1,000 by electrophoresis 2) It has cysteine or half cystine or methionine as the amino terminal amino acid.

 3) H— Lys— Arg— Lys— Arg— Ser— Gin— Met— Leu— Phe— Arg—

 Binds to a monoclonal antibody against Gly-Arg-Arg-Ala-Ser-Gln-ΟΗ.

 According to the present invention, the D-purified human immune interface protein can be obtained by conventional methods.

• Use the same usage for the same purpose as the I-I FN obtained by the method! ) It can be used, but it has less contaminating proteins and pyrogens than conventional products, so it can be used safely as an injection drug substance.

 The human I-IFN protein of the present invention has anti-inoles, anti-tumor, cell growth inhibitory and immunopotentiating effects. The human I-IFN protein of the present invention can be mixed with sterilized water, human serum albumin (HSA), physiological saline or other known physiologically acceptable carriers, and can be administered parenterally or topically. it can. In the case of Kiyoshi, a normal person can receive 100,000 to 100 million ut, preferably 50 to 60 million ut, by intravenous injection or intramuscular injection. The preparation containing the human I-IFN protein of the present invention may contain other physiologically acceptable activities such as salts, diluents, adjuvants, other carriers, buffers, binders, surfactants, and preservatives. Components may also be included. Preparations for parenteral administration may be sterile aqueous solutions or suspension ampoules in physiologically acceptable solvents, or sterile powders (usually I-I), which can be used after dilution in physiologically acceptable diluents. It is provided as an amplifier (obtained by freeze-drying FN solution).

 Further, the above-mentioned preparation containing the human immune interferon protein contains 1 to 99 other active ingredients relative to the substance of the present invention, such as IFN-β or IFN- (9 or interleukin 2 or any other lymphokine). May be.

OMPI — I 8—

 In the present specification, when abbreviations for amino acids, peptides, protecting groups, active groups, and their ponds are indicated by abbreviations, they are based on the abbreviations by IΠPAC—I 订 BCGommisaion on Biological Nomenclature or common abbreviations in the art. The following is an example. When there is an optical isomer with respect to amino acids, etc.]), the L-isomer shall be indicated unless otherwise specified.

 DN A: Deoki 5 Riho: Baby Acid

 A: Adenine ''

 T: Thymin

 G-: Gwanin

 C cytosine

 RN A; ribonucleic acid

 d A T P; Dexyadenosine triphosphate

 d T T P: Deoxythymidine triphosphate

 dG T P

 Π T P '亍' o J

 A P; Adenosine triphosphate

 E D T A; Ethylenediaminetetrasulfonic acid

 S O S Sodium sodium dodecyl sulfate

 G y: Glycine

 Al a '.-Aranin

 Vaa: Paris

 Leu; Leucine '

 11 e r isoleucine

S ex: Se! / -1 y-

T h r;

 Cy s: Sistine

 M e t;

 Glu: Klutamic acid

 A S J) Asno laginic acid

 Lys resin

 Ar g Arginine

 Hi s: Histidine

 P θ Pheninolealanine

 T 7-r: Chiro. Shin

 T r p Trif. Tohwan

 Pro *.

 A s n · Asno La Gin

 Gin

 z: Carbobenzoxy

 • Boc: t-butoxycarbo ^:

 H "br: 4-Methoxy-2,3,6-trimethylbenzenesoni

Pme ： Ventamechi Pengens Hoji ^

ΟΒτι ： Shichibuchi esthetic

 0ΝΒ: 1Τ—Hydroxy, n—5-no Bonen 2,3 -Zika ^ Boxy

 Midoste

 T> C C: ΪΓ, Ν'-disc hex '/ V force Bozimid

D CTJ: ίΓ, dicyclohexyl ゥ rare

 E0NB: P-Droxy 5-Nobo / ^ Ne 2,3 -Zika Boxy

Ο ΪΙ HOB: U—Hydroxypenzoto Liazoi

 CHA: Awesome mouth

 DCE A: Exit mouth

 TEA: Triethylamine

 TFA: G! ;

 USA: Methanesulfonic acid

 THF: Tetohito, Roblanc

 DMF: Dimethiformamide

 MeOE: Methaneau

 AeOEt: firewood acid

Easy drawing

Fig. 1 shows the elution pattern of the monoclonal antibody of the present invention on the DEAE cellose column, and Fig. 2 shows the elution pattern of the monoclonal antibody of the present invention. ₀ indicating the result

FIG. 3 shows the results of the electrophoresis of the human immunointerferon-feron protein obtained by the purification method of the present invention shown after Example 13, and FIG. 4 shows the molecular weight variation similarly shown after Example i3. The result is shown. Figure 5 shows the primary structure (Tong group arrangement) of pHIT 3709 plasmid obtained in Reference Example II (V8), Figure 6 shows the reference structure 2), ptrp 701 and ptrp 771 in <<), and Figure 7 shows the reference example 2 (iii) pHIT trp 1101 and Fig. 8 shows the construction of Reference Example 2 (iV) (pEIT cell rp 2101), and Fig. 9 shows the ω recombinant i FN-r And fc) show competition with I FIT-r immobilized on the solid phase. FIGS. 10 and 11 show (a) Escherichia coli extract containing or not containing IFN-r and (b) enzyme against the Monica-Nanole antibody according to Examples 20- (i) and 20- (ii), respectively. 1 shows the competition with the paired peptide of the present invention which was recognized. --Best mode for carrying out the invention

 In the following examples, thin-layer chromatography was performed using the following developing solvents, using Mek's Sili-Ige Plate 60 F £ 54 or Funakoshi Pharmaceutical's Cell-Port-Splate, Abysse SF. Was.

Ef ¹ : Chlorophonorem: Methano -〃: 鏵 acid-9: 1: 0.5

Bf ^2:醉酸E Chi: pyridinium Jin: acetic acid: water - 30: 1 0: 3: 5

 Bf ^: Kuho Rohonolem: Methanol: k = 7: 3: 0.5

 Pf: n-butanoyl: viridine: acid: water-30: 20: 6: 24

Bf ⁵ : 齚 酸 チ: _n —butano-: 齚 jung: water = 1: 1: 1: 1: 1

 Example 1

 (ί) Manufacture of Z- Ser- Gin-OBu 7

 Z-Gln-OBu * 10! Is dissolved in methano

The black was reduced as a catalyst in a stream of hydrogen. The catalyst was removed by filtration and the solvent was distilled off.

¾J was dissolved together with Z-Ser-OH 7.5 ^, 30273 6.8 in DMF 250 W and cooled on ice. Under eternal cooling! ) C C 7. 15? And add 0 at room temperature for 4 hours

Stirred for 12 hours. Were removed and precipitated DC Y, the residue after the evaporation of the solvent was extracted into AcOUt 3 0 0 W, 4% aHC0 3 water, 0.2 IT hydrochloride, washed with water and dried over anhydrous sulfate Na Application Benefits um. The solvent was distilled off, and the precipitated crystals were collected by filtration with an ether. After drying, the crystals were recrystallized from c 『5Ci. Yield 6.5, Yield

O PI

, ^{5 1.2 ¾, Rf 1 0.6 4} mp 9 7 -. 1 0 0 Wa, [a] ¾ ⁵ - 24.1.

 (c = 0.40, methanol)

Elemental analysis as C ₂ oS ₂₉ 0 ₇ li3

 Calculated: C, 56.72; H.6.90; N, 9.92

 Experimental values: C, 56.21; H, 6.72; N, 9.76

 Production of (β) Z-Ala-Ser-Gln-OB *

Dissolve Z-Ser-Gln-OBu ¹¹ 4.2 3 in methanol 30 and mix. Radium black was reduced as a catalyst in a stream of hydrogen. The catalyst was removed by filtration, and the residue obtained by evaporating the solvent was purified to give Z—Ala—OH 2.34? , HONB 2.2

It was dissolved in a mixed solvent of AcOEt 200 *, dioxane 200 », and DMF 100» and cooled with ice. Under cooling! CC 2.38? After stirring at 0 for 4 hours and at room temperature for 12 hours, the DCU was filtered off and the solvent was distilled off. CH ₃ C and ether were added to the residue, and the crystals were collected by filtration, dried, and then recrystallized from CH ₃ CN. Yield 3.7 2, Yield 75.3 R, Rf ¹ 0.60, mp. 16 5 — 17 0 t, [a] ¾ ^a —4 1.2 ° (c = 0.50, Methanol)

Elemental analysis as C ₂₃ H ₃₄ 0 ₈ N ₄

 Calculated: C, 55.86; H, 6.93; N.11.33

 Experimental values: C, 55.58; Η, 6 · 74; Ν, 11 · 12

(1) Production of Z-Arg (Pme) -Ala- Ser-Gln-OBu t

Z-A 1 a-S β rG 1 η-0 Βu ⁴³ · ⁴⁶ was dissolved in methanol ³⁰⁰ W and reduced with palladium black as a catalyst in a hydrogen stream. The catalyst was removed by filtration, and the residue obtained by removing the solvent was prepared from Z-Arg (Pme) -OH-CHA 4.549.

With _{Z-Arg (Pme) -OH f} HOB 1.9 f was cooled and dissolved in DMF 1 5 0 permanent. Under cooling, DCC 1.9 was added, and the mixture was stirred at 0 for 4 hours and at room temperature for 15 hours. DC DC was removed by filtration, the solvent was distilled off, AcOEt was added to the residue, and --A precipitate was obtained and filtered off. After drying, it was reprecipitated with methano- and AcOEt. Yield 4.5 5 75.5% ^{yield, Si "1 0.5 7, mp} 1 3 0 -. 1 34 ° C, [] ^{5 - 24. Γ (c = 0.26} , methanol 〃)

Elemental analysis as C ₄₀ H ₆₀ 0 _{1] L} N ₈ S-H ₂₀

 Calculated: c, 55.22; Η, 7.00; if, 12.88; S, 3,69

 Experimental values: c, 55.23; H, 6.93; 12.54; s, 3.48

 (iV) Z-Arg (: Pme) -Arg (: Pme)-Ala— Ser-Gln-OBu

 Z— Arg (: Pme) — Ala— Ser— (Jin— OBu 7 4.3

 It was dissolved in W and the palladium black was reduced in a stream of hydrogen. The ^^ was removed by filtration, and the residue obtained by evaporating the solvent was purified using Z-Arg (Pme) -OH-CHA 3.24.

 Prepared Z—Arg (Pme) —OH, EOB with 1.42 nt

 And cooled forever. Add ID C C 1.4 1 under cooling at 0 for 4 hours at room temperature

 For 20 hours. The DCU was removed by filtration and the solvent was distilled off.

Was added to obtain a precipitate, which was collected by filtration. After drying, it was reprecipitated. IKfi4,4, yield ^{7 1.7, Ef 1 0.63, mp} . 1 25-

1 3 0, [] ^{^ 3 - 1 8.0 (c =} 0.4 0, methanol)

Elemental analysis C ₅ 7H ₈₆ 0 i ₄₂ Ti ₂ S ₂ -H ₂₀

 Calculated values: c, 54.9S; H, 7.12; N, t3.50; S, 5.15

 Experimental values: C, 54.66; H, 6.92; N, 13.32; S, 5.34

 (V) Manufacture of Z-Arg (Pme) -Gly-OBu

 Z-Gl OBu 7 13.0 was converted to MeOH 500: and catalytically reduced in a stream of hydrogen using black as a catalyst. The solvent was separated off, the aqueous solution was decompressed and concentrated and the residue was dissolved in DMF200W. In addition, Z-Arg (Pme) — 0Η ·

■ CHA 20.0 ^ Y]? The prepared Z-Arg (Pme) -OH, HOB 75.4 ^ was added, ice-cooled, and DCC8.2 was added, followed by stirring for 48 hours. Precipitated DC

OP After concentration, the mixture was concentrated and dissolved in AcOEt 50. The AcOEt layer 4 HaHC0 ₃ water, 1 0? 6 Kuen acid water in washing净後, Ha ₂ and dried S0 ₄ ^ After condensation, was collected as沈凝added petroleum Penjin. Yield 19.8 (96.8). mp. end 1-73, [] ²³ + 0.2. (c = 0.9, DMP), Bf ¹ 0.62

Elemental analysis (as 3 ₃ ιΗ45 θ ₇ Ή ₅ S

 Calculated: c, 58.93; H, 7.18; ir, 11.03; s, 5.08

 Experimental value: C, 53 * 42i H, R. 58; HO. 95; S, 4.84

(Vi) Manufacture of Z-Piie-Ar CPme -Gl -OBu ¹¹

 Z-Arg (me) -Gly-OB ^ 1Q.0 was catalytically reduced in MeOH 500 W, and then transferred to DMF300 <J. This with Z-: Plie-OH 4.72,

Add HOBt ZZ 59, cool with ice, add D C C 3 «59, and! Stir for 5 hours. The separated DC was separated and the solution was concentrated.

40. The AcOEt layer was washed with 4HTaHC0 water, 10 $ citric acid water, and dried with ifa ₂ SO. After concentration, -te ^ was added and the mixture was collected as crystals and recrystallized from MeOE- ヱ -te. Yield 1 G.5 ^

(85.3). mp .1 Q 1-103 ° C, C «^ °-8.3 ° (c = 0.9,

D M F), Ε Q.64

Elemental analysis C ₄₀ H ₅₄ 0 ₈ as ₆ S

 Calculated: c, 61.67; H, 6.93; N, 10.79; s, 4.12.

 Experimental values: c, 61.66;: s, 6.56; 10.93; s, 4.14

 (Vii) Production of Z-Le -Bhe-Arg (Pme) -Gl -OBu *

Z-Phe-Arg (Pme) -Gl-0 But 5.5 was contact-reduced in MeOH 300 * ?; then, it was transferred to DMF 300 W. This Z-Le-OH DCHA 3.3 1 ^ 7] Z-Leu-OH, HOiiB 1.47 — 20—

Cool, add DCC 1,6 8 and stir for 48 hours. The precipitated DC Y and泸別, concentrated, OAcOEt was dissolved in AcOEt 3 0 0 layer was诜狰with 4% HaHC0 ₃ water〗 0 Kuen acid solution and dried with Na ₂ S0 _4. After enrichment, ₃ yield was庐取as a powder by adding ether & 2 f (98.3 ^) m P, 1 7 1 - 1 7 3 Ό, C «¾ 6 - 1 5.5 (c = 0.9, DMF), Bf 1 0.64

Element removal C ₆ H ₆₅ 0 ₉ 2T ₇ , S

 Calculated: C, 61.93; H, 7.34; iT, 10.99; S, 3.59

 Experimental value: C, 62. (32; H, 7.37; 11.08; s, 3.59

 (Vfii) Boc—Michiichi: Production of Leu—Phe—Arg (Piae) —illy—OBu

Z—Leu—Phe—Arg (Pnie) —Gly—OBu 76.0 was reduced by corrosion in 200 W of MeOH and then transferred to DMF 150 ”. Boc-Me 7-11 0H.DCEA 3.0 ] Add the prepared Boc Me Shichiichi OH, H0 1.39 ^, cool on ice, add Dcc 1.59, stir for 15 hours, discriminate ₃ folded DCs, concentrate, and concentrate BicQH- AcOE dissolved seven, 1 056 and washed with click E phosphate solution, then dried and concentrated in Na ₂ S0 _4, E -.. Tenore was泸取as crystals by adding yield 6.2 ^ (9 3.1%). mp. 1 9 2— ί 95 ³ C,

〔¾1 9. (c = 1.0, DMF) ₅ Rf ¹ 0.6 4

Elemental analysis C ₄₈ H ₇₆ 0 ₁₀ W ₈ S ₂

 Total male: c, 5.8.27; H, 7.74; I.33; s, 6.48

 Experimental values: c, 58.48; H, 7.79; 1] .- 34; S, 5.98

 (IX) Production of Boc-Gln-Met-Le-Phe-Arg (Pme) -Gl7-0H

Boc-Met-Le -Phe-Arg (Pme) -Gl7-0Bu ^t 5,5 ^ TFA

Was added and shaken at room temperature for about 0 minutes, followed by concentration, addition of ether, and extraction and drying. This was dissolved in DMF 5 and cooled on ice. One 2 O —

1.8 * was added. To this; Boc-Gln-OH 1.85 H0 1.49, DCC 1.90]? The prepared Boc-Gln-Ol was added and mixed at 15 o'clock. After concentration, AcOH was added, and AcOEt was added to obtain a precipitate, and the Tto yield was 5.3 ^ (89.8%). mp. 181-183X: (decomposition),! : A] 1 1 9. S (c = 1.0, DHF), Ήί 丄 0.30

Elemental analysis C49 Η ₇ 6012 Κ 10 S ₂

 Calculated: C, 55.45; H, 7.22; IT, 13.20; s, 6.04

 Experimental value: C, 55.33; H, 7.17i ir, 13.34; S, 6.20

 (X) Production of Z-Ser-iTHliH-Boc

 Z— Ser— OH 1 0.0, septum force bathet 6.2 y Dlii "

15 Cool forever, add Η01 Β 8.0 and DCC 9.3

Stir for 15 hours. The separated DCs were separated, concentrated, and then dissolved in AcOEt. AcOEt layer, 4% NaHC0 ₃ ', and washed铮1 0% Kuen acid solution, and dried over li s 304. After contraction, add ether and extract as crystals.

OR o Yield. 3 (50.45S) mp. 95-98 Ό, C «-6.2 (c = 0.8 _i D MF), Ef ¹ 0.62

Elemental analysis C ₁₆ H ₂₃ 0 _{s 3}

 Calculated: c, 54.38; Η, 6.56; ίΓ, Π.89

 Experimental values: C, 54.77; H, 6.88; 12.29

 (Χί) Production of Z-Arg (Pme) -Ser-S "HNH-Boc

 Z-Ser-iTHlTH-Boc3 was transferred to DMF50O after the thigh was placed in MeOH300. To this, add Z-Arg (Pme) -OH, ΗΟΒΐ1.5 prepared from Z-Arg (Pme) -OE-CHA S.29], cool on ice,

Add DCC 2.3 and stir for 15 hours. The precipitated DC was separated, concentrated and reduced to AcOE. AeOE layer, 4% iTaHC0 ₃ water, 1 0 - '- and it washed净in Knin acid water: dried with ITA ₂ S0 _4. After concentration, add ethanol and collect as a precipitate. Yield 7.45 (93.7). mp. 100-1 (Π XT, C « _D -0.9 ° C c = 1.2, DMF), Bf ¹ 0.5 1 Elemental analysis As CssH ^^ O ^ I ^ S

 Calculated value: C, 55. (36; Η, 6.86; H3.62; S, 4.46 Experimental value: c, 55.49; H, 6.94; 13.14; S, 3.86 (fl) Z-L3TS (li " r) -Arg (Pme) —Production of Ser-3iH: ErH-: Boc

Z-Ar g (Pme) -Ser-Ι ΕΙΓΗ-Β oc 3.9 was subjected to catalytic reduction with MeOH 300 0 and then transferred to DMI “50”. Z-I rs (M "r) -0H, HOB

After adding 0.88, the mixture was cooled on ice, and: Dec1, 34 was added and stirred for 20 hours. Precipitated DC13: was separated and concentrated, and then AcOE was added to obtain a powder, which was recrystallized from MeOH-AcOEt. Yield 5.1 ^ (9 3.4 $ 6)

 g

mp. 10 3-0 5, [a-7.2 ° (c = 0.8, DMF), Bf ¹ 0.57 Elemental analysis C ₄₉ H ₇₃ 0 ₁₃ H _g S ₂

 Calculated: 55.50; H, 6.94; if, 11.89; s, 6.05 Experimental: c, 55.70; H, 7.15; IT, Π.60; s, 5.63

(Xiil) Z-Arg (Pme) -Lys (M7r) -Arg (Pme)-Ser-NMTH-: Boc

 Z-Lys (Mtr) -Arg (Pme) -Ser-HHiJ "H-Boc 4.8 f was dissolved in MeOH 300 f and subjected to catalytic reduction, and then dissolved in DMF7. Arg (Pme) -OH'CHA 2.8 Add prepared Z—Arg (Pme) — '0Η, add HOBt 0.74 ^, cool on ice, add: DCCU 2 ^, and stir for 15 hours. Separately concentrate and dissolve in AcOE

OMPI -2B- AcOEt layer 4% lTaHC0 _3-permanent, and净1 0 Kuen acid 7_R, and dried over Na2S0 _4. After concentration, ether was added and the precipitate was collected and reprecipitated from MeOH. Yield 5.8 f (89 · 8). mp.136-137,

(A) ^2S -6.6 ° (c = 1.0, DMF), Ef ¹ 0.58

 D

Calculated elemental folding C ₆ 6H ₉₉ 0 E _{6 ίί 13 S 3:. C} , 55.56; Η 3 6.99; ΐί, 12.78; S, 6 Ryo 4 Found: C, 55.34; H, 7.Q7 ; ir , 12.50; S, 6.59

(XiV) Z-L73 (Mtr) -ArgCPme) 1 11 ァ 3CM r) 1 Arg C Pme) 1 Ser-- Production of KH H-Boc Z-Arg (Pme) -Lys (r) -Arg (Pme) -Ser-UHiTH-Boc 3.0] was dissolved in ieOH 15, reduced by sensitization, and then dissolved in DMF60. To this, -Lys (Mtr) -OH-DCHA 1.54]? Prepared Z-I ^ sCMtr -aE, HOBt 0.31 was added, ice-cooled, DCC 0.57 ^ was added, and the mixture was stirred for 15 hours. And泸別the DC Y that issued folding, after concentration, and in AcOEt, and CLEANING The AcOEt layer 4% aHCO water, 1 0 $ Kuen acid solution and dried with Na _n S0 _4. After concentration, ヱ -tel was added thereto to collect crystals, which were recrystallized from AcOEt. Yield 2.70 (72.8 $)

P. 1 23- 1 25, [of ⁶ - 5.9. (c = 1.1, DMF), Ef ¹ 0.57 Elemental analysis c _{82 3} o ₂₀ iri ₅ s ₄ Calculated: c, 55.73 Η, 7.02; N, 11.89; s, 7.26 Experimental: c, 55.89; H , 7.3Q; u, 11.72; S, 7.Q8

(XV) Bo c Gin-Met— Leu-: Phe-Arg (Pme)-Gly-Arg (Pme)-ArgC me)-Ala-Ser-Gin-OBu 7 Production Z-Arg (Pme) -Arg (Pme ) -Ala-Ser-G-ln-OBu ^t 1.0 --Dissolved in MeOH 100, catalytically reduced, and then dissolved in DMF 2 flW. Then, add Boc-Gln-Met-Leu-Phe-Arg (Pme)-(JI7-OH 0.85, HOBt135 5 ^), cool with ice, and add: DCC210 ^ The obtained DC TJ was separated and concentrated, and then MeOH was added to the precipitate to remove the precipitate, yielding 1.50 f (87.4%).

m _P. 2 16-21 C (decomposition), [Do: ¾ ⁶ - 1 1.8. (C-1.0,

DMF), Bf ¹ 0.4 3

Elemental analysis C ₉₈ Hi54 023 as 22

 Calculated values: c, 55.09; H, 7.27i, 14.42; S, 6,00

 Found: C, 54.81; H.7.33; U, 14.23; S, 5.79

 (Production of XVDH-Ljs-Arg-Lys—Arg—Ser—Gln-Met-Leu-Phe-Arg—Gl-Arg-Arg-Ala-Ser-Gln-OH

Boc -Gln-Me ~ LeU'— Phe -Arg (me) -Glj-Arg (Pme) -Arg (Pme) -Ala-Ser-Gln-OBut]. Q 9 ΚΤ Έ A10 After 5 vibration 0 min] was mixed with warm, concentrated, E -? it was collected泸a precipitate by addition of Te and dried ₃

 On the other hand, add TFA | 0 «f to -Lya, tr) -Ar (Pme) -Ls (M7r) -Ar (Pme)-Ser-ITHITH-Boc 0.83 ^ and shake at room temperature for 10 minutes. After mixing, the mixture was concentrated, added with ether and collected as a precipitate, and dried. Combine this: DMF, cool with dry ice-acetone,

6.2 N HCi / AcOEt 0.23 W and isoamyl nitrite (0.075 W) were added, and kept at −25 to −20 for 20 minutes (hydrazine test: insult). This was again cooled with dry ice-aceton and neutralized by adding TEA 0.25 «f. Dissolve the amine component in DM540β, cool on ice, add TEA 0.16 ^, add the azide solution prepared earlier, and allow 4 hours for 72 hours.

OMPI I mixed. The reaction solution was poured into dilute acetic acid water, and the deposit was removed.

Washed with CH ₃ cir. Yield ί.40 (8 1.5 ^), Bf 1 0.0 9. Among them, 40 OW ^ rO.15 5 MM s A is melted into τ FA—thioa-y-〃-one-methyl sulfide (8: ί ·· 1) 60, shaken at room temperature for 2 hours, and mixed. AcOiT¾400 ^ was added, and the mixture was concentrated. Ether was added, the precipitate was collected and dried. This was dissolved in a small amount of 1 AcOH and applied to a column of Sephadex G-25 '(2.2 X 12 Ocw). The fraction was eluted with 1 H AcOE, and the fractions of 160 words-28 were collected and freeze-dried, then passed through a column of Amberlite IBA-410 (Seul) and freeze-dried. Furthermore, purification was performed by HPLC using a column of ESE-: LS-410 (2J47.5c + 2.1430) to obtain the objective. Yield 45 ^

 , 24 o

 [N: ζ— 45.9 (c = 0.7, 0.1 N AcOH)

Bf ⁴ (cellulose) 0.20 '

 Amino acid analysis: Irs1, 71, Arg 4.71, Ser 1.69, Glu

 2.12, Gly 1.00, Ala 1.03, Met 0.32, Leu 1.30,

 Phe 1.08 (average recovery rate 77)

Example 2-Synthesis of Carrier-protein and Polypeptide Complex II

 Example · The combination of holibeptide obtained in (χνί) and Cycloglobulin (hereinafter G-) was performed according to the method of Goodfriend et al. (Science,

144, 1334, 1964). That is, the e. Lipeptide 2.5 was mixed with TG 3.75 ^, 2 50 mM phosphate buffer was added, and the mixture was stirred well in ice water. To this, a solution prepared by dissolving canolebodiimide hydrochloride ¾30.4¾ in distilled water 200 was added drop by drop to D. Then, the mixture was reacted for 3 hours while stirring in ice water. After the reaction, make sure that it is transparent with distilled water.

OMPI One one

 The mixture was freeze-dried to obtain 4.7 W of the protein complex.

 Example 3.

 Preparation of antigen for EIA for antibody detection

 The antigen for EIA was prepared according to the method of Kitagawa et al. (Janab Biochemistry, Vol. 79, p. 233, 1976).

 Ii) Introduction of maleimide groups into polypeptides

 E obtained in Example 1 (XVI). Rebeptide (3.50 nmoles)

 1 Dissolve in OO mM phosphate buffer pH 6.8, and add IT-hydroxysuccinate-midoester of 1- (4-forced hexoxime / V) maleimide 5 85 g (1. 5 moles) 70 «of — dimethyl chromamide solution After stirring for 30 minutes at 30, the mixture was fractionated using a Sephadex G-25 column to obtain a polypeptide fraction having a maleimide group introduced therein, and thus yielded 185 nmoles.

 0Ό Binding of maleimide-introduced polypeptide to / 9-D galactosidase Example 3 Maleimide-introduced polypeptide obtained in Π) ί 6.511 moles and β-T) Combined with 3.3 nmoles of lautosidase After 4 hours of reaction at 4 hours, the reaction was stopped with 4 12.5 nmoles of ^ S menolecaptoetano. /?-D-Galacto combined with Toshida-ze. Ribeptide was fractionated on a Sepharose 6B column and used in the following experiments.

Example 4

 EIA method

Antibody injection in serum or hybridoma supernatants immunized with the protein complex obtained in Example 2 was detected by the EIA method (Immunobu-Macologie, Vol. 1, p. 3, 19). 1978) to Wachi, serum some Haiburido - the Ma supernatant buffer _{- a (20 m M Na 2} HP0 4, 10 OmM -2-JTaCl, 0.1 Na¾, 1 mM MgCl ₂ , pH 7.0), diluted with 100 / ^])), the e obtained in Example 3. The mixture was thoroughly mixed with the peptide derivative 100, and was subjected to anti-JS at 24 for 24 hours. After the reaction, 3 celloses 100 conjugated with egrat anti-mouse IgG were added, and the mixture was reacted for 24 or 4 hours. After the reaction, the case was thoroughly washed with Puffer A containing 0.5% Tween 20, and 4-methyleneperi- f j-1: D-galactoside 20

5 (3 0 ^ added and after 2 hours at 3 Ryo, l OO mM force - 'sulfonate buffer - C _P H 10.5) Stop W 3 W addition reaction, fluorescence and fluorescence intensity in the supernatant (Excitation-365 nm, Exciton 450 nm).

Example 5

 Exemption 2¾

Each of 6 6-8 week-old BALBZC female mice was well mixed with Freund's complete adjuvant as a protein, and inoculated subcutaneously (primary immunization). Two weeks after the first immunization, the same amount of antigen was mixed well with Freund's complete adjuvant, and subcutaneously inoculated (secondary immunization). after 6 days of immunization rows Natsuta ₃ tertiary immune, blood was partially collected from the mice, was boss measure the anti钵価in serum by EIA method real ¾ example 4 described. Of these, r-2 mice with high antibody titers were inoculated intravenously with 120 g of antigen dissolved in 5 ^ of drinking water to achieve final immunization. anti-^ value of each mouse are shown in Table 1 ₃

OMPI --Immunized ft-mass anti-peptide antibody titer

(%)

 1) Serum dilution is 1 Z 1 000

 2) Serum dilution is 1 ZS 300

 3) Serum dilution is 1 end 800

 4) I: Undetectable

 5) 'Search ^ f

 BX: (Bound enzyme activity Z plus total enzyme activity) X 100 Example 6

 Cell fusion

 Immunization was performed by the method described in Example 5, and 3 days after the final immunization, the spleen was excised from the ^ 2 mouse, squeezed with a stainless steel mesh, filtered, and placed in an e-Gluz Minimum 'Etsencia Medium (MEM). The cells were suspended to obtain a cell suspension. A cell used for cell fusion: BAL BZC mouse myeloma-ma cell P3-63.A 8. ΐ (Ρ31) was used (current topix-in-micro / kusaiguchi-g-and-immunology).

'' 翥 81 1st page, 1st, 13 years 8 years). The vesicle fusion was carried out according to the original method (Neichia, 256 ', p. 495, 1975). That is, spleen cells and 3: 1 each contain serum and are washed three times with MEM and mixed so that the ratio of the number of stored cells to the number of P 3 LM is 5: 1. after thoroughly removing the ₃ supernatant centrifuged for 15 minutes to precipitate the means pursuant cells 800 rpm, precipitated ¾: disentangling籙Ku, 45% polyethylene glycol 〃 (: PEG) 6000 scratch (Koch light Co.) The fusion was performed by adding 0.3 «f and leaving it to stand in a warm water bath for 30 minutes. After fusion, HEM was added to the cells at a rate of 2 、 f per minute, and a total of 12? Of 3 112 112 ぇ was added, followed by centrifugation at 600 rpm for 15 minutes, and the supernatant was removed.環 Μ に 1 δ40 medium containing 10% fetal calf serum (Β Ρ ΐί Ι 1 640—10 FCS) Ρ 31 Μ ί W 640 2 ifl. The cells were inoculated into a 24-well multi-layered nut, Nu (manufactured by Limbu), and seeded in I-44 wells at a time. After the incubation, the cells were cultured at 37 ° C. in 5 CO 2 -furan apparatus. after time, HAT (hypoxanthine ί X 1 0 one ⁴ Micromax, Aminoputeri down 4 X 1 Cf ^'7 Μ, thymidine ^{ί.6 X ί 0 "5) ί} ) containing PBMI 1 6 40 - 1 0 FCS medium ( HA 0? Medium)! By adding each 1M solution, the selective culture of HA Τ was started. In the HAT selective culture, the old solution was discarded for 1 s after 3, 5, and 7 days from the start of culture, and

U suturing was performed by adding HAT medium. Proliferation of hybridomas was observed 10 to 14 days after fusion, and when the culture broth turned yellow (about 1 to 14 days).

1 0 ⁶⁾ supernatant was collected, in the EIA method, a search for the presence or absence of antibody. In this way, when the supernatant of 141 wells in which the growth of the hybridoma was recognized was examined, the antibody activity was strong in 2 wells (a 2-1 and r 2-! 00), and 2 wells (r 2 -62). , r 2 - 70) ₃ real discussions showed a weak Katsuchu 7 --Claw: =: Ng

 Each of three hybridomas (a2-! 1, 62, 100) showing positive antibody activity was cloned by limiting dilution. That is, two hybridomas were suspended in HPMI1S40-20FCS so as to reach 2 cells / cell, and the suspension was dispensed in a 96-well microphone plate (manufactured by Nunc Corporation) at a ratio of 0.1 per 1 kg. At the time of segregation, B mouse thymocytes were added as puper cells to make up to 5 x 10 "cells. Thus, after about two weeks, cell proliferation was observed. The supernatant was probed and the presence or absence of the antibody was examined by the EA method described in Example 4. As a result, in r2-11, 8 clones out of 19 clones, r2- In 62, 3 out of 54 clones, and in r2-100, 5 out of 47 clones showed anti-honey injection (Table 2).

OMPI 3δ-Table 2

 Anti-peptide activity of cloned hybridoma

実施例 8 一 —

Example 8 One —

 Monoclonal Antibody F F

 The binding ability of the monoclonal antibody to IFH-r was examined by the following method. That is, a culture of 2 to 3 clones of r2-ll, r2-62 and r2--00 cells, respectively, was added to a three-sense solution 300 bound with a rabbit egan anti-mouse IgG antibody. Add 300 pieces of Kiyoshi at room temperature

The reaction was performed for 18 to 20 hours. After the reaction, the cells were mixed well with physiological saline, and 550 cells of the IFN-a obtained in the following manner were added, and the mixture was reacted for 3 to 4 hours. After the reaction, the supernatant was collected, and the IFIT-r activity in the supernatant was measured by the cell transfusion effect (c) reading method using a microplate. (Applied Microbiology, Vol. 16, pp. 1706, 1968). That is, 50 μm was put into all the 96-well microplates (manufactured by Nunc Corporation), and IFFT sample was added to the first layer at 5 μm, followed by serial 2-fold dilution. Each © e you this Yo, the cell suspension 50 that so that adjusting the WISH cells in I W those 4 1 0 ⁵ to 20 ^ FCS-containing MEM was added, 24 hours, 37, cultured in a carbonate Gasufuran unit did. After culturing, the vesicular stomatitis virus (Niyuja-shi-strain)

Adjust with MEM to 200 TCID ₅₀ (Tissues-Cuche Infection Dose 50), add 50 to each well, and incubate at 37 ° C in a CO 2 gas flannel. did. Approximately 35 hours later, when the cells in the wells to which the iFir sample was added developed I00 CPE, the CPE in each well was observed with a cranioscope and 50% C: PE was developed. The IF if titer was the reciprocal of the dilution number of the sample.

 The FN-r sample used was a human peripheral lymphocyte, which had a naphthalin A 40 / ul and a 12-0-tetradecanoinyl phorbo-1-13 acetate 1 2 hours after stimulation with 5 ng,

O PI — Ϋ́ δ—

 The culture medium contained human cultures (acid, unstable in ρ Ε processing) and 4400 cuts. -If an antibody capable of binding to ιΐΊΤ-r is present in the culture supernatant of the cloned cells, IFIT-r activity added later binds to the antibody on cellose. In addition, the activity in the supernatant is reduced:> Yugo shows relatively strong IN-τ binding ability in the supernatant of r2-111 clone, and the added IP IT -r (550

Of the antibodies bound to the antibody (Table 3).

 Table 3

 Absorption ability of IF-T activity by Monoclonal antibody

実施例 9

Example 9

 Ascites in hybridomas producing monoclonal antibodies

BAL BZc mice that have been administered intraperitoneally with 1 x 10 ° clone cells in advance of mineral oil intraperitoneally, producing r 2-1 1. Antibodies capable of binding to IF ir -r By inoculating intraperitoneally I made it. Hybrid -〗 0 days after the administration of Ma intraperitoneally, where the anti钵価was collected ascites was measured by EIA method, showed antibody activity up to I 0 ^7-fold dilution. , The black - anti钵活拴in the culture supernatant of the emission cells are recognized to〗 0 ^4-fold dilution, about 1 0 0 0 times antibody activity by to ascites rises.

 Example 10

 Monochrome-Purification of Na ^ body

Using 4 W of the peristalsis obtained in Example 9 as a starting material, according to the method of Stalin et al. (Jana Biob Zika Chemistry, 256, 9975, 13.81). Monoclonal antibodies were tested. First After centrifugation 1 0, 0 0 0 Rotation 1 for 5 minutes to remove Fuiburi down like universal quality from ascites, phosphate buffer one meal brine (PBS: 8, ί m -lTaH 2 P0 4, 1.5m3i ΕΗ _{2 Ρ0 4, 2.7ηιΜ KCl, 137mM} kaCl, H 7.2) 2 80 nm <D purple Metropolitan absorption (a 280) is 1 2 ~! Diluted to a degree that indicates 度 = After dilution Add saturated ammonium sulfate solution to Sampnole to a concentration of 47%, perform stirring for 60 minutes while stirring at 4 ° C, and then centrifuge ( 1 G, C) (30 rotations, 15 minutes) to obtain a precipitate ^. The precipitate was dissolved in 20 mM Tris buffer solution (pH 7.9) containing 50 mM liaCl, and the solution 2 was subjected to filtration. Two hours later, two new same dialysates were used and dialysis was performed for another 5 hours. After dialysis, 100,000 rotations to remove precipitate

Centrifuge for 15 minutes, was adjusted so that the value of the supernatant Alpha ₂₈₀ is a concentration of 2 0-3 0. This sun: 7 m with a sufficient amount of 50 m Μ-Tris buffer containing ITaCl >> |

'DΞ52), and fractionation was carried out using a Tris buffer solution containing ₅₀ mM NaCl at an outflow rate of 1.5 a. Under these conditions, antibody activity is primarily

O PI 通] was recognized in the fractionation (Fig.〗). Antibodies were confirmed by SDS-polyacrylamide gel electrophoresis (SS-PAGE) according to the method of Laemli et al. (Naturia, Vol. 227, p. 680, ί970). That is, several fractions fractionated on a cellulose ammonium sulfate column were reduced with 2-methabetethanol, respectively, and subjected to 17% SDS gel; 30 volts, 24 hours electrophoresis. As a result, two bands, an E-chain and a I-chain, were found at a molecular weight of about 55 kDa and about 28 kDa, respectively (Fig. 2). The antibody fraction purified as described above was compared with — _r , and the binding ability was determined by adding IF-r 2200 σ according to the method of Example 8 to find that the binding ability was about 50%. Showed binding & to the antibody (Table 4). Table 4

 Example 11

 Subclass to which monoclonal antibodies belong

OMPI --The IgG subclass to which the τ2-\-nocchi-null antibody belongs was 10-fold diluted the fraction 17 purified by the method of Example 10 to obtain goat anti-mouse IgG〗, G2a, G2b, Precipitation reaction in agar with G3 antibody (Miles) (Octa-orchid-method: immunologica method., ^{ Γ? R Ge fusion fusion technique, black black, oxford,

1964). The result was a monoclonal antibody and goat anti-mouse.

One prominent band was observed with the IgG2b antibody, and no band formation was observed with the other anti-antibodies. Therefore, the monoclonal antibody was found to belong to IgG2b ('Table 5).

 Table 5

 Monochrome; ^ class belonging to ^ 侔

Example 12

? Example Motodori purified in the manner of .10] fractions mono click polyclonal antibodies 2 5 I (6 5.3 ^) the 0.1 M NaHC0 _3, pH 8: having conducted an晚dialyzed against 3 solution . After washing the Abuigel 10 (Bio'Rad) 25 thoroughly with a glass filter, float it in a 0.1 M NafiCO ₃ pH 8.3 solution and mix with the antibody. 4 hours at 'C.] The reaction was carried out with stirring. After that, let it stand at 4 C for-晚 and then use a glass filter

O PI The well was washed well with a 0.1 M liaHCOg pH 8.3 solution. A solution containing 0.1 M ethanolamine and 0.15 M NaCl (H 8.0) ¾: 25 ^ was added to the reacted gel, and shaken for 4.1 hour to block unreacted active groups that might remain. Thereafter, the gel was thoroughly washed with PBS, suspended in 0.1 l PBS 25 containing HaN ₃ and stored in 4. From the amount of antibody added and the amount of antibody in the recovered solution, 1 * equivalent! It turned out that 2.35% of the antibodies were bound. The reaction product thus obtained was packed in a column and used as an antibody column.

Example 13.

 Supernatant 60 obtained in Reference Example 4 contains 1 mM EDTA and 0.15 M NaCl

After dilution to 15 with 20 m Tris-HC1, pH 7.6 (TEH), the mixture was applied to an anti-IFN-r antibody column (prepared by the method of Example 12).

After thorough washing with TEN, an additional 0.01 «

 (Shell) Washed with TEN containing 0.5 M NaCl. Next, IFN-r was eluted with 0.1 M vinegar containing 0.25 M NaCl, and the eluate was immediately neutralized with 1 M Tris HC1 pH 7.6. The resulting solution was dialyzed against distilled water for 4 hours and 16 hours, frozen with acetone-dry ice, and freeze-dried to obtain a powder.

 The result was iiTF?].

OMPI ― ―

Protein (W) Total activity (ϋ) Specific activity (Ιί / ^) Recovery (Lysate supernatant 250.8 4 XI 0 ⁸ 1.6 x10 ⁶ 1 Antibody column treatment 2.0 1.5 x10 ⁸ 7.5 X10 ⁷ 37.5 (by viral activity measurement by cytopathic effect inhibition test of VSV for WISH cells (supra)) the specific activity of the human immunodeficiency I centers Feron protein was 7.5 X 1 0 ⁷ Xi /. of the protein sex shape below Too]?

 Properties of human immune interferon protein

 (0 molecular weight

 The protein obtained in Example 13 was treated with 2-butane ethanol.

The protein was identified as a single band when subjected to SDS-boriatari / u amide (17.5 *) electrophoresis (15 mV, 6 hours) and stained with Coomassie blue (FIG. 3). From the relationship between the migration distance of the molecular weight marker and the migration distance of the protein, which were simultaneously electrophoresed, the molecular weight of the protein was estimated to be 17,000 soil 1,000 (Fig. 4). On the other hand, another band was detected at a molecular weight of 33,000 ± 2,000 for the protein that was not treated with 2-meadow ethanol. Since this value corresponds to about twice the molecular weight of IFN-r of 17,000 ± 1,000, this band is considered to be derived from the IFN-r dimer.

(B) Amino acid analysis

Transfer the protein obtained in Example 13 to a glass hydrolysis test tube], add 200 volumes (V / W) of constant boiling constant hydrochloric acid containing 4 thioglycolic acid, and seal the tube under reduced pressure. After that, the mixture was hydrolyzed for 24, 48, and 72 hours with 110 watts. After hydrolysis, open the tube, remove the salt under reduced pressure, dissolve the residue in 0.02 N acid, and use Hitachi 8335 high-speed amino acid analyzer to perform the amino acid analysis. Carried out.

 Cystine and cystine were subjected to the formic acid oxidation of the above protein according to the method of Hirs [Methods EnzymoL 11, 197 (1967)], and then hydrolyzed for 24 hours in the same manner as described above to analyze amino acids. By all means]? Quantified as Sistine II. Amino acid analysis values were determined by averaging the values obtained after 24, 48 and 72 hours of hydrolysis. On the other hand, the values of serine, threon, tilidine and tributane were determined by extrapolating the hydrolysis time to 0 hours. Table 6 shows the results. Table 6 Detected Amino Acids) Detected Amino Acids 1 .3 Aspartic acid 13.4 Methine 2.9 Sleinine 3.6 Isoleucine 4.8 Serine 7.4 Leucine 6.8 Gutamate 12.5 Tyrosine 3.4 Purin •: | Hue = Panin 6.7 Glyin 3.8: Lysine 13.5 Phala = 5.3 5.3 Histidine 1.5 Cysteinic acid 1.3 Arginine 5.4 Parin 5.7 Tryptophan 0.9

ΟΜΡΙ One b— (Picture) Amino terminal amino acid analysis

 The protein obtained in Example 5 was used according to the method of Hirs [Methods EnzymoL 11,

197 (1967)], and the amino acid amino acid analysis of the Edman degradation method by Iwanaga et al. [Eur. J. Bioohem., 8, 189 (1969)] was performed. The produced thio-thiohydantoin-amino acid (PTH-amino acid) was purified using an Ultrasphere 0 DS column (manufactured by Atex (USA). 46 x 250 ram, particle phantom 5) using the Archer method [Altex Chromatogram, 3 , 8 (1980)]]), and were quantified using a Varian high-performance liquid chromatograph Model 5040 (US). As a result, PTH-methionone sulfone and PTH monocystic acid were detected. As is clear from the above examples, according to the purification method of the present invention, a substantially pure human immunointerferon protein having a specific activity of 7.5 × 10 ⁷ or more can be produced.

OMPI IFO The following shows the results obtained using the Noclona II anti-fly. Abrupt 1

 Immunity

 Fourth immunization was performed on the r-3 mice described in Table 1 of Example 5 in the same manner as in the second and third immunizations, two days after the fourth immunization, and 120 ig as in the fifth example. It was inoculated in the thymus with a solution prepared by dissolving the raw material in 0.5% saline.].

Example 15

 Cell fusion

 Three days after the final immunization described in Example I-4, spleen was excised from the mouse, and cell fusion was performed and HAT selective culture was performed by the method described in Example 6. As a result, in March 43, hybridoma was found to be buried, and the presence or absence of antibodies was searched for by the EIA method described in Jeongseon-jo 4. The result was 2 ゥ (r3-11, r3—19). In addition, strong antibody activity was observed.

Example 16

 13_

Each hybridoma (r3-1 !, a3-! 9) showing strongly positive antibody activity was cloned by the method described in Example 7. As a result, 9 * clones out of 21 clones in 7 * 3-11, and〗 5 clones out of 25 clones in 3-3- and 99 showed anti-tumor activity (Table 7). — —

 Of cloned highgly dorma

 Anti-peptide antibody activity Noiburi K--ma ot B / T ()

 o

 T 3 1 I I

 7 7

 0 1 I

 Λ

 C 7

 0 o

 7 7 f g Q

8 1 9

H 78

1 7 1 1 I

16 8 1

r 3-1 9 2 85

 3 88

 5 83

 7 80

 3. 77

 12 75

 13 T 3

 1 76

 16 80

 17 8 1

 18 90

 19 85

 20 9 1

 23 8 t

 24 82

i ⁰ Example 17

 Monoclonal anti-IF 21-A binding ability

 The binding ability of the monoclonal antibody to IFH-r was examined by the method described in Example 8. However, the 15 · κ-r sambu used in this example was obtained by combining human IFN-transducer with brassmid and working on E. coli.

(Recombinant i Fir -r; see Reference Example 4). As a result, strong I Fir -r binding ability was observed during the purification of Γ3-1-1..1 and Γ3-192 Bound to the antibody (Table 8). Table 8 Recombination with Monoclonal Antibody

 iFir-r activity

実施例 18

Example 18

 Monoclonal-Subclass to which the antibody belongs

 As shown in Example 9 for Asa-1, .1, Asa 13.20 钿 跑, ascites can be created by the method of *! ), The ascites obtained was purified by the method described in Example I, Example 0, and the obtained subclass of Monoclonal-anti-Fyle was defeated. . The results showed that a significant band was observed between goat anti-mouse Ig (Jl antibody) and that of both 7-3- -1.1 and r3-19-1.20 noclonal antibodies. The formation of Pando

OMPI -4-I didn't see it. Therefore, the r3-11.1 r3-f9.20 monoclonal antibody was found to belong to igGl (Table 9). Table 9

 Monoclonal-Subclass to which the antibody belongs

実施例 19

Example 19

 Determination of IFN-Γ by EIA method I

 Quantitative method by competition between fej I Fr for monoclonal antibody and (b) I FW-Γ immobilized on solid phase

 Phosphate buffer containing 0.1 M sodium bicarbonate to express i F K- ^ 15 gZW expressed in E. coli as shown in Example 13

(pH 8.0), this 100 was dispensed to each tube of a 96-well microphone plate, and reacted at 4 for 24 hours. After the reaction, dissolve 100 μl / volume of phosphate buffer containing 2 cow beabumin in 100 / <each to block the excess binding site of the gel, process at 4 C for 24 hours, and use it for ELISA.

O? I Made a brat. More in Bureto, prepared Yo, 1 contains about 50 amount of the antibody of an antibody that maximum amount 00 ^ antibody r 3 -! 1 1.1) solution with various concentrations ^{(75, 3 X 0 2,} 1.25 10 3, 5 1 0 ³ ,

F ΙΪ - r含有液中の I F ir - r が ¾aされることが判明した。 A mixture of the recombinant I PK— r prepared at 2 × 10 ⁴ and 8 × 10 ⁴ / W) was allowed to react at 37 ° C. for 3 hours, and reacted at room temperature for 3 hours. After the reaction, the well was washed well with phosphate buffer, 100 g of a goat anti-mouse igG antibody labeled with horseradish oxidase was added, and the mixture was reacted at room temperature for 3 hours. After the completion of the reaction, wash the well with phosphoric acid 3 »mouth liquid, and add a substrate solution obtained by adding 22 5? -Year-old soap-diamine and 1 hydrogen peroxide to 1 10,1 1-citrate buffer. After adding greed 100, the enzyme reaction was carried out at room temperature for 15 minutes, and the reaction was suspended with 4N sulfuric acid. After detention, the amount of dye was quantified at 492 nm using Matiskian (Tertec, Thailand). Figure 9 shows the results. Using this ELISA method, 3 and 0 ² to 5 10 ³

It was found that IF ir -r in the F ΙΪ -r-containing solution was ¾a.

Real 20

 Quantitative method of IPN-r by EIA method H

 Monoclonal-na Quantitative determination of fe) IF 2i-r sambu and fc) Xuesin-labeled ぺ -butide for anti-honey

Jeongjeong example 20 -Π)

 The recombinant Ι ΡΪΤ-Τ obtained in Reference Example 4 was converted to various degrees (64,

^{320, 1.6 10 3, 8 1} 0 3, 5 x 10 4, 2 10 5 S / ml) to prepare, the 1 00 * a certain amount of r 3 - 1 1.1, r 3 S.20 antibody-containing solution The reaction was carried out with 100 at room temperature for 2 hours. The enzyme-conjugated polysaccharide obtained in Example 3 was added to this, and added for 4 hours. I responded. After the completion of the reaction, this mixture was added to 3ί cellulose solution 00 conjugated with a perforated anti-mouse IgG antibody, and reacted at room temperature for 4 hours. After the reaction, the reaction was repeated in the same manner as described in Example 4, and the fluorescence intensity was measured. As a result, monochrome - Na antibodies (r 3 - 1 1.1, r 3 - 19.20) binding enzyme-linked Boripe tides quietness conductor to the, IPN of 320TT - slightly inhibited in the presence, 8 1 0 ³ u presence Below, it was almost completely inhibited (Figure 0). Thus by the present method ^ Then door] ?, 320 - 8 x 1 0 3 S / mi of τ i * if - possible the quantification of I i ¹ r of § containing solution and ¾ ivy.

 (Example 20 -ίϊ)

 Although the principle is the same as that of Example 20-1), a modified method under JK was carried out as a method for automatically and quickly measuring a large number of sambs using a multi-scan.

 (1) Preparation of mouse I sG adsorption plate

9 D play for microtest holes bets I Nunc one Imm noplate I: Nunc mouse to each well of (made in Denmark) I g G solution _{(1 Q μ S Zmi 0.0 1} M borate瑷衝solution p H) injected 20 0 Then left for 4 minutes. After removing the solution, a 0.02 M buffer containing 1 BSA (pH

7.4) 300 was injected and left for 3 hours in a chamber filter, and the liquid was removed.

 (2) Preparation of Anticrona antibody r 3-I 1.1 adsorption plate

 Prepare a 350,000-fold dilution of r3-〗 1.1 in 0.02 M phosphate buffer 0.1 (pH 7) containing 0.1% BSA, and use the mouse Ig prepared in (1) (each well of the J adsorption plate). Then, after injecting 50ί, leave it at 1 て for 1 て.

Wash three times with 0.01 μl buffer ( _Ρ 7.4) to produce antibody-adhered plate.

Ο ΡΙ -¾2- (3) Operation

The test sample or standard IFH-Γ is adjusted to an appropriate concentration with buffered narrow A (0.02 M phosphoric acid buffer at pH 7 containing 0.1 M HaCl, 1 mM MgCl ₂ , 0.1 Si BSA, 0.1 Na). Dilute and inject 75 into each hole of the plate prepared in (2). Next, 5 ^ of the enzyme sample of Example 3 diluted 30 times with buffer A is injected into each well of the plate. The reaction was carried out for 3 hours with the addition of a bulk. The reaction was continued at room temperature for 3 hours. After removing the reaction 硖, the plate was washed four times with 0.01 M phosphate buffer 硖 (pH 7.4) 75.

 For the color development reaction, each well of the plate was prepared with buffer 硖 A in each of the wells of 1.5 4 trofu-1-β-1D-galactobino s / do (Wako fine, special grade)

Inject 1 50 / ^ at room temperature!晚 Reacted. 0.4 Ii carbonate耰街greed (ρΗ 1 0.5) 1 0 0 / * was added to each well, sealed accompanied color reaction, 405 nm of absorbance microtiter Tracey _¾ Nbureto for photometer Titertek

It was measured by Multiskan (manufactured by U-One (USA)).

 Fig. 11 shows the standard curve of iFir-r. Table 10 includes i FH-r

For S test samples, the results obtained by this method were compared with the antiviral activity (AVA). Table 11 shows the daily variation of this method. The variation coefficient was 7 * 4, giving good results.

One OM—PI one 1

 Table 10

 Ten

 Table 11

Fifteen

30

twenty five The crude I FN -α content used in Examples 13, 19 and 20 (i), (ii) was determined by the method shown in the following Reference Example! ? I got it.

Reference example〗

 (\) Isolation of mBlTA encoding human I-IF F

 Human lymphocytes]) prepared lymphocytes were treated with 12-0-tetradecanoylhobo 13-acetate (TSA) (15 ag / ml) and concanavalin A (40 ^ gZ *). The cells were cultured in tfBPMl-l640 medium (including fetal bovine serum of ί0) for 3 hours to induce. 24 hours later, this induced one! 0 human lymphocytes were converted to a solution of guanidine (5 II guanidine, 5 liters of ethanol), 50 mΜ.

After destruction and denaturation with a Teflon homogenizer in Tris (玨 H7.6, ίOmM EDT A), N-lau mouth iriza sodium succinate was added to the mixture, and the quenched mixture was added to 5,7 Cesium cesium lust

[Layered on 5.7 M cesium chloride, 0.1 M ethylenediaminetetraacetic acid (SDTA) QMI, and centrifuged at 4,000 rpm for 30 hours at ί5 using a Beckman SW 27 rotor to obtain SNA strain.

The BU precipitate was dissolved in a 0.25 K-lauroyza / u-sodium sodium cosinate solution and precipitated with ethanol to obtain 8.3 W of BIfA. this! ? The NA was adsorbed on an oligo (dT) Senorelose column with a high salt solution [0.5 K NaC, 1 OmM Tris · EC £ pH 7.6, ImM EDTA, 0.3 SDS].

OMPI One

 A solution of mRNA containing poly (A) in a low salt solution (1 OmM Tr-HCl-pH 7.

By eluting with 1 m EDTAjO.3% SDS), mSNA 70 Dz was collected from the solution (A).

This mENA is further precipitated with ethanol, dissolved in a solution of α2 ″ (1Οαα Kris-HC1 pH 7.ό, 2 mMED, 3% SDS), and treated with 65 for 2 minutes to obtain 1 Q to 35 % sucrose density gradient centrifugation (at ₂ 0 using a rotor of Beckman ST727, 25000rpm-C2 hour centrifugation) by the the child]? to obtain a fractionated by 2 2 fractions. EN per each fraction A portion of A is injected into oocytes of African frogs, and the interferon activity in the synthesized protein (cytopathic vesicular stomatitis virus (VST) on SH cells derived from human amniotic membrane) Measured using an effect inhibition test: Anti-inoles activity (Stewart. W. Ξ. THe interferon -Sys em, (

Springer Berlinl 97 was measured, and the activity of 1-5 juniftono gENA was detected in fraction 12 (sedimentation constant S value showed 12-14 S). The mRNA of the fraction 12 thus obtained was about 20.

'' (ϋ) Synthesis of single-chain DNA

Using the mHNA and reverse transcriptase obtained above, a reaction solution of 10 O (5 g of mfNA, 50 ori '(dT), 100 units of reverse transcriptase, 1 dATP, 1 dCTP, dGTP and iTTP, 8 mM MgCl _2l 50 mM PC 1, 1 Q mM dithionite, 50 mM Tris-HC 1 pH 8.3) 42 * C Incubate for 1 hour, then extract with phenol Then, the cells were treated with 0.1 N NaOH at 70'C for 20 minutes to decompose and remove RNA.

 (iii) Synthesis of double-stranded DNA_

Complementary DN A ⁵ 0 ^ of the reaction solution of the single chain synthesized here (Mena and O Li

OMPI The reaction was carried out for 42 hours in the same reaction solution as described above except that it contained no dT, thereby synthesizing a double-stranded D1A.

 (iV) Add d C tile

The duplex 3) nuclease S 1 5 0 <a reaction solution NA (duplex]) NA 0.1 M Kokusan sodium ρΗ4.5, 0.25 M NaCl, 1.521 ZnS0 4, 60 Interview - Tsu City of SI Nuclease) at room temperature for 30 minutes, deproteinize with phenol, precipitate DNA with ethanol, and add terminator transferase to 5 Q fig reaction mixture (double-stranded DMA, 0.14 M codylic acid). Cali, 0.3MTris (base) pS 7.6, 2 Jichiosureito _{'ImM C 0 C1 2, 0.15} mM dCTP, 3 0 Interview - Tsu Tota! - Mina transferase peptidase) by use ^ 3 min 3 7 in duplex) NA Approximately 20 dextrin 'chains were extended at the 3 ^ end of the DNA. In these series of reactions, about 300 ng of double-stranded DNA having a deoxycytidine chain was obtained. .

 (V) Cleavage of Escherichia coli plasmid

 On the other hand, a restriction enzyme Pste was added to a reaction mixture of 50/50 (NaOH, 6 mM Tris-HCl (pH 7.4), 6 mM MgClo. 6 mM 2-mcapto) with 10 / «of E. coli plasmid pBR322 DNA. Ethanol, 100

Bovine serum albumin, 20 units of Pstl] was used for 3 hours at 37 t to excise one Ps11 recognition site in PBR322 DNA, deproteinized with phenol, and then terminated with terminal transferase. 5 0 / ί ^ of reaction (:. DNA 1 0 a, 0.14M Kakoji acid strength Li, 0.3 M Tris (base) pH 7.6, 2 mM Jichiosurei _{toe, 1 mM C 0 C1 2 0.15mM} dGTP, 3 0 Interview -Butardina transferase) for 3 minutes at 37 t! To extend about 8 deoxyguan chains at both ends of 3T of the above plasmid pBR322 DNA.

Ο ΡΙ --(Vi) CDNA association and transformation of E. coli

The synthesis obtained in this way! 0.1 μ9 of heavy chain DNA and 0.5 μ9 of the above plasmid pBR322 are heated in a solution of 0.1 NaCl, 50 mM Tris-HC1 pH 7.6, ImM EDTA for 65 2 minutes and 45 2 hours, and then cooled. E. coli 117776 was transformed according to the method of Enea et al. [XOL BioL 96 _t 495 (1975)].

 (Vii) Isolation of CDNλ-containing brasmid

)をト リ： cステノレ法〔 Crea. R.ら、 Proc.Approximately 8,500 tetracycline-resistant strains were isolated in this way, and each of these DΝDΝ was immobilized on a -Troserose filter. (Grunstein ML and flogness, DS Proc. NatL Acad. Sci. USA 72, 3961 (1975)) —D.7. Goeddel et al.'S report [Nature, 295.503 (1982)]:!: — IF amino acids Amino acid NO. L-5 (Cys-Tyr-Cys.Gln-Asp) and amino acid NO. 77-82 (Lya-Gin-Asp-Met-Asn-Val) based on the sequence]? Postal (TCCTGGCA TA C ³ 'and 5' respectively)

) To Tri: c Stenole method [Crea. R. et al., Proc.

Natl. Acad. Soi. USA, 5765 (1978)]. A reaction solution of 50 β was prepared using the T4 oligonucleotide (Kinase) for this lignonucleotide (oligonucleotide 0.2 «, 5 OmM Tris' HC1 pH, 1 OmM MgCl ₂ , 10 mM mercaptoethanol, 50 μαγ -3 ^c, P ATP, 3 ^ = · T4 Polynucleotide (1) 37 hours, 37 ', and 5'-end was labeled with ³² 。. Using this labeled oligonucleotide as a probe, Lsuwn et al. A strain that is associated with the D- し た fixed on the above-mentioned trocellulose filter according to the method [Nucleic Acids Res., 9, 6103 (1981)] and reacts with the two oligonucleotide probes by autoradiography. Were isolated. -S8- Plasmid DNA was isolated from the cells of each of these strains by the Anorecali method [Birn-boim II C. & Doly, r. Nucleic Acids Res .. 7, 1513 (1979)]. Next, insert the plasmid DNA insert with the restriction enzyme Pstl]), cut out)), select the separated plasmids containing the longest fragment of the insert, and replace the plasmid with PHIT3709. It was named. Next, the primary structure (base sequence) of the mRliA sequence inserted into this pSIT3709 plasmid was determined by the dinucleotide synthesis chain termination method and the Maxam-Gilbert method. Its primary structure is shown in Figure 2.

Reference example 2

 (I) The promoter part of tributophan synthesis in the colon bud [Expression plasmid] [Promoter, D If A fragment including operator, 276 base pairs, Bennett, G. Ν · et al., J. Μοΐ · Biol., 121, 113 (1978)] was constructed to construct a plasmid ptrp601 (vector is pBR322).

 —Plasmid PBR322 with restriction enzymes EcoRI and Ava

Of the EcoRI-Ava I fragment containing the Tet-cyclin resistance gene The white part is DNA. I chose Limerase I large fragment. This fragment was ligated to the cleavage site of PvuII of ptrp601 using T4DNA ligase to construct ptrp701 (FIG. 6).

(B) Next, ptrp701 was partially digested with Clal to eliminate one of the two restriction enzyme Clal cleavage sites present in ptrp701 to obtain a fragment in which only one of the two Clal cleavage sites was cleaved. . The resulting portion was filled in with DN JL volimase I page fragment and then religated with T4DNA ligase to obtain ptrp771 (FIG. 6).

(Hi) pHIT3709 is cut with the restriction enzyme Pst I to obtain the IFN-r structural gene. --The obtained Pstl fragment was obtained. This fragment was partially digested with the restriction enzyme BstNI to obtain a BstNI-: Pstl fragment in which the BstNI site in the IFN- structural gene was cut. After filling the j) margin of the BstNI cleavage site with DNA polymerase I difragment, an oligonucleoside adapter containing a protein synthesis initiation codon ATG chemically synthesized by the above-mentioned triester method CG AT AA TGTG TACTGCC

 TA TT AC A C AA TG A C GG

 Was ligated with T4DNA ligase.

 On the other hand, a fragment obtained by cutting the ptrp771 with the restriction enzyme Pstl and the restriction enzyme Clal into the IFN-Γ gene to which the above-mentioned adapter was bound was inserted into the downstream of one of the motors of T4 and T4. The DNA was ligated using DNA ligase to construct an Ffi-r expression plasmid pHIT trplIOl (.12 W).

 (ίν) pHITtrplOl was further improved and the following passage was established.)) pHITtrp2101 was constructed.

 First, ptrp601 was treated with restriction enzymes Clal and Hpall to obtain a Clal-Hpa! I fragment 0.333 Kb containing a trp promoter. This fragment was cut with Clal and treated with ethanol phosphatase.

Trp promoter and combined using T 4 DNA ligase to obtain a pHITtr _P 2101 entered the two series 1 ((2l ^).

 Escherichia coli 294 was transformed with this plasmid ρΗΙΤ · trp2101 according to the method of Cohen et al.

 E. 'coli294ZpHIT · trp2101 was obtained.

Ginseng example ¾

 -E. coli 294ZpHITtrp2101 containing 8 μ9 / ηί >> tracycline, 0.4% casamino acid, and 0.1% glucose.

Ο ΡΙ

WIPO, The cells were cultured in a Meyer at 37 t, and when the growth reached KU220, 3β-indolino-inorea-clinoleic acid (IAA) was added to 30 S ¹ / ^, and the cells were further cultured for 4 hours.

Reference example 4

 The culture solution 1.2 obtained in Reference Example 3 was centrifuged to collect bacterial cells, and the cells were suspended in 0.05 Tris-HC1 pH 7.6 containing 60i of 10% sucrose. 0.2 M feninole methinolace refoni phnoleorate, (PSF) 0.3 m, 5 M NaCl 2.4 nl, 0.2 チ レ ン ジ ア ア ミ ミ ア （（（(Ε D Ε A) 2.4 M, 1 M Sunoremidin 2: 4 W, 5 ^ Z lysozyme. Add 2.4 «, leave at 0 for 1 hour, treat with 3 7 for 5 minutes, and further apply ARTEK (USA) ultrasonic It was processed at 0'C for 30 seconds with the Xiao Xian.

 The lysate was centrifuged at 105,000x for 1 hour to collect the supernatant 66. Industrial applicability

 The novel polypeptide of the present invention can be used to produce a monoclonal antibody that binds to a human interferon. The monoclonal antibody is useful for detection and purification of human Γ-type interferon.

O PI

 V / IPO

Claims

— Scope of the request 1 set  (M) '' (C) H-X-Lys Arg Ser Gin Met Leu Phe Arg Gly-Y-OH  (N)  (Where X is a bond or lie Gin Val Met Ala Glu Leu  (C) I  Ser Pro Ala Ala Lys Thr Gly Lys Arg. It has from 1 to 16 amino acids counted from its C-terminus in the tide chain  (N)  Y represents Arg Arg Ala Ser '' (C)  A peptide chain having 1 to 5 amino acids counted from its N-terminus in a peptide chain bound by Gin. 2. The polypeptide according to claim 1, wherein Y is Arg Arg Ala Ser G In. 3. The polypeptide according to claim 1 or claim 2, which is Lys Arg. 4. A complex obtained by chemically bonding the polypeptide of claim 1 to a carrier protein.  5. The complex according to claim 4, wherein the carrier protein is bovine thyroglobulin.  6. A cloned hybridoma comprising mammalian kidney cells and homologous or heterologous lymphocyte-like cells immunized with folibeptide or the protein complex thereof according to claims 1 to 5. . 7. The hybridoma according to claim 6, wherein the mammal is a mouse. 8. The hybrid-doma of claim 6, wherein the lymphoid cells are myeloma. . * 9. Monochrome for the polypeptides described in claims 1 to 3 Null antibody o  10. The monoclonal antibody according to claim 9, which binds to human r-type interferon.  11. The monoclonal antibody according to claim 9, which belongs to the subclass of Ig G2b or Ig (J1).  12. Monoclonal antibody comprising culturing the hybridoma according to claim 6 in a greedy medium or inoculating a mammal to produce and accumulate monoclonal antibodies, and collecting the monoclonal antibodies. Production methods for antibodies.  13. The production method according to claim 12, wherein the mammal is a mouse. . 14. A method for purifying human r-type interferon, which comprises purifying a substance containing crude human r-type interferon using the monoclonal antibody according to claims 9 to 1 \. . 15. The ~ range ninth of claims as an antibody, of〗 claim wherein monochrome - Detection of human § interferon by radio I Takeno Apu Si method or Enzaimu Imunoassi method with Toku徼the use Rukoto naphthalocyanine antibody _Λ Ο Η_,