JPH02485A - Novel human interleukin-4, recombination vector for manifesting the same factor and transformant transformed by the same vector - Google Patents

Abstract

PURPOSE:To efficiently produce novel human interleukin-4 containing sugar chain having specific physicochemical properties by combining manifestation vector containing SV40 initial promoter and CHO cell. CONSTITUTION:A cell derived from ovarium of Chinese hamster deficient of dihydrofolate reductase (CHO cell) is transformed by IL-4 manifesting vector composed by co-existence of DNA fragment having gene of human interleukin-4(IL-4) in downstream of SV40 initial promoter and DNA fragment having gene of dihydrofolate reductase in downstream of a promoter fulfilling a function in eucaryon cell and resultant transformant is cultured to obtain IL-4. Said IL-4 has 17000-19000 molecular weight, 2000-4000, preferably 2500-3500 molecular weight of sugar chain part and 8.5-10.5, preferably 9.8-10.1 isoelectric point.

Description

Detailed Description of the Invention [Industrial Application Field] The present invention provides (1) a novel human interleukin-4 having an immunostimulatory effect, (2) a novel recombinant vector for expressing the factor, and ( 3) It concerns a novel transformant transformed with the vector.

[Conventional techniques and (problems)] Interleukin 4 (hereinafter abbreviated as IL4) is a lectin yaphorbol ester or other protein produced by T lymphocytes stimulated by antigens, and is a protein produced by B lymphocytes. Differentiation/proliferation, T lymphocyte differentiation/proliferation, mast cell differentiation/proliferation
It has various functions important for the immune response of the living body, such as proliferation [Y, Noma et al., Natur
e, 319.640-646.1986 (hereinafter referred to as Document A); F. Lee et al., Pr.
oc.

Natl, Acad, Sci, USA, 83.20
61-2065.1986 (hereinafter referred to as Document B)
:E, 5everinson et al., Eu.
r, J.

Immunol, 17.67-72. 1987
and T. R. Hosmann et al., Proc.
Natl, Acad, Sci, USA, 8
3. 5654-5658° 1986]. Therefore, due to its strong immunostimulatory effect, it is effective against infectious diseases, AIDS, cancer, etc.
Application to the treatment and prevention of immunodeficiency diseases is eagerly desired [R, Fernandez-Botran et al.
, Proc, Natl, AcadSCi, US8,
83.9689-9693, 1986; R, Pa1aC
iO3et a.

See EHBOJ, 6.91-95.1987].

Natural human IL4 can be obtained by drug treatment of normal human peripheral blood lymphocytes, cancerous human T cell lines, or human T cell hybridomas, but its production amount is extremely low, and the physical chemistry of the substance itself is limited. Even the physical properties (e.g., total molecular weight, molecular weight of sugar chains, isoelectric point, etc.) have not been confirmed. Furthermore, it has been difficult to produce purified human IL4 in a sufficient amount for clinical use. Therefore, we have applied genetic recombination technology, which has made rapid progress in recent years, to produce large amounts of these physiologically active substances in trace amounts in vivo. It is expected that a method to obtain this information will be established.

Recently, mouse IL4 CDNA was cloned (References and Reference B), and human IL4 CDNA was also cloned using this DNA as a probe [T., Yoko et al.
ta et al, Proc, Natl, 8cad,
Sci.

USA, 83.5894-5898.1986 (hereinafter referred to as Document C)], its nucleotide sequence was revealed, and its expression in monkey cells was reported (References C and PC
(See International Patent Publication No. WO87102990). An expression system using yeast as a host has also been reported (J, EXp, 1 (ed, 166).
476 (1987)). However, the expression by monkey cells is transient, and the cells used for production are human r.
Since the cells die after producing L4, the cells cannot be reused and new cells must be constantly transformed. Furthermore, transformation requires a large amount of vector DNA and a great deal of labor, making it unsuitable as a method for supplying sufficient amounts for clinical use. If it were possible to establish an expression vector and a host cell line that can continuously produce human 1-IL4 with high production efficiency using genetic recombination technology, the above-mentioned drawbacks would be eliminated, and a complete protein with constant physicochemical properties could be established. Although it is expected that the novel human IL4 can be produced in large quantities, there have been no reports to date of the creation of such an effective expression system.

[Means for solving the problem] To date, many expression vectors and hosts for expressing the vectors (e.g., Escherichia coli, Bacteria,
This includes prokaryotes such as actinomycetes and lactic acid bacteria, as well as eukaryotic cells such as yeast, mold, and animal cells. ) has been found.

However, it has recently become clear that in order to obtain a specific gene product, especially to produce it with high production efficiency that can be used at an industrial level, it is necessary to select the optimal expression vector and host for that gene. I've come to understand.

Therefore, the present inventors conducted intensive research to develop the optimal expression vector and host for efficiently producing human IL4, and as a result, a DNA fragment containing the human IL4 gene downstream of the SV40 early promoter was found. , dihydrofolate reductase (hereinafter referred to as DHF) downstream of an appropriate promoter.
It is abbreviated as R. )) has constant physicochemical properties by using an expression vector in which DNA fragments containing oral genes coexist and Chinese hamster ovary-derived cells deficient in DHF R (hereinafter abbreviated as CHO cells). They discovered that a completely new human IL4 could be obtained, and completed the present invention.

The present inventors first investigated the mouse IL4 expression system as an initial stage of IL4 research. At first, we chose Escherichia coli as a host and tried to express it. The reason for this is that among the large number of hosts,
This is because E. coli has the fastest growth rate and can be cultured efficiently, and when considering a new expression system for a certain target gene, it is common to first try using Escherichia coli.

However, with E. coli, this goal could not be achieved using conventional genetic recombination techniques. For example, tr
A fusion protein of the signal peptide of 01111)F and mouse IL4 was expressed under the control of the p promoter, but the amount was extremely small. Also tac.

Even transformants could not be obtained under the control of promoters such as PL and ompF.

Next, using yeast, which is a eukaryotic cell, as a host, GALlo,
Mouse IL under the control of promoters such as Pf-105
4 was attempted, but the objective could not be achieved using conventional genetic recombination techniques.

Therefore, as a result of continuing research by limiting the host to animal cells,
Expression vector containing the SV40 early promoter and CHO
By using a combination of cells, they were able to discover for the first time a highly productive expression system. When examining the expression system for human IL4, we took into account the homology between the mouse IL4 nucleotide sequence and that of human IL4, and
As with the m gene 6 mouse IL4, we thought that it would be difficult to efficiently produce it using E. coli or M mothers, so we developed S from the beginning.
Expression vector containing V40 early promoter and C)-10
Using different combinations of cells, we can roughly express D H
It has been found that by incorporating the FR gene and simultaneously using a DHFR-deficient strain, an expression system with high production efficiency can be obtained.

Furthermore, novel human ILs produced by this expression system
It was confirmed that No. 4 has characteristic physicochemical properties (molecular weight of sugar chain moiety, etc.).

In other words, expression systems using CHO cells as hosts have been attempted for several physiologically active proteins, but the sugar chains that bind to the amino acid moieties differ from each other in terms of their binding position, chain length, sugar composition, etc. They are completely different depending on the physiologically active substance. This fact is comprehensively manifested as a difference in the molecular weight of the sugar chain moiety.

For example, as shown in the table below.

Table ■ 1) Roger Dijkmans et al.
, J. Biol, Chem.

262, 2528-2535.19872) Fu
-Uen Lin et al, Proc.
Natl, Acad, Sci.

USA, 82. 7580-7584. 19853)
Randal J, Kaufman et al.
al, Mo1ecular andCell
ular Biology, 5, 1750-1
759.1985. Therefore, it cannot be said that if the CHO cell expression system is used, a glycoprotein with certain characteristics can be obtained regardless of the type of physiologically active substance. This time, it was completely unexpected that human IL4 with sugar chains having specific physicochemical properties was obtained.

[Structure of the Invention] Therefore, the present invention provides (1) a novel human [4, ( 2>(i) SV40 early 7omo J-cD [to human I
t4 gene, and (ii) a D fragment downstream of a promoter that functions in eukaryotic cells.
A novel human IL4 expression vector characterized by the coexistence of a DNA fragment having the HFR gene (3) A DHFR-deficient CHO cell line is transformed with the human IL4 expression vector shown in (2) above, and jW and (4) a novel human IL4 produced using the transformant shown in (3) above as a host.

The amino acid sequence of the protein portion of human IL4 is shown by the formula 8 (see Document C) a In the above formula, the IL4 of the present invention is
It was confirmed that up to the 4th glycine was missing, and there were 129 amino acids from the 25th hisdecine to the 15353rd phosphorus (see Examples 8 and 9).

The human IL4 of the present invention is completely novel in that the sugar chain moiety is different from previously known human IL4.

The molecular weight of human IL4 of the present invention is 17 to 19 kd (kilodaltons). The molecular weight of the protein part alone is 149B2
Therefore, the difference of 2000 to 4000, preferably 2500 to 3500, is considered to be a sugar chain.

On the other hand, the molecular weight of human IL4 expressed in monkey cells described in PCT Patent International Publication No. 487102990 is 22 kd, and therefore the carbohydrate is considered to be about 7 kd. From this, human IL4 expressed in monkey cells has more than twice as many carbohydrates as the human IL4 of the present invention,
It was found that the molecular weights of the sugar chain moieties in both cases were significantly different. Furthermore, the molecular weight of yeast-expressed human IL4 is 60k.
d, and the molecular weight of the sugar chain portion is expected to be 45 kd, so the difference in molecular weight from the sugar chain of IL4 of the present invention is extremely large.

Among the humans [4] of the present invention, preferred ones have an isoelectric point of 8.
5 to 10.5, more preferably 9.8 to 10.1.

Among the human IL4s of the present invention, those containing at least mannose, fucose, and galactose as neutral sugars constituting sugar chains are more preferable. Furthermore, the amine sugar contains at least glucosamine. It contains at least N-acetylneuraminic acid as sialic acid.

Among the human IL4s of the present invention, those having a content of mannose, fucose, galactose, glucosamine, and N-acetylneuraminic acid of 3.0 to 4.0 and 1.0 to 1.0, respectively, per total mole of sugar chain moieties are generally preferred. 2.0, 2.5~
3.5, L5 to 5,5 and 1,5 to 2.5 moles.

Although the human IL4 of the present invention is sufficiently pure, it does not have to be a single compound. The present invention includes homogeneous human IL4 and heterogeneous human IL4 [4] which have slightly different sugar chains within the above-described gyrus.

The novel human [4] shown in (1) of the present invention is produced using the human It4 expression vector shown below and a transformant transformed with the vector.

The present invention includes the human expression vector [(2) above] and the transformant [(3) above].

The human IL4 expression vector of the present invention comprises the above (i) and (
In addition to the DNA fragment shown in ii), it is produced by joining (iii) a DNA fragment containing an E. coli replication origin and a drug resistance gene.

The human IL4 gene is obtained by preparing messenger RNA (mRNA) from cells capable of producing IL4, such as normal human lymphocytes, and cloning the cDNA synthesized from this using reverse transcriptase into Escherichia coli (cDNA library). I can do it.

For example, a part of mouse IL4 cDNA (described in Documents A and B) can be used as a probe for selection by cross-hybridization, and human IL4 cDNA can be selected by cross-hybridization.
The activity of human IL4 can also be used as an indicator (q). Also, by using these methods together, efficient selection can be made.
It is also possible to select by hybridization using a synthetic A ribomer having a part of the base sequence (described in Document C) as a probe.

Recently, αlRN of lymphokine genes such as IL4
It has been reported that the base sequence rich in adenine (A> and uracil (U)) present in the 3' untranslated part of A is involved in the instability of these mRNAs [G, Slaw e
tal,, Ce1l, 46.659-667.19
86]. According to the present inventors, human IL4 has a base sequence (472
There are three locations: 476th to 476th, 506th to 510th, and 514th to 518th. ) is considered to be equivalent. Therefore, it is thought that the production efficiency of the target substance will be improved by culturing using a vector-transformed host in which the nucleotide sequences involved in these destabilizations have been removed.

As a result of repeated studies based on these assumptions, the present inventors developed a vector in which the nucleotide sequence downstream of the nucleotide sequence encoding human IL4 translation termination (i.e., the nucleotide sequence after 464th A) was removed. It was confirmed that when using this vector, the production amount was several ten times higher than when using a vector that did not remove the corresponding base sequence. The present inventors are the first to demonstrate this fact through experiments. Of course, the same effect can be fully expected using a vector in which only the three base sequences involved in destabilization are removed.

A human IL4 gene from which base sequences involved in destabilization have been removed is prepared as follows. That is, human IL
The vector DNA containing the 4 genes is cut with a restriction enzyme that recognizes a specific base sequence that exists singly downstream of those sequences, such as 5aNI, and then digested with an ecrinuclease such as Ba or Q31. This is done by cloning DNA fragments into E. coli.

Therefore, the object of the present invention can be achieved as the human IL4 gene used in the present invention, whether it is a gene in which the nucleotide sequence involved in destabilization is not removed, or a gene in which at least the nucleotide sequence is removed. . here,"
"A gene from which at least the nucleotide sequence involved in destabilization has been removed" means a gene in which all three nucleotide sequences involved in destabilization, indicated by "A T T T A ," have been removed, and the 3' side is untranslated. It means a gene in which all or part of the remaining base sequence has been removed, or it has not been removed. Preferably, it is a gene from which at least a base sequence involved in destabilization has been removed.

Any vector containing the SV40 early promoter may be used as a vector for expressing human IL4m Fuko in animal cells. For example, ps2 vector (R, C, H
ull+gan et al.

Sc 1ence, 209.1422-1427.1
980) YapKCR# H2 vector (K, 0°fl
are et al, Proc, Natl, Acad.
.

Sci,, USA, 78.1527-1531.19
81), but pKCR-82 vector is preferred.

The pKcR Machi vector contains the SV40 early promoter as well as a splicing site and a polyadenylation site derived from the eel β-globin gene, so it is suitable for expressing the target gene in animal cells. or,
Since the vector contains a DNA replication origin that enables replication in E. coli and an ampicillin resistance gene as a selection marker, vector DNA can be prepared in large amounts and in a pure manner using E. coli.

The DHFR gene was obtained using a known method [R, J, Kaufman
etat,, ) lolecular and Ce
1lular Biology, 2.1304-131
9'+1982. Furthermore, any promoter for expressing the DHFR gene may be used as long as it can function in eukaryotic cells, such as the SV40 early promoter, metallothionein gene promoter, adenovirus gene promoter, and retrovirus gene promoter.

A human IL4 expression vector can be constructed by joining three DNA fragments having the above-mentioned characteristics, but the vector DNA can be prepared by a general method [Ding, Hanjatis et al. , Mo
1ecular Cloning, ColdSp
ring trough laboratory,
1982].

DHF R-deficient Chinese hamster ovary-derived cells (hereinafter referred to as C
) Abbreviated as lQdMr-cell. ) is G, Urlau
b et al, Proc, Natl, Acad
, Sci, USA.

77, 4216-4220.1980. Vector DNA can be introduced into CHOdMr cells using known methods, such as the calcium phosphate method [F, L,
Graha...et al., V+rology, 54
, 536-539.1973. ] is carried out.

The above-mentioned cells into which the expression vector of the present invention has been introduced are able to grow even in a selective culture medium lacking thymidine and hypoxanthine, in which the parent cells before transformation, CHOdMr- cells, cannot survive. Can be sorted.

In addition, due to the action of a competitive inhibitor of D)-IFR, DHFR
Cells amplified by J. Fuko can be easily identified because they can be grown in a selective culture medium containing a competitive inhibitor of DHF R, such as methotrexate.

One of the transformants of the present invention thus obtained has been deposited as CHO-891 cells with the Fermentation Research Institute, Japan with deposit number I FO50137, and with the Microbial Technology Research Institute with deposit number BP-1752. It has been deposited in

The human IL4 high-producing cell line of the present invention is cultured in a conventional culture medium (e.g., α-MEM containing 10% fetal bovine serum) in the presence or absence of methotrexate, and the Proteins can be prepared in a common way, e.g.
Purification can be performed by salting out, ion exchange chromatography, gel filtration, hydrophobic chromatography, affinity chromatography, or the like.

The present invention also includes human IL4 produced using the novel transformant shown in (3) above as a host.

[Effect] Human IL4 obtained by the present invention can be used to improve the immune response of the living body, and can be used to treat diseases for which such improvement is desired, such as cancer, infectious diseases, AIDS, and functional immunodeficiency. It can be used for prevention. In such cases, the dosage will vary depending on the symptoms, age, weight, etc.
It can be administered intravenously in a range of 0.1μ to 1 mFl.

The immunostimulatory activity and activity of human IL4 of the present invention were measured using human lymphocyte proliferation stimulating activity as an index.

Human lymphocyte proliferation stimulating activity was measured by the following method.

Lymphocytes prepared from human peripheral blood were cultured in a culture medium containing phytohemagglutinin (10 μg/d) [RP containing 10% fetal bovine serum, 5X10-5M2-mercaptoethanol].
M1-1640] and cultured at 37°C in the presence of 15% CO2 for 4 to 6 days.

Lymphocytes that have been thoroughly washed (3 or more times) with culture medium are
The cells were resuspended so that 5×104 cells were contained in 00 μg of culture solution, the test lymple was added, and the culture was continued for an additional 3 days.

Tritiated thymidine (0.25 μCi) for the last 12 hours
/culture), and its uptake into the macromolecular fraction was measured using a liquid scintillation counter.

The specific activity obtained as a result is 2,5~ 10x 10” IJ/my protein T:-Atta.

In general, human IL4 obtained by 1q according to the present invention can be used as an antigen to create an antibody against human IL4. Furthermore, the factor can be used for various purposes such as separating and purifying human IL4 receptor using the factor as a ligand.

[Example] The present invention will be described in more detail with reference to the following examples, but these are not intended to limit the scope of the present invention.Example 1 Preparation of human 114-fold gene (i) Preparation of human IL4 Isolation of encoding mRNA Lymphocytes prepared from human peripheral blood were treated with phorbol-12-myristate-13-acetate (PMA) (1μ9/〃
culture solution [10% fetal bovine serum, 5X10'M
RPM l-1640 with 2-mercaptoethanol
] and cultured at 37°C in the presence of 15% CO2 for 12 hours. The lymphocytes, which had been washed twice with culture medium to remove PMA, were resuspended in a culture medium containing concanavalin A (>3 μg/ml) and further cultured to induce human IL4. After 6 hours, the lymphocytes were washed twice with culture medium to remove PMA. RNA was collected from (6 x 109 pieces) using the guanidium thiocyanate method, and oligo(
dT) 89 μm of polyA-containing mRNA was fractionated by Ffi”A using a cellulose column.

A portion of the mRNA obtained in this way was extracted from a mature female African frog (Xenopus, 1eavis).
oocytes injected into modified Barth “S” culture medium (Colman, A., Transcripti).
on and on and on a pr
actual approach PP, 291
Edited by HameS, B, D.

and 1liqc+ins, S, J,, IRL Pres.
S., 1984) at 20°C for 136 hours. After this, the human IL4 activity in the culture supernatant was determined by the human lymphocyte proliferation-stimulating activity (lymphocytes prepared from human peripheral blood) in a culture medium containing phytohemagglutinin (10 μg/weight) [10% fetal bovine serum, 5X10-5M2-mercaptoethanol]. [RPM l-1640] at 37°C, 5% CO
The cells were cultured in the presence of 2 for 4 to 6 days. The lymphocytes, which had been sufficiently washed (three or more times) with the culture solution, were resuspended in 200 μg of the culture solution to a concentration of 5×10 4 ff6], the test sample was added, and the culture was continued for an additional 3 days. Tritium thymidine (0,25 μCi/culture) for the last 12 hours
After pulsing with water, the amount incorporated into the polymer fraction was measured using a liquid scintillation counter. ) and human tonsil B lymphocyte proliferation stimulating activity (lymphocytes prepared from human tonsils were treated with 2-amineethylisothiouronium bromide, and T lymphocytes were removed by the rosette formation method using sheep red blood cells. In this way, The obtained B lymphocytes were suspended in a culture medium [RPM r-1640 containing 10% fetal bovine serum, 5 x 10'' 5M2-mercaptoethanol] containing anti-fc]M antibody-bound biz (5 μg/d),
The cells were cultured at 37°C in the presence of 15% CO2 for 2 to 3 days. Anti-IgM antibody-bound beads were removed by specific gravity centrifugation using Lymphoprep (manufactured by Nyegaard), and the B lymphocytes washed with coarse culture medium were resuspended so that 5 x 10' cells were contained in 200 μg of culture medium. A test sample was added and cultured for an additional 2 days. The amount of tritiated thymidine incorporated into the polymer fraction was measured using a liquid scintillation counter. ) was confirmed by measurement.

(ii) Construction of cDNA library Plasmid I
) CDVl-PL [described in Document A] by restriction enzyme 1Kpn
Digested with l. A dT strand with an average length of 45 bases was added to the KpnI fragment of DNA using terminal transferase. Next, this DNA was digested with EC0RI and the larger fragment was collected. The obtained DNA fragment was purified using an oligo dA column and used as vector primer DNA. Plasmid pSP62-2 [Reference A
] was digested with 3acl and S aCI IJfrE
A dQ chain with an average length of 14 bases was added to the DNA using terminal transferase. After digesting this DNA with old ndll, the smaller fragment was collected and added to the linker-DNA.
I got it.

Using polyA RNA6μ9 obtained in Example 1(i),
Complementary DNA was synthesized from 2μ9 vector primers using 10 units of reverse transcriptase. Furthermore, an aC chain with an average length of 14 bases was added to the DNA end using terminal transferase. This DNA was cut with old ndI[I and ligated with the above linker DNA. Subsequent E.coli DNA polymerase J.E.coli
i Using RNASe H, replace the RNA portion with DNA, and use the obtained plasmid DNA containing the CDNA to transform Escherichia coli HBIO1 using a conventional method.
A CDNA library consisting of one million independent transformed bacteria was constructed.

(iii) Probe preparation Plasmid (pSP6Km) encoding mouse IL4
Restriction enzyme 3a from IL4-374> (described in document A)
Cut out an insert of about 800 bp with mHI, and roughly add it to 40 μg of reaction solution [35 μy of DNA, 12
mM CaCN2.12mM MqCl)2.0.
2M NaCJ! , 20mM Tris salt'W (
pH 8,0>, 1mM EDTA, Ba, Il 31
0.3 units] The reaction was carried out at 30°C for 3 minutes. After phenol extraction, 3amt-II was added to the ethanol precipitated product.
The linker was attached with T4-ligase. Furthermore, BamHI
After treatment, the purified reduced inrete was added to Ba1 of lcl 8.
psP6mlL4Δ was prepared by integrating into the llHI site. 5' side BamHI/ obtained from this 1 lasmid
The RsaI fragment does not contain a portion of the SP6 vector and can be used as a probe. At the same time, Rsa I/Rsa f covering almost the entire area from this vector
Fragments were also prepared separately.

(iv) Isolation of cDNA clones Four replicas were prepared using the transformant obtained on GSNSN Film (manufactured by Gel-N Science Inc.) using a conventional method. A BamHI/Rsa I fragment of mouse IL4 (120 bp
), using RsaI/Rsal fragment (373 bp),
Add two filters each to the reaction solution [50mM l~Lis-HCl (pH 7,8), 10mM EDTA, 1M
NaC (J, 0.1% BSA, 0.1% polyvinylpyrrolidone, 0.1% Ficoll 400.0.1% 5DS
Hybridization was carried out at 37° C. for 24 hours in Rike semen DNA treated with 150 μg/d sonication. After the reaction, the filter was 6XSSC (1xSSC: 0.
15 MNaCI, 0.015M sodium citrate)
, then washed 3 times in 0.1% SDS at room temperature for 1 hour, and then washed in 2XSSC, 0.1% SDS at 37°C.
Washing was performed twice with 120 min. BamHI/R3al,
Of the two filters each using the RSa I/RSa I probe, one was further coated with 2XSSC10.
, 42°C in 1% SDS for 20 minutes. As a result of odoradiography, 258 clones with signals relatively stronger than the background were selected from BamHT/Rsal and 68 from RSa I/RSa J. 2 to 10 clos near the signal were scraped off with a platinum loop and suspended in 100 μp of L medium. At this time, clones randomly selected from 10 independent sites were collected in one tube, and in the end, 26 pools were obtained using the 8amHI/RsaI probe, and 1q of 71-L clones were obtained using the RsaI/RsaI probe. Plasmid DNA was isolated from each pool, cut with Sa and IN, used as a template, and rnR using 5P6 RNA polymerase.
NA was synthesized (1.5-2.5 μg>, mRNA was treated with phenol and chloroform, and precipitated with ethanol.
It was dissolved in PBS and adjusted to 0.3 μq/μg.

Each of the respective RNAs was injected into African frog oocytes (20 cells (70 n, l)/feed). Oocytes were incubated at 20℃30 in modified Perth culture medium (10μg/cell).
After culturing for an hour, the culture supernatant was collected and the activity of 114 was measured. As a result, Il4 activity was observed in pool 6 and pool 15. From this back pool 6, each plasmid DNA
A was collected and transformed into E. coli To13101, of which 3
00 clones were randomly selected and 30 pulls of 10 clones were created. These 30 pools were screened in the same manner as above, and Il4 activity was found in 5 pools. Two of these pools consist of loan 2.
Plasmid DNA was collected from 0 and screened using the same procedure, and as a result, one [p
14 activities were observed in clones of SP (6-127> and psP (6-230)). pSP (6-127)
The base sequences of pSP (6-230) and pSP (6-230) were each determined by the dideoxy method. As a result, it was found that pSP (6-127) and pSP (6-230) encode the same content. Base sequence of insert The amino acid sequence derived from this is shown in Figure 1.

Example 2 Construction of vector for expressing human IL4 Plasmid ps
A 0.8 kb BamHI fragment containing the human 14 coding region was cut out from P (6-127) using agarose gel, and both ends were blunted with Klenow fragment.
[I linker added. This was transformed into the animal cell expression vector pKCRH2 [, 0'Hare et at,
Proc, Natl, Acad, Sci, USA.

78, 1527-1531.1981]
nd[ll night, plasmid I) KCRh
IL4 (qta.

Next, a vector plasmid DSV 2 ne that can express the neomycin resistance gene neo in animal cells.

(R,C,)lulligan et al, 5ci
ence, 209.1422-1427゜1980), was excised with enzymes Acc I + EcoRI, blunted with Klenow fragment, and 3 aml-
II clinker was added. This is used as a vector plasmid p to ID0 (S, Kim
et al., Ce1l, 42. 129-138.
A plasmid p[)neo was prepared in which the DHF RjM gene and neoO were transcribed in the same direction. g
[) neo was digested with EC0Rr, blunted with Klenow fragment, and 3a and Qi clinkers were added. This was digested with Saρ, the 6.5 kb fragment was purified with agarose gel, and this was inserted into the 5ajI site of pKCRhlL4.

Thus, the human fL4-derived Jff vector DKCRh I L4-[) with DHFR and EO as selectable markers.
constructed n.

Example 3 Plasmid pSP (6-127> was opened with Sad I and digested with 0526 units of 8a, I!31 at a concentration of 3.5 μg/40 μg. The reaction was carried out at 30° C.
μg each at 2 minutes, 4 minutes, 6 minutes, and 10 minutes, followed by 1 Mg.
Stopped by sampling into 2M EGTA. A portion (2 μg) of this was digested with ECORI, and the remaining 8 μg of the pool in which DNA was observed around 100 bp in PAGE (4 minute reaction) was blunted with Klenow fragment and an Xho I linker was added. After ring closure, this was transformed into E. coli D H1, resulting in approximately 2
Plasmids were obtained for 120 of the 000 strains. Of these, 28 strains that produced EcoRI + XhoI fragments of about 100 bp were partially sequenced.
A plasmid psP(6-127)Δ was obtained in which the TGA stop codon was directly followed by an XhoI site. This indicates that the entire nucleotide sequence downstream of the nucleotide sequence encoding the translation termination of human IL4 has been removed.

BamHI from plasmid psP (6-127)△
The 0.5 kb fragment excised with +Xhol was blunted with the Klenow fragment, and the old nd [[ linker was added. This is p
Insert into the old nd[l site of KCRH2 and create plasmid p.
KCRh14Δ (q.

Plasmid pMDo, described in Example 2, was digested with ECO, blunted with Klenow fragment, and a 5aOI linker was added. Here, the 3.5 kb Sa,III fragment containing the queried DHF RM gene was integrated into the 3a, Q site of pKCRhlL4Δ, and the plasmid pKcRh
lL4Δ-D was obtained.

Example 4 Creation of transformed cells Plasmid pKCRh 114-Dn was transformed with restriction enzyme pvu
It was digested with [and made into a linear chain. Approximately 1 x 106 CHOs
dMr-strain cells [G, Urlaub et at.
Proc.

Natl, Acad, Sci, USA, 7
7, 4216-4220.1980], 20 μq of the above linear pKCRhIL4-DnDNA was subjected to calcium phosphate precipitation method [F, L, Graham et al.
l,, Virology, 54.536-539.1
Transfection was carried out by 973@].

After 2 days, culture medium (αMEM containing 10% fetal bovine serum) was added.
) was added to the selection culture medium [10% dialyzed fetal bovine serum (GIBC)].
Hyboxanthin and thymidine-free αME containing (manufactured by Company O)
M (manufactured by F.I.O.W.)
The cells were transferred to 5 petri dishes and cultured for 2 weeks. 100 cells were cloned from the approximately 1×10 3 colonies obtained, and the human TL4 activity (the above-mentioned human lymphocyte proliferation stimulating activity) of each clone was measured. this house,
Ten clones that proliferate relatively quickly are shown in Table ■.

Table ■ IL4 activity was measured. Among these, 10 clones that proliferate relatively quickly are shown in the table.

Table ■ Next, plasmid pKCRt11L4Δ-Rinite was transduced into CI-Cl-10d strain cells in the same manner as plasmid pKCRh IL, 4-Dn, and 100 colonies were selected from surviving colonies in the selection medium. cells of human pKCRhIL4 of each clone.
pKCRh I L4-
Human IL4-producing cells with an average production efficiency of about 30 times higher than when transduced with Dn were obtained.

Next, 2g from clone A (clone NO, A-8°-1
0) and 6 from clone B (clone N(lB1, -2
．． -3. -4. -9. -10> and get 50 each
nM methotrexate (hereinafter abbreviated as MTX)
7X10”I per 10cm plate in selective medium containing
Cells are plated to contain viable cells for 2 weeks to 1
After culturing for several months, IL4 activity secreted into the medium was assayed.

A large increase in IL4 production was observed after MTX treatment in the eight inner loans A-106 and B-1. So 50
Two OfFM clones were isolated from a group of cells derived from clones A-10 and B-1 that survived in the presence of nM MTX.

In addition, in clone B-9, no enhancement of IL4 activity was observed in the presence of 50 nM MTX;
A similar experiment was conducted in the presence of M MTX, and 10 subclones were isolated from the surviving clones.

Among the IJ clones derived from clones A-10, 8-1, and B-9, the human IL4 activities of 15 clones with relatively fast proliferation rates are shown in Table Iv.

Even after 2 months of culture, 11 of these 15 clones remained
-4 production was maintained at a rapid rate. Among these, the clone with high IL4 production η, B-9-1, was named CHO-891 cell and was deposited with the Fermentation Research Institute, No. I FO50137, and with the Zarani Institute of Microbial Technology, No. 3p-17.
It has been deposited under No. 52.

Example 5 Culture of transformants A 1.0 volume microcarrier spinner flask [manufactured by Bet Ico] was charged with
After adding 3 g of Cytodex 3 (manufactured by Pharmacia) and swelling it in a phosphate buffer solution containing physiological saline (DH7,3> [hereinafter abbreviated as PBS (-))]. , autoclave sterilization was performed.

The PBS (-) in the flask was diluted with 1% tallow serum (FB).
After replacing with 400 rnll of serum-free medium containing S) (trade name: SFMlol, manufactured by Hinaga "AM"), CH
O-891 cells (prepared in Example 4) 7X107 (
The flask was stirred at 20 rpm for 2 minutes in a 5% CO2 incubator at 37°C and then allowed to stand for 1 hour. This operation was repeated 4 times to allow the cells to adhere to the microcarriers.

100ml of SFMIOI medium containing 1% FBS in the flask
ml! After culturing for 2 days at 2 Orpm in a 5% CO2 incubator at 37°C, add the above medium 5
00ml was added.

After culturing for 4 days, it was confirmed that the cells were in a monolayer on each microchire layer, so the culture medium was replaced with SFMIOI medium that does not contain F B S and culture was continued.

The culture medium was renewed at a rate of 500 d/day, and the culture was continued for 24F1 without collecting the culture supernatant.

Example 6 Purification of human IL4 12 g of the culture supernatant obtained in Example 5 was incubated with cold IJ at 4'C.
j, 10% trifluorochloric acid (hereinafter abbreviated as TFA) 1207! After adding acid ↑; [, Hybraly I-C1 (
120 g of Sepralyte C-1 (trade name) (manufactured by Analyte Chem International Co., Ltd.) was added, and the mixture was slowly stirred for 2 hours. After standing for 30 minutes, remove the supernatant and pass the beads through a glass filter (3G).
Collected above. At 4 °C, the beads were washed in several batches with a total amount of 2 g of 30% acetonitrile (containing 0,1% TEA), and then washed with a total amount of 400 d of 50% acetonitrile (containing 0,1% TFA). ) to wash out the adsorbed matter.

The obtained solution was concentrated under reduced pressure at 40°C or lower to remove acetonitrile, and then 50mM phosphoric acid tries solution (DH
It was adsorbed onto a cation exchange column [trade name: Mono S (manufactured by Pharmacia Rei)] which had been equilibrated at 6.0>. 50rr 1M phosphate buffer (pH 6,0)
After washing for 10 minutes at a rate of 0.
50mM phosphate buffer containing 3M NaC,Q (
1) Absorbance (280 μTrL) is 0.1 at >16.0
Washed until below. Next, 0.4M Na(J)
50mM phosphorus iy2 buffer (0116,0) containing
As the eluent, Fast System (Phast S) was used as the eluent.
System, trade name) (manufactured by Pharmacia) was eluted while monitoring, and fractions with a band at 18 to 20 kd were collected.

The dyed fraction was diluted with 50mM phosphorus solution (
After diluting it 10 times at pH 6.0>, it was again adsorbed onto a cation exchange column. Absorbance (280
μm) is 0.02 or less, and Na(fJj
A linear gradient with R degree from 0.3M to 0.5M was performed over 30 minutes, and the fractions were similarly subjected to ultraviolet absorption and sodium dodecyl lylphate/polyacrylamide gel electrophoresis (hereinafter abbreviated as SDS/PAGE). .

) was monitored.

Ammonium sulfate was added to the collected fractions to a final concentration of 1.
After melting to 7M, 1.7Mti+! 50rr 1M phosphate buffer containing ammonium L-acid (pt16.
The ammonium sulfate concentration ranged from 1.7M to O(pyro)M.
A linear gradient was applied over a period of 60 minutes or more, and fractions containing a band of 18 to 2 Qkd were collected while monitoring the fractions by ultraviolet absorption and SDS/PAGE. A wide fraction (mainly 0.5-0.3M) was preserved. Next, the Li-band portion was collected and concentrated using an ultrafiltration membrane [trade name: YM-10 (manufactured by 7 Mikon Co., Ltd.)]. did.

This was equilibrated with 50rTIM phosphorus-MiN buffer (ρi 16.0) to form a supercolumn 12 column (2.6x 80
When separated at 0.74 force ram volume (cm), a single peak centered at 0.74 force ram volume was obtained (q).
10> to give the final preparation (q.

The final preparation was prepared using a dye method using bovine serum aluminum as the standard.
(manufactured by Biorat). the result,
The yield was 5 my. Furthermore, the recovery rate using activity as an index was 23% based on the initial culture supernatant.

Example 7 Human IL4 molecules
H6, 8, 4% SDS, 18% glycerol, 1.4M
2-mercaptomethanolphenol blue) was mixed and heated at 100°C for 3 minutes, and then subjected to SDS-PAGE [10-20% thickness 111II
n Slab Glue (manufactured by Dai-ichi Chemical Co., Ltd.)]. A band was observed at the molecule @18kd by Coomassie blue staining. A single band was confirmed by silver staining, and 411 was detected using a densitometer (Model CS-910, manufactured by Shimadzu Corporation).
As a result of measuring 4 times, the peak purity was found to be 98% or more (see Figure 4).

Example 8 N-terminus analysis of human I[-4] Liquid phase protein sequencer (-E del 477A,
The following single sea fence was confirmed using Applied By A System Demne 1).

II+s-Lys-X-8sp-rle-Thr-Le
u-Gln-Glulle-fle-Lys-Thr-
Lcu-Asn-Ser-Leu-Thr-Glu-G
ln-Lys-Thr-Leu-X-rhr-Glu-
Leu (in the above formula, the X portion corresponds to Cys). This N-terminal sea fence completely matched that of human I-114 using COS-7 cells [Proc.
Natl. Acad. Sci. , 83
．． 5394 (1986)]. From this result, 241F1 chrysin and 2
It can be seen that the signal peptide is completely separated between the histidines of 51[-J.

Example 9 Amino acid silk production of human IL4 100 μU of rimple was dissolved in 6NmM and 4% diA glycolic acid and hydrolyzed at 110° C. for 22 hours under a vacuum seal. This was used for amino acid analysis1 (Hitachi 83
5), and the results are 17.

Table V Amino Acid Analysis Example 10 The molecular weight of the protein trade portion predicted from the sugar-containing CDNA of human 11-4 is 14
Since it is 9B2, the difference of about 3000 (about 17%) is carbohydrates.

Example 11 Sugar component (a) of human [4] 2N trifluoroacetic acid was added to 30 μ9 of a neutral sugar sample, and 1
After hydrolyzing by heating at 00°C for 6 hours, the mixture was dried under reduced pressure. This was dissolved in distilled water and subjected to HP I C analysis.

0.5M borate buffer (pt18.7 > medium, TSK-
(Iel 5IJC1ar AXG column (Toyo gta i
The analysis was carried out using the following method (manufactured by Ill. As a result, mannose,
It was confirmed that it contained fucose and galactose.

(b) Add 4N hydrochloric acid to the amino sugar rimple and prepare the analytical rimple in the same manner as for the neutral sugar.

TSK-g in 0.16M borate buffer (pH 7.6>)
1- using etSCX column (Toyo Kashitatsu (1Shu Manufacturing)
IPLC analysis was performed. As a result, it was confirmed that it contained glucosamine.

(c) 0.01M sulfuric acid was added to the sialic acid sample, and the sample was heated at 80° C. for 1 hour for hydrolysis, and then dried under reduced pressure. This was dissolved in distilled water and subjected to IPLC analysis.

Gelpak C- in 0.3% phosphoric acid
The analysis was performed using a 620-10 (Type 1) column (manufactured by Hitachi). As a result, below N-purneuraminic acid,
Abbreviated as NANA. 〉 was confirmed to be included.

Roughly speaking, the proportions of each sugar component were as shown in the table below.

Example 12 Using 0.2 m9 of isoelectric point sample of human TL4, ) lono P column (
HR5/20. Chromatofocusing was performed using a chromatography system (manufactured by Pharmacia).

25rTIM triethylamine buffer (all 11.
0 > to 1745 diluted formalite 8 to 10.3
(pH 8.0, manufactured by Pharmacia), a single peak was eluted at pH 9.96. Therefore, it is thought that the isoelectric point must be around 10.

Example 13 A glycosylated sample of human I[-4 5μ'J (Im'j/d) was heated to RKM 29 degrees 0
．． After treatment with 5% SO8 and 0.1M 2-mercaptoethanol at 100°C for 13 min, the final concentration was 0.2M.
of fat phosphate buffer (Oft 8.6), 1
0mM 1.10-nandroline, 1.25% Nonidet P-40 (NP-40>, 10 units/m
1 of N-Glycab (manufactured by Shiku Dienzyme) and 3
The reaction was carried out at 7°C for 16 hours. Part of the υ sample is SDS
Separation was performed using PAGE [20% slab gel with a thickness of 1 mm]. Molecule ♀15kd with Coomassie blue staining
allowed a single hand.

[Brief explanation of the drawing]

FIG. 1 shows the structure of human IL4 cDNA and the structure of the polypeptide derived therefrom, FIG. 2 shows the construction of the human ■[-4 expression vector DKCRil IL4-Drl of the present invention, and FIG. Invention Human [4 Expression Vector D
Figure 4 shows the construction of K(fllL-4Δ-D,
This is a diagram obtained by measuring human f4 (f4) using a densitometer after separating it by 5DS-PAG.

Claims (1)

[Scope of Claims] 1) Human interleukin 4, characterized in that (a) the molecular weight is 11,000 to 19,000, and (b) the molecular weight of the sugar chain moiety is 2,000 to 4,000. 2) Human interleukin 4 according to claim 1, wherein the sugar chain moiety has a molecular weight of 2,500 to 3,500. 3) A patent characterized in that (a) the molecular weight is 17,000 to 19,000, (b) the molecular weight of the sugar chain moiety is 2,000 to 4,000, and (c) the isoelectric point is 8.5 to 10.5. Human interleukin 4 according to claim 1. 4) Human interleukin 4 according to claim 3, which has an isoelectric point of 9.8 to 10.1. 5) (a) The molecular weight is 11000 to 19000, (b) The molecular weight of the sugar chain moiety is 2000 to 4000, (c) The isoelectric point is 8.5 to 10.5, (d) The sugar chain Human interleukin-4 according to claim 1, which contains at least mannose, fucose, and galactose as neutral sugars constituting the human interleukin-4. 6) (a) The molecular weight is 17,000 to 19,000, (b) The molecular weight of the sugar chain moiety is 2,000 to 4,000, (c) The isoelectric point is 8.5 to 10.5, (e) The sugar chain Human interleukin 4 according to claim 1, which contains at least glucosamine as the amino sugar constituting the human interleukin 4. 7) (a) The molecular weight is 17,000 to 19,000, (b) The molecular weight of the sugar chain moiety is 2,000 to 4,000, (c) The isoelectric point is 8.5 to 10.5, (f) The sugar chain Human interleukin-4 according to claim 1, characterized in that it contains at least N-acetylneuraminic acid as a sialic acid constituting the human interleukin-4. 8) (a) The molecular weight is 17,000 to 19,000, (b) The molecular weight of the sugar chain portion is 2,000 to 4,000, (c) The isoelectric point is 8.5 to 10.5, (d) The sugar chain (e) contains at least glucosamine as an amino sugar forming the sugar chain; (f) at least N-acetylneuraminic acid as a sialic acid forming the sugar chain; Human interleukin 4 according to claim 1, characterized in that it contains. 9) (a) The molecular weight is 17,000 to 19,000, (b) The molecular weight of the sugar chain moiety is 2,000 to 4,000, (c) The isoelectric point is 8.5 to 10.5, (d) The sugar chain (e) contains at least glucosamine as an amino sugar forming the sugar chain; (f) at least N-acetylneuraminic acid as a sialic acid forming the sugar chain; (g) per total mole of sugar chain moiety, mannose, fucose,
Galactose, glucosamine and N-acetylneuraminic acid are 3.0-4.0, 1.0-2.0 and 2.0, respectively.
Human interleukin 4 according to claim 1, characterized in that the human interleukin 4 is contained in a proportion of 5 to 3.5, 4.5 to 5.5, and 1.5 to 2.5 mol. 10) A human coexisting with (i) a DNA fragment having the human interleukin 4 gene downstream of the SV40 early promoter and (ii) a DNA fragment having the dihydrofolate reductase gene downstream of a promoter that functions in eukaryotic cells. Human interleukin 4 is produced using a transformant obtained by transforming a dihydrofolate reductase-deficient Chinese hamster ovary cell line with an interleukin 4 expression vector as a host. 11) A patent claim characterized in that the human interleukin 4 gene in the human interleukin 4 expression vector set forth in claim 10 is a gene from which at least a base sequence involved in destabilization has been removed. Range 10th
Human interleukin 4 as described in section. 12) Human interleukin 4 according to claim 10, characterized in that (a) the molecular weight is (17,000 to 19,000), and (b) the molecular weight of the sugar chain moiety is 2,000 to 4,000. 13) ( Claims characterized in that a) the molecular weight is 17,000 to 19,000, (b) the molecular weight of the sugar chain moiety is 2,000 to 4,000, and (c) the isoelectric point is 8.5 to 10.5. Human interleukin 4 according to item 10. 14) (a) The molecular weight is 17,000 to 19,000, (b) The molecular weight of the sugar chain moiety is 2,000 to 4,000, (c) The isoelectric point is 8.5 to 10.5, (d) The sugar chain Human interleukin-4 according to claim 10, which contains at least mannose, fucose, and galactose as neutral sugars constituting the human interleukin-4. 15) (a) The molecular weight is 17,000 to 19,000, (b) The molecular weight of the sugar chain moiety is 2,000 to 4,000, (c) The isoelectric point is 8.5 to 10.5, (e) The sugar chain Human interleukin 4 according to claim 10, which contains at least glucosamine as an amino sugar constituting the human interleukin 4. 16) (a) The molecular weight is 17,000 to 19,000, (b) The molecular weight of the sugar chain portion is 2,000 to 4,000, (c) The isoelectric point is 8.5 to 10.5, (f) The sugar chain Human interleukin-4 according to claim 10, characterized in that the sialic acid constituting the human interleukin-4 contains at least N-acetylneuraminic acid. 17) (a) The molecular weight is 17,000 to 19,000, (b) The molecular weight of the sugar chain moiety is 2,000 to 4,000, (c) The isoelectric point is 8.5 to 10.5, (d) The sugar chain (e) contains at least glucosamine as an amino sugar forming the sugar chain; (f) at least N-acetylneuraminic acid as a sialic acid forming the sugar chain; Human interleukin 4 according to claim 10, characterized in that it contains. 18) (a) The molecular weight is 17,000 to 19,000, (b) The molecular weight of the sugar chain portion is 2,000 to 4,000, (c) The isoelectric point is 8.5 to 10.5, (d) The sugar chain (e) contains at least glucosamine as an amino sugar forming the sugar chain; (f) at least N-acetylneuraminic acid as a sialic acid forming the sugar chain; (g) per total mole of sugar chain moiety, mannose, fucose,
Galactose, glucosamine and N-acetylneuraminic acid are 3.0-4.0, 1.0-2.0 and 2.0, respectively.
Human interleukin 4 according to claim 10, characterized in that it is contained in a proportion of 5 to 3.5, 4.5 to 5.5, and 1.5 to 2.5 mol. 19) Coexistence of (i) a DNA fragment having the human interleukin 4 gene downstream of the SV40 early promoter and (ii) a DNA fragment having the dihydrofolate reductase gene downstream of a promoter that functions in eukaryotic cells. A human interleukin 4 expression vector characterized by: 20) The expression vector according to claim 19, wherein the human interleukin-4 gene is a gene from which at least a base sequence involved in destabilization has been removed. 21) A human in which (i) a DNA fragment having the human interleukin 4 gene downstream of the SV40 early promoter and (ii) a DNA fragment having the dihydrofolate reductase gene downstream of a promoter that functions in eukaryotic cells coexist. A transformant obtained by transforming a dihydrofolate reductase-deficient Chinese hamster ovary-derived cell line with an interleukin-4 expression vector. 22) The transformant according to claim 21, which is obtained by transforming the human interleukin 4 gene with a human interleukin 4 expression vector in which at least a base sequence involved in destabilization has been removed. .