JPS6232885A - Human interleukin 2 manifestation vector and eucaryotic cell strain containing same - Google Patents

Abstract

PURPOSE:To produce human interleukin 2 (IL2) added with saccharide in high production efficiency, by linking a gene coding human IL2 to the downstream of the promoter sequence in a vector which can be replicated and manifested in a eucaryotic cell. CONSTITUTION:A gene sequence 1 having a gene coding human IL2 containing signal peptide gene necessary for secretion and a polyrA signal linked to its downstream is prepared under the control of a promoter functioning in a eucaryotic cell. Separately, a gene sequence 2 containing a PVUII incision long-chain fragment, etc., of bovine papilloma virus type-I gene is prepared under the control of a promoter functioning in a eucaryotic cell. The objective IL2-manifestation vector is produced by linking the gene sequences 1, 2 and the replication initiating point and drug-resistance gene sequence of Escherichia coli.

Description

[Detailed Description of the Invention] [Industrial Application Field] The present invention is a novel expression vector aimed at producing human interleukin 2 using animal cells as a host by transgenic recombination technology, and the present invention comprises the vector. The present invention relates to an animal cell line and a method for producing human interleukin 2 using the cell line.

[Conventional technology]

Interleukin 2 (hereinafter abbreviated as IL2) is a type of lymphokine produced by T lymphocytes stimulated by lectins and antigens, and its functions include differentiation and proliferation of T cells, differentiation and proliferation of B cells, and natural killer. It is said to promote cell proliferation, and is expected to be used in clinical applications as immunostimulants and immunotherapeutic agents, as well as in diagnostic agents and reagents.

Production of IL2 has traditionally relied on cell culture technology, but this method is difficult to defeat, and the production concentration is low, so a complicated purification process is required to produce pure 112. Ta. For this reason, attempts are currently being made to obtain IL2 in humans by cloning the DNA encoding the IL2 polypeptide using genetic engineering techniques.

The cDNΔ of IL2 was first cloned by Noguchi et al., and its expression in monkey cells was reported (Nature
, 302.305 <1983)).

Subsequently, Rene [)evos et al.
AcidsRes, 11.4307 (198
3)), 5teven A, Rosenberg et al. (3science, 223, 1412 (19
84)) have reported the cloning of cDNA and expression in E. coli.

However, it is known that some natural human IL2 proteins have added sugars. When human IL2 is synthesized using prokaryotes such as Escherichia coli and Bacillus subtilis, the substance synthesized is only the protein portion of human IL2 and no sugar chains are attached.

As a method for producing natural human IL2, there is a method of treating normal human lymphocytes or cancerous human lymphocytes with drugs, but this method has the following drawbacks.

1. Normal lymphocytes do not have the ability to proliferate, so they must be supplied from blood.

2. The amount of human IL2 produced by normal lymphocytes is low.

3.Culture of human lymphocytes requires expensive serum;
Production costs are high.

Genetic engineering techniques using animal cells as hosts are expected to eliminate the above-mentioned drawbacks and produce glycoproteins that are indistinguishable from natural human IL2 containing sugar chains.

As an attempt to express foreign genes using genetic manipulation techniques using eukaryotic cells such as animal cells as hosts, Dihydrophylla 1! [! Chinese hamster ovary-derived cells that are deficient in 2-reductase: dhfr(-) CHO cells (G, UrlaUb etal, Pro℃, N
a-mouth, Acad, Sci, USA.

77.4216.1980), but to date there have been no reports of the establishment of eukaryotic cells that constitutively produce human IL2 with high production efficiency.

[Problem that the invention seeks to solve]

The purpose of the present invention is to develop a cell line that constantly produces glycosylated human IL2 with high production efficiency, has a fast proliferation rate, grows in an inexpensive medium, and has an unlimited number of passages. The purpose is to establish it using manipulation technology.

[Means for solving problems]

The above object is achieved by the present invention as described below.

That is, the present invention provides human 112
The present invention relates to a human IL2 expression vector obtained by ligating a gene encoding a human IL2 expression vector, and to a eukaryotic cell line obtained by transforming eukaryotic cells with the vector.

Preferred expression vectors of the present invention will be described below, but the present invention is not limited thereto.

The construction of the IL2 expression vector of the present invention consists of: 1. Under the control of a promoter that functions in eukaryotic cells, a gene encoding human IL2 containing a signal peptide gene necessary for secretion, and a polyrA addition signal downstream thereof; Under the control of a promoter that functions in eukaryotic cells, the genetic sequence placed in the bovine papillomavirus type (hereinafter referred to as
(abbreviated as BPV-1) gene pvu [cleaved long-chain fragment or gene sequence containing the dihydrofolate reductase (hereinafter abbreviated as DHFR) gene, consisting of ■, replication origin in E. coli, and drug resistance gene This is achieved by joining gene sequences.

The gene encoding rL2 has already been developed by Natuguchi et al.
re, 302.305 (1983)).

Rene Oevos et al.
ids Res,.

21.4307 (1983)), 5teven A.

Rosenberg et al. (Science, 223
, 1412 (1984)). In other words, poly(A) mRNA is prepared from cells capable of producing 112, such as human leukemia T cell lines, human tile cells, and human peripheral blood lymphocytes, and CD N A is synthesized from this using reverse transcriptase. and cloned into E. coli. For the obtained E. coli strain, IL2Q DNA could be selected by mRNA hybridization translation assay method.
DevOS et al.), and the previously reported IL2cD
IL2 cDNA can also be obtained by selecting positive strains by colony hybridization using a DNA oligomer having a partial base sequence of NA as a probe (
3 Teven A, Rosenberg et al.).

The amino acid sequence of the protein portion of IL2 is, for example, as shown below, and the gene encoding it has, for example, the following sequence.

Ala pro 7hr 3er S
er 3er ThrGCA C:CT
ACT TCA AGT TCT ACA
s lys Thr Qln leu
Gln 1euAAG AAA ACA
GAG CTA CAA CTGGlu
His leu 1-eu Leu
Asp 1euGAG CAT TTA
CTG CTG GAT TTAGln M
et Ile Leu Asn Gl
y 1ieCAG ATG ATT TTG
AAT GGA ATTAsn Asn
Tyr Lys Asn Pro
LysAAT AAT TACAAG AAT
CCCAALeu Thr Arp
Met Leu Thr PheCTC
ACCAGG ATG CTCACA TTTL
JS Phe Tyr 1ylet P
ro Lys LysAAG TTT T
ACATG CCCAAG AAGAla T
hr Glu Leu LVs Hi
s LeuGCCACA GAA CTG
AAA CAT CTTQln Cys
leu Glu Glu Glu 1
euCAG TGT CTA GAA GAA
GAA CTC ys pro leu
Qlu Qlu Val L-eu
AAA CCT CTG GAG GAA
GTG CTAAsn Leu Ala
Gln Ser Lys AsnAAT
TTA GCT CAA AGCAAA
AACPhe His 1-eu Arg
Pro ArOASDTTT CACTT
A AGA CCCAGG GACl-eu
lle 3er Asn Ile
AsN ValTTA ATCAGCAAT A
TCAACGTAArle Val Leu
Glu 1-co Lys GlyATA
GTT CTG GAA CTA AAG
GGA Ser Glu Thr Thr
Phe Met CysTC Ding GAA A
CA ACA TTCATCTGTGlu T
yr Ala ASpGlu Thr Al
aGAA TAT GCT GAT GAG
ACA GCAThr lie Val
Glu Phe Leu AsnΔC
CATT GTA GAA TTT CTG
AACAra Trp Ile Thr
Phe Cys GlnAGA TGG
ATT ACCTTT TGT CAASe
r Ile Ile Ser Thr
Leu Wang “AGCATCATCTCA
ACA CTG ACT The signal peptide gene of the present invention is preferably human IL2, but any signal peptide gene whose signal peptide portion and structural protein portion are separated at a precise position during secretion can be similarly used.

The promoter of the present invention that functions in eukaryotic cells refers to a promoter of an animal virus gene or a promoter of an animal cell gene, which is a promoter of a downstream human Il gene in an animal cell.
Any material may be used as long as it has the function of transcribing the L2 gene sequence into mRNA.

As an example, the 401st stage promoter of the SV40 first-born promoter, the adenovirus gene promoter,
Examples of animal virus gene promoters include HB virus gene promoters and retrovirus gene promoters. Further, promoters of animal cell genes include thymidine kinase gene promoters, metallothionein gene promoters, interferon gene promoters, and the like. Among these, SV40 early morning promoter and SV 401M phase promoter are preferred.

The pvu [truncated long-chain fragment of the BPV-1 gene of the present invention can be produced by the method described in Japanese Patent Application No. 59-170351. In the vicinity of BamHI of BPV-1, there are two restriction enzyme cleavage sites close to each other, Pvul
Since there is no I cleavage site, pvu digestion yields linear long fragments. In the present invention, this p
In addition to the long fragment digested with vulJ, BPV-
1 can also be used with BalllHI and HindI [lr digested long chain fragments. In addition, the DHFRm gene has been described by R, J., Kaul'mar et al., Mo.
1ecular and Ce1lular 3 i
ology vol, 2, p1304-1319
(1982) or by the method described in Japanese Patent Application No. 59-170350.

Linking an origin of replication in E. coli and a drug resistance gene is useful for preparing the vector DNA simply, in large quantities, and in a pure manner.

The replication origin is colicin E1, which is known to be an inhibitor of E. coli chromosomal DNA synthesis and cause replication amplification.
Those derived from plasmids, such as pBR322 and related plasmids, are preferred, but are not limited thereto.

Drug resistance genes include ampicillin resistance gene, tetracycline resistance gene, kanamycin resistance gene,
Examples include streptomycin resistance gene and neomycin resistance gene.

However, when introducing these gene sequences that function in E. coli into animal cells, it is necessary to introduce harmful sequences (p.

1sonou 5equence: 1vlonic
alusky et al, Naturevol
, 293. p79-81.1981) is important.

The above three DNs ■, ■, ■ with the above properties
Human 112 in animal cells by joining the A fragment.
Expression vectors can be created.

Vector DNA can be prepared by common methods. (T, Maniatis et al, M
□ olecular C1oninq, p86-9
6. (1982) Vector DNA can be introduced into eukaryotic cells using existing methods. (F, L, Grah
am etat, Virology vol, 5
4. The eukaryotic cells into which the p536-539° vector is introduced are not particularly limited, as long as they can be transformed with BPV-1 when a PvuII-cleaved long fragment of the BPV-11 gene is introduced. As a specific example, mouse-derived C127-1 cells are often used, and cells into which a BPV-based vector has been introduced and expressed have an increased proliferation rate and can be easily identified by the formation of foci.

Cells transformed with BPV-based vectors are grown in normal culture medium,
For example, it grows in [) u l Becco's MEM medium and secretes interleukin 2 into the medium. Many of the transformed cells obtained by introducing BPV vectors have 50 or more copies of B per cell extrachromosomally.
Because it has a PV-based vector, human JL per cell
A high titer of human 112 with high 2 production is detected in the culture medium.

In addition, for example, the cells into which the DHFRi gene is introduced are those that are deficient in the DHFR3i gene and the cells into which the DHFRi gene is introduced.
Cells in which the HFR3u gene is known to be amplified by the action of a competitive inhibitor of DI-IFR are usually used. As a specific example, D)-IFR-deficient Chinese hamster ovary-derived cells (dhfr (-) CHO cells:
G, Urlaub etal, Proc.
N at, △cad, Sci, USA, v.

1, 77, p4216-4220.1980) is often used.

Cells into which the DHFR vector has been introduced and the DHFR31 gene is expressed are dhfr (
-) Thymidine, adenosine, which CH○ cells cannot survive,
It can be easily identified by taking advantage of its ability to grow on a selective medium lacking deoxyadenosine and its size of +9 L.

Cells in which the DI-IFR gene has been amplified due to the action of a competitive inhibitor of DHFR can be easily identified because they can be grown in a selective medium containing a competitive inhibitor of DHFR, such as metol-rexade (MTX). can.

Many of the cells in which the DHF R gene obtained in this way is amplified also have the human l12m gene amplified at the same time, so the human l12 production per cell is high, and therefore a high titer of human l12m is produced. 12 is detected in the culture medium.

Human IL2 synthesized from cells transformed with DI-IFR vector or BPV vector is
It can be purified from culture fluid using general purification methods. for example,
By using an antibody column bound with anti-112 antibody, nearly pure human 112 can be obtained in one step. Various methods used to purify human IL2 can also be applied as they are.

〔Effect of the invention〕

By the present invention, it is possible to establish a cell line that constantly produces human IL2 that closely resembles the natural type with high production efficiency, has a fast proliferation rate, grows in an inexpensive medium, and has an unlimited number of passages. was completed.

The details of the present invention will be described below with reference to Examples.

Example 1 pvu of BPV-1 gene [vector psVh I L2/BPV9 in which human l12m gene was linked to the cleavage site
The method for producing 7C is shown below. The basic genetic manipulation method is “Molecular at Oning” (
Maniati S et al. (1982) Coldspring
Harbor Laboratories).

(1) Preparation of vector pIL2-28.3 Human IL
2 Vector pIL2-28 for cloning the iM gene
．． No. 3 was prepared as follows. Lymphocytes were isolated from human tonsils and treated with 2% fetal bovine serum and 10mM Hep.
es(N-2-hydroxyethyl pipe
razine -N' -2-ethanesuNo
nic acid) (DH7, 2>, 5X
5X10'' in RPM I 1 640 medium containing 10'M2-mercaptoethanol 0 units/d penicillin, 100μ9/mR streptomycin
They were suspended so that the number of particles per d was obtained.

Next, PHA (phytohemaggluti
nin) (Difc.

Laboratories) at 1%, TPA (1%
2-0-tetra clecanoyl ph
orbor-1 3-acetate>
(Sigma) was added at 5ng/7 (Y
．． In, Yip et al., Inrection and 1
mmunity. 34.131 (1981), Hir.
ano et al., Microbiology and I
mmunol. 2 7.

87 (1983)). By this method, IL2 and IFN-
Production of physiologically active substances such as γ is induced. PI-IA
and 16-24 hours after addition of TPA, cells were collected in mR.
Adjusted NA.

Adjustment of mRNA, production of cDNA, and cloning into plasmids were performed using known methods (Okayama et al., Mole
cular and cellular biolo
oy.

280 (1983)). The resulting CD
Known IL2C DNA (
Noguchi et al., NatlJre, 302.305 (1
983)) Complementary chlorine sequence GAT GCT T
By colony hybridization using TG ACA AAA GGT as a probe (Q runs
Tein et al., proc.

Natl. Acad. Sci., 72.396
1 (1975)) positive strains were selected and plasmid p1
12-28.3 was obtained. (FIG. 1) 300 μ9 of pIL2-28.3 was digested with 3 μm H1 and subjected to agarose gel electrophoresis. A short fragment band containing the human IL2i gene was cut out, and the DNA fragment was recovered and purified from the gel by electroelution. Add this further to B
Human I was digested with a1I and analyzed by agarose gel electrophoresis.
A long chain fragment containing the L2 structural gene was isolated and purified. Then T
a Make the ends of this DNA blunt ends with DNA polymerase, and add BΩ1 to these ends with T4[)N△ligase.
■Added a linker. Furthermore, B(III and Rs
After digestion with aI, the fragment containing the IL2 structural gene was separated and purified by agarose gel electrophoresis. This is called fragment a.

On the other hand, pSV 2 rabbit β glob
in (R.

C. Mulligan et al. 3science 209.
1422 (1980)) Hind III, B
(Of the two DNA fragments obtained by digestion with III and 1q, the longer DNA fragment was separated and purified by agarose gel electrophoresis and electroelution as described above.

Let's cut this into pieces. Furthermore, the following oligomers were chemically synthesized using known methods.

5'AGCTTTGCAAAGATGT3'...
...d 3'AACGTTTCTACA5' ...... e Next 0.1μ! Fragment a of 7, 0.1μ! ? Fragment of bSO
, 18μJ of oligomer d, and 0.09μJ of oligomer 〇 were mixed and reacted with DNA 4 DNA ligase at 15°C for 18 hours to connect them.
MC1061 (M, J, Ca5adab
an et al., J, 1ylol.

Biol., 138.179 (1980). Plasmid DNA was extracted from a strain showing resistance to ampicillin 100/l19/d, and 1-1indll [base sequence around the cleavage site was converted to Maxa+++G11ber].
t method (Meth Enzym, 65, 499 (
1980)) and the desired vector psvh
Obtained IL2. (Figure 2) (3) Vector psVh
pdBPV-1 (142-6>) was used as a vector containing the BPV-1 gene produced by IL2/BPV97 (7) (P.

M, l-10Wlel/et al., proc, Na order, A
cad, sci.

79.7147 <1982)). This pdBPV-1
<142-6) was digested with pvu[, and long chain fragments were separated and purified by agarose gel electrophoresis. This is called fragment a.

On the other hand, 1) After digesting SVhlL2 with AccI, the longest chain fragment was separated and purified by agarose gel electrophoresis.

Let's cut this into pieces.

Next, 0.5 µm fragment a and 0.5 µm fragment a were mixed and reacted with T4 DNA ligase at 15°C for 18 hours to ligate them, resulting in E. coli MC1.
061 was transformed. Plasmid DNA was prepared from the obtained ampicillin-resistant colonies, a restriction enzyme cleavage map was created, and the desired vector psvh I L2/BPV was prepared.
I got 97. G of HoaI site of BPV-I DNA
When 1, the pVU1 site is 2 of 4783.5042.
There are 504 fragments of the SV40 early gene.
PVU of 2 [The one connected to the site is 1) SVhlL2
/BPV97C, 4783 pvu[ site connected to psVh I L2/BPV97T. (Figure 3) Example 2 A vector pAdDh112 expressing the mouse DHFR gene and human IL2m gene was prepared by the following method.

Origin of replication of SV40, E. coli plasmid pBR322
origin of replication and the tetracycline resistance gene, and DHF linked to the adenovirus major late promoter.
α6 known plasmid pAdD26SVA from Ra gene
#3 (RJ, Kaufman et al., Molecule
ar and Cellular Biology 2
．． 1304 (1982)) was digested with EC0RI,
It was treated with DNA polymerase (■) to make the EC0RI cleavage site blunt-ended. Example 1
The mixture was mixed with 0.6 μ9 of the ACCII-digested longest chain fragment of psVhIL2 obtained in (2), and ligated by reacting with T4 DNA ligase at 15° C. for 18 hours. Using 20 ul of this reaction mixture, E,
Plasmid DNA was obtained from colonies exhibiting tetracycline resistance by transforming E. coli MC1061.
Prepare a restriction enzyme cleavage map and select the desired plasmid 1.
) AdDhlL2 (q). Since there are two orientations of 112 contained in this plasmid, [12 genes are read in the same direction as the D t-l F R gene is read in the same direction as pAdDh I L2T. , and the different one was named pAdDh I 12G. (Figure 4) Example 3 Expression of IL2 polypeptide (1) Expression of human 1 and 2 polypeptides in CO8m cells Vector pI L2-28゜ shown in Figure 1 3 is 11
It has the SV40 early gene promoter and replication origin upstream of the two-way gene, and the poly rA addition signal of the SV40 late gene downstream thereof. It also contains the replication origin of pBR322 and the ampicillin resistance gene.

This plasmid can express the IL2 gene under the control of the SV40 Hatsumei promoter in mammalian cells.

Therefore, the monkey-derived cell line CO8-1 (
Y, Gluzman, Ce11.13.175 (
1981)) 2.5μ to about 5X105 pIL2-
28.3 using the calcium phosphate method (F, l, Qr
aham et al., virology) dan 4.536 (1
973)). Human IL2 activity in the cultured cell solution 3 days after introduction was measured. CTL for IL2 titer measurement
G11lis et al. (S, G11liS et al., J. 1 mmunol, 120.2027 (1
978) ) method or T. Mosmann's method (J. Immunological Met.
Hods, 65, 55 (1983)).

As shown in Table 1, the culture supernatant of C08-1 cells transfected with p112-28.3 had IL2 activity. plFNγ-15 (Patent application 1987-139398
The culture supernatant of cells transfected with IL
2 activity could not be detected.

Table 1 Transfected IL2 active plasmid DNA unit/ml pIFNγ-15×4 The example shown in Example 3 (1) is a transient expression. I
In order to constitutively express L2, the vector psVhIL2/BPV97G1μ9 shown in Example 1 (3) was injected into approximately 5×10 5 Cl27I cells (D, R, LOWV et al., J, ■1ro1., 26°) by the calcium phosphate method. 1
91, (1978)). The next day, the cells were sown on 9,747,213 sheets of 10 cm diameter, and 15
After culturing for one day, transformants forming about 40 foci were obtained. These 12 clones are 2
Separated into 4-well plates. When the cells were cultured until they became C0nfluent and the 11-2 activity of the culture supernatant was measured, it was found to be 810 units/rd! A recombinant cell line <B Ori''E) producing human IL2 was isolated. The results are shown in Table 2.

Below Margin Table-2 Clone rL2 titer No., unit/d BOri' E-3,1180 3,2810 3,3180 3,4540 3,53Q 3,6 9Q 3.7 270 3.8 540 3,9 9Q 3.10 810 3.11 30 3.12 90 Among these clones, BOri” E3-2 and BO
ri” E3-8, 1 x 106 pieces each, 25 cd
The cells were cultured in a flask containing 10 ml of Dulbecco's modified Eagle's medium containing 101/1 fetal bovine serum, and the medium was replaced with 10 ml of fresh medium every 3 days, and the IL2 activity in the medium was measured. The results are shown in Figure 5. A high titer of IL was obtained.

(3) pAdDh I L2C d h f r-
Human 112 expression vector p obtained in Cell Transformation Example 2
AdDhlL2G, 4 μ3 was added to approximately 1×10′ dhfr-CH○ cells DXBI [strain (
G, Ll rlaub et al., proC, Na mouth;
Acad, Sci, USA, 77.421
6 (1980)), and 2 days later, cells with a diameter of
When the seedlings were reseeded in five 0 cm dishes and cultured for 10 days, about i ooo d Fl f r .proud transformants were obtained. 24 clones of 12 of these
The cells were separated into a well plate, cultured until confluent, and the IL2 activity of the culture solution was measured. (Table 3) Table-3 Clone IL2 titer No., unit/m1 CHOIL2-1 16 (4) d h fr” Cloned cells of Example 3 (3) that became human IL dhfr” introduced into CHO cells CHOIL2-1 was grown in selective medium and grown to a diameter of 10
Approximately 2 x 105 cells per cm dish of 0.02
MM was cultured in a selective medium containing MTX. After 14 days of culture, 17 clones were separated into 24-well plates and 0.
The cells were cultured in a selective medium containing 0.02 MM of MTX. IL2 activity of the culture supernatant was measured for five strains with fast growth rates.

Margin Table-4 Clone IL2 titer No. (unit/fd CHO [L2 1-1 370C1-10I
L2 1' 50 As shown in Table 4, CHOI L2-1 cultured in MTX-free medium
Clones selected on medium containing MTX were 3-1
It showed 0 times higher IL2 activity.

Example 4 Sex of human IL2 produced by BOri”E3-2 (1) I
Maintenance of L2-dependent CTLL2 cells 10% fetal bovine serum;
5X10'M2-mercaptoethanol, 100 units/
80 units/d of IL2 in RPM11640 medium containing snake penicillin and 100 μa/mfl streptomycin.
CTLL2 cells could be subcultured for more than 6 months in a medium to which 3Qri"E3-2 culture supernatant was added.

(2) Purified BOri”E2 ig culture supernatant 112 and Example 1
As described in (1), lymphocytes were isolated from human tonsils, and 112, which was produced without serum, was first precipitated with 80% saturated ammonium sulfate, and the precipitate was diluted with 10 mM Tr.
is dissolved in HCl (pH 7,5) and diluted with 10
The mixture was thoroughly dialyzed with mMTris HCI (t)H7,5). This was mixed with 10mM 7ris)-ICI(
It was adsorbed onto Madrex Blue Agarose (Amicon) flattened with DH7゜5) and 10mM Tris.
HCl (Dt-l 7, 5 > Te washing, 0.5
10 mM Tris HCI (p
IL2 was eluted with H7,5). This was added to 10mM
Dialyzed with Tris HCI (DH7,5>, 0.0
10mM TrisHCI with 5M NaCl
< 1) Apply to DEAE Sephacell planarized with H7,5), and adsorb the non-adsorbed fraction again to Madrex Blue agarose equilibrated with 10mM Tris HCI (pH 7,5) containing 0.05M NaCl, and equilibrate. Wash with the buffer used for
10 mM Tris HCI (pH
7,5>, IL2 was eluted.

These were separated by 5DS-polyacrylamide electrophoresis (10
~20% polyacrylamide gel) and blotted onto a nitrocellulose membrane according to a conventional method (circle area-
Shi, Cell Engineering 2.1061 (1983)). 11 of the proteins transferred to nitrocellulose
In order to specifically stain only 2, we would like to use an enzyme-antibody staining method. Fumi Enbe, Cell Engineering 2.1061 (198
3)). - An anti-human IL2 mouse monoclonal antibody (QenZyme) was used as the secondary antibody, a peroxidase-labeled anti-mouse I (] rabbit antibody (Cappel 1)) was used as the secondary antibody, and color was developed with 3,3'-diaminobenzidine. As shown in Figure 6, the molecule ω is approximately 16,400 Daltons and 1
A band corresponding to 4800 Daltons was recognized. This is described in Robb et al. (Mo1. Immunol, 18.1
087 (1981)) is 16,200 Daltons and 1
This is in excellent agreement with the results reported to be 4,600 Daltons.

From the above results, IL2 produced by BOri”E3-2
It was speculated that IL2 contains molecules to which sugars have been added, similar to IL2 produced by human lymphocytes.

[Brief explanation of drawings]

FIG. 1 shows the construction of vector pIL2-28.3. FIG. 2 shows the construction of vector psVhlL2. Figure 3 shows vector psVh I L2/BPV97
G oyo (f D S V h [L 2 / B P
V 97 T 17) The production method is shown below. Figure 4 shows vectors pAdDh I L2C and pAd
A method for producing Dh I L2T is shown. Figure 5 shows a clone of mouse C127I cells transformed with BSVh I m2/BPV97C, BOri'
IL2 production by E3-2 and BORI E3-8 cells is shown. Figure 6 shows IL2(b) produced by 13Qri''E3-2.
) Shows the results of plotting and staining by enzyme antibody method of IL2(a) produced by human tonsil-derived lymphocytes. Patent applicant Toshi Co., Ltd. Figure 1 pIL
2-28.3 Figure 2 psVIIL2 Number of days Figure 5 b ri - Figure 6

Claims (7)

[Claims] (1) Human interleukin 2 is present downstream of the promoter sequence in a vector capable of replication and expression in eukaryotic cells.
A human interleukin 2 expression vector obtained by linking a gene encoding. (2) A vector capable of replicating and expressing in eukaryotic cells,
The expression according to claim (1), which is a vector containing a replication origin necessary for replication in E. coli, a PvuII-cleaved long fragment of the bovine papillomavirus type I gene, and a poly r A addition signal. vector. (3) A vector capable of replicating and expressing in eukaryotic cells,
The expression vector according to claim (1), which is a vector containing a replication origin necessary for replication in E. coli, a gene for dihydrofolate reductase, and a poly r A addition signal. (4) The expression vector according to claim (1), wherein the promoter sequence is an SV40 early promoter or SV40 late promoter sequence. (5) Human interleukin 2 is present downstream of the promoter sequence in a vector capable of replication and expression in eukaryotic cells.
A eukaryotic cell line obtained by transforming eukaryotic cells with an expression vector linked to a gene encoding the . (6) A vector capable of replicating and expressing in eukaryotic cells,
The eukaryotic cell according to claim (5), which is a vector containing a replication origin necessary for replication in E. coli, a PvuII-cleaved long fragment of bovine papillomavirus type I DNA, and a poly r A addition signal. KK. (7) A vector capable of replicating and expressing in eukaryotic cells,
Claim No. 5, which is a vector containing a replication origin necessary for replication in E. coli, a DNA encoding dihydrofolate reductase, and a poly r A addition signal.
Eukaryotic cell lines as described in Section.