JPS62175182A - Expression vector for animal cell and use thereof - Google Patents

Abstract

PURPOSE:To secrete a protein produced in a cell to the outside of the cell and facilitate the collection of the protein, by using a DNA, coding a signal peptide and linked to the upstream side of a DNA sequence coding a protein. CONSTITUTION:A DNA sequence coding a signal peptide of a protein, derived from an animal cell and different from the animal cell is linked to the upstream side of a DNA sequence coding a desired protein by completing reading frames. An animal cell is transformed by the resultant expression vector having the resultant DNA and promoter DNA and cultivated to secrete and accumulate a desired protein in a culture medium. The resultant protein is then collected. When the desrired protein is interferon, interleukin or B cell growth factor, human interleukin-2, interferon-gamma, etc., may be cited as the DNA sequence coding the signal peptide.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to expression vectors for animal cells and their uses.
Regarding this.

BACKGROUND OF THE INVENTION Secretory proteins synthesized within cells (proteins secreted from cells through the cell membrane) must necessarily pass through the cell membrane during the secretion process. In order for these proteins, which are normally water-soluble, to pass through hydrophobic membranes, binding between liposomes and endoplasmic reticulum membranes is first required. Secretory proteins are generally synthesized intracellularly as pre-proteins with an additional peptide sequence (called a signal peptide), which functions as a signal that directs liposomes to bind to the endoplasmic reticulum membrane. It is thought that there are some.

It is also predicted that there are receptors on the endoplasmic reticulum membrane that recognize pre-proteins. As soon as the preprotein is synthesized, it passes through the endoplasmic reticulum membrane with the signal peptide at the beginning. At this time, the signal peptide is excised by a membrane-associated endopeptidase called ngnar peptidase, and the mature protein with the final hydrophilic folded structure is secreted into the lumen of the endoplasmic reticulum. This protein is exported to the outside of the cell via the Golgi apparatus [Natural
e) 28-3433 (1980)].

Signal peptides of secreted proteins have some similar parts (J) for each individual protein, but their primary structures are usually quite different
y)-(J, Mo1. Biol,) 167:391
(1983)]. It is thought that this is probably the result of signal peptides evolving into forms that allow individual proteins to best secrete the secretion.

Therefore, when expressing useful genes using animal cells,
Even if only the gene encoding the mature protein portion is constructed in an expression form, the synthesized protein will accumulate within the cell and will not be secreted into the cell culture medium. In other words, in such a case, the D N
It is necessary to add A (leader sequence) to the 5' end of the gene. However, there are many genes for which the leader sequences are still unknown. In addition, in the case of epidermal cell proliferation η-1 factor (EGF) [Xience (
Science) 221-:236 (1983)], when the target protein is a large mature protein that is produced by cleavage from a small part of the C-terminus.
cDNA based on Metsenger RNAΔ It is extremely difficult to obtain cl)NA containing up to the 5' end of the leader sequence using DNA because RNA is huge. In order to mass produce a known protein whose genetic structure is not clear, even if the gene is chemically synthesized using the second structure of the protein, it is not possible to express it in a secreted form in animal cells because the leader sequence is unknown. Accumulated shellfish inside cells - no choice. This (the amount of production of the desired protein may be extremely reduced) and it takes a great deal of effort to extract the desired protein from cells and purify it from the extract. In i-j (instead of j,
We realize that it is extremely difficult to obtain the desired substance in its pure form.

Problems to be Solved by the Invention Until now, it has been nearly impossible to express secreted genes in animal cells for which the leader sequence is unclear or is extremely difficult to obtain. They recognized that the gene product can be efficiently secreted and accumulated in the culture medium in animal cells by using the gene's unique leader sequence, and conducted further research to complete the present invention.

...Kozuharu J Subuta Iv61 - Yoshiro Ichiriki Hirozoko Fuyu and Azaki The present invention is based on (1) the signal peptides of different proteins derived from animal cells in one sequence of DNA sequences encoding proteins; DNA sequences encoding the D
Expression hector for animal cells with NA.

(2) Upstream of the DNA sequence encoding the protein, =
4-An animal cell transformed with an expression hector for animal cells having DNA formed by linking DNA sequences encoding signal peptides of different proteins produced in animal cells with their reading frames aligned, and DNA of an animal cell promoter; (3) DNA in which DNA sequences encoding signal peptides of different proteins derived from animal cells are linked in the same reading frame in the same way as DNA sequences that coat proteins, and DNA of promoters for animal cells. This is a method for producing a protein, which is characterized by culturing transformed animal cells with an expression hectare for animal cells having the above-mentioned protein, secreting and accumulating the protein in the medium, and collecting the protein each time.

Regarding the DNA sequence (gene) encoding the protein mentioned above, it is more desirable that the gene encoding the protein used for secretory expression has a known base sequence, and the gene isolated from the chromosome, metsusenger RNAΔ (mrjNA, )
It may be anything, such as cDNAΔ synthesized based on , or a chemically synthesized gene. Specifically, genes for various physiologically active proteins are mentioned, such as interferon (for example, IFN, IFN-α, IFN-β,
IFN-γ, etc.), interleukins (interleugin-1, inter[l-kin-2, etc.), B cell growth factor (BGF), B cell differentiation factor (BJ) F), macrophage activating factor (MAF) , Lymhotoginn (1,T
), cytokines such as tumor necrosis factor (TNF), transforming growth factor ('I'C)
F”-α); erythroboedin 2 fibroblast growth factor (
FOP), epidermal growth factor (EGF), insulin,
h)・Growth hormone natobebuti)・Protein hormone:
Hepatitis B virus antigen, influenza antigen 1, foot disease virus antigen.

Pathogenic microbial antigen proteins such as malaria parasite antigens; peptides such as pebutigase (e.g., teinkuplasminogen agitator, nokinase 1 serratio peptidase, etc.) and lysodeme; immunoglobulin proteins (IgG
, IgE, etc.), and blood protein components such as human serum albumin (H9A). Above all,
Hepatitis B virus gene [JP-A-5? -209298 publication], immunoglobulin E gene [Nuclic Ac1ds Rcs
, ) lInia 19 (1983) l, immune interferon (IFN-γ) gene] - JP-A-58-18919
Publication No. 7'', Hi)・Epidermal growth factor (EGF) gene [Special application 1977! ]-210502 (October 9, 1982)
The specification corresponds to Japanese Patent Application Laid-Open No. 61-88881. ], Human fibroblast growth factor (FGF) [Patent application 1986
Patent Application No. 57919 (filed on March 14, 1985), Japanese Patent Application No. 82699 (filed on April 9, 1986), and all or part of the structural gene of the protein. may be used. Furthermore, when constructing a secretory expression plasmid by inserting these genes downstream of the leader sequence or the like of an expression vector, an appropriate synthetic oligonucleotide may be linked to the genes as necessary.

A DNA sequence encoding a sogenic peptide of a different protein derived from animal cell 1] 1 is an I) NA sequence encoding a sogenic peptide of a protein derived from an animal cell different from the protein related to the above gene, and includes, for example, HiI interleugin-2 (IL-2)
No. 528], IPN-γ [Unexamined Japanese Patent Publication No. 58-18919
Publication No. 7], human tumor necrosis factor (TNP) [NEID-1
--(Nature), 312, 724 (1984)
], Human Link (L, T)
(NaLure), 312 Near 2], (1984)]
Examples include DNA sequences encoding signal peptides such as.

Preferably, a DNA sequence encoding the IL-2 signal peptide ζ represented by the formula % is particularly preferred, and a leader sequence of II, -2 represented by the formula % is particularly preferred.

] The DNA sequence encoding the sogenic peptide of the protein mentioned above may have at its 3' end a DNA encoding 1 to 20 amino acids of the N-terminal polypeptide of the protein.

The DNA sequences encoding the sogenic peptides of different proteins derived from animal cells of the present invention can be synthesized as cDNA (leader sequence) synthesized based on mRNA or chemically synthesized by methods known per se, such as the phosphite amide method. Fully synthetic or semi-synthetic [Tetrahedron Letters (T
etrahed'ron 1. etLers) 22:
1859 (1981)].

When producing a protein leader sequence based on mRNA, a plasmid containing the protein leader sequence DNA can be obtained by, for example, (a) isolating mRNA encoding a protein derived from animal cells, and (b) extracting a single strand from the RNA. cDNA, then double-stranded D
(c) integrate the cDNA into a plasmid, (d) transform a host with the obtained recombinant plasmid, and (p) culture the obtained transformant, and then extract the protein from the transformant. Isolating a plasmid containing the leader sequence, (f) cutting out the desired cloned leader sequence DNA from the plasmid, and (g) producing it by ligating the cloned DNA downstream of a promoter in a vehicle. I can do it.

This plasmid can be introduced into a suitable host, such as E. coli. From the transformants thus obtained, clones can be selected using methods known per se, such as the colony hybridization method. These series of basic operations are well known and are described in Methods in Enzymolog.
y) 68 (+979) and Molecular Cloning (198
2).

Co1d Spring Harbor Labora
It is detailed in the story.

The leader sequence DNA is obtained by producing a plasmid from the transformant by a method known per se, and then cutting the plasmid with, for example, a restriction enzyme, and then using a method known per se such as polyacrylamide gel electrophoresis or acarose gel electrophoresis. Be able to let go. Furthermore, the base sequence of the isolated DNA fragment can be determined by a method known per se, for example, by a method known per se.
-1y method “Proc, Nat.”
Acad, Sci, USA).

74:5463 (1977)].

The animal cell promoter used in the present invention includes:
Any promoter suitable for the host used for gene expression may be used.)

For example, monkey cells CO3-7, vero; Chinese hamster cells c++o, mouse cells r, etc.
The SV40 native promoter, suitable for expression,
One example is the promoter of the virus.

The expression vector for animal cells of the present invention has, for example, upstream of the NΔ sequence encoding the protein I) D encoding the signal peptide j of a different protein derived from animal cells.
DN formed by concatenating NA sequences with their reading frames aligned.
It can be produced by constructing a known vector such as pBR322 by inserting the DNA of A and an animal cell promoter into a known vector such as pBR322 using a restriction enzyme or case.

In addition, the transformed animal cells of the present invention can be produced by transforming animal cells with the expression vector for animal cells described in Section 2, and selecting and harvesting the cells as necessary.

The animal cell mentioned above may be any animal cell that can express a protein leader sequence, such as mouse (e.g. L cell, Balb/3T3 cell), human (e.g. PL cell), hamster (e.g. CI cell), etc. -TO cells), etc.

Transformation is, for example, conformal transformation [Cell 1
6+777 (1979)], the desired animal cell transformant can be advantageously selected and isolated (cloned).

The method for producing the protein of the present invention can be carried out by culturing the above-mentioned transformed animal cells, secreting and accumulating the protein in the medium, and collecting the protein.

The animal cells are cultured in a known animal cell medium, for example, MEM medium containing 5 to 20% fetal bovine serum.
It is carried out for 10-10 days at 0°C.

The target protein produced and accumulated in the culture medium can be used by removing the cells and concentrating and drying the culture solution as it is, but it can be used by appropriately combining known partial purification methods. Be able to separate and purify the protein that you want.

According to the method for producing proteins using animal cell transformants of the present invention, glycolylated proteins can be easily produced in large quantities depending on the purpose. In this case, compared to proteins produced by E. coli using genetic recombination techniques, glycosylated proteins are more stable and highly soluble in water, and are also secreted, so they can be purified and used advantageously.

Glyconolated proteins produced according to the present invention have physiological activities similar to those of known proteins, and can be used as pharmaceuticals and the like. It can be used in the same manner as these known proteins.

Furthermore, cell growth factor genes (e.g. r=EGF, TGF
Animal cells (J) transformed with an expression vector for animal cells described in an unknown device that expresses a gene such as FGF, FGF, etc. Effects similar to those observed when cell growth factors are added to the cells should be observed.In other words, an increase in proliferation rate and a decrease in serum requirement should be observed in these transformed cells. When applied to cells, non-n is advantageous in terms of producing useful substances.

In the specification and drawings of this application, when bases, etc. are indicated by abbreviations, IU P A C-I U B Comm
1ssionon Biochemical Nome
Abbreviation by nclanrre or 13 in the field
It is based on commonly used abbreviations, examples of which are listed below.

1) NA Deogyne ribonucleic acid CD N A Complementary deogine ribonucleic acid A
Adenine 1゛ Dimine G Guanine C Noton RNA Ribonucleic acid The present invention will be explained in more detail with reference to the following Examples, but the present invention is not limited thereto.

The transformants disclosed in the Examples were obtained from the Institute of FermcnLa.
Lion, OsakaiFo) under the following deposit number.

Mouse 1, Δ9-1sII EGP-2 cells IP
O-50057 (deposited July 18, 1985) Mouse L-1sII IgE 9 cells/TP O-
50056 (Deposited July 1985 1.81F) Mouse Δ3]-1s1.1. EGF-2 cell 1, FO
-50093 (Deposited July 30, 1985) Example 1 Expression vector for human EGF secretion and human E
Animal cell transformant producing GF (1) Plasmid pT8406.1) 'I' B /
Construction of 110 and pTB411 for animal cells 11. , -2 gene expression vector p'1. '
BI06F Patent Application No. 1986-133490 (June 1985
The specification (corresponding to Japanese Patent Application Laid-Open No. 61-63282) Example 1 (1)] was cut with restriction enzyme 11g1AI, and I1. , -2 gene leader sequence containing i, O
A kb DNAI specimen was isolated. The sticky ends were made blunt by T4 DNA polymerase reaction, and EcoRI linkers were added.
After adding CCGGAATTCCGG with T41) NA ligase, EcoRI and 1lindll
Complete digestion was performed to isolate a 64 kb DNA fragment consisting of the 5V40 DNA [lJ sequence (promoter and splice site) and the IL-2 gene leader sequence. Also, the above p'r B 106 is separately Bam
Cut with HI and old ndlll to create plasmid pBR3.
DNA for replication in E. coli of DNA derived from 22
2.6 containing the replication initiation site, the sequence containing the ampinilis resistance gene, and the polyA addition site derived from 5V40 DNA.
kb DNA fragment was isolated. On the other hand, the human 1-EGF synthesis gene was cloned I) T-836], [Patent Application No. 1988-21. 0.18 kb E encoding human BGF) from Example 4]
A coRl-BamHI DNA fragment was produced. These 3
The seed DNA fragments were ligated by T4DNA ligase reaction to construct pTB406. p'J” B 406
is the nucleotide number of the IL-2 gene DNA, ]
Leader sequence up to J23 (Figure 1) and human EGF
EcoRI linker and EGFI)N in which the synthetic gene DNA was inserted so that the reading frames of both matched.
It has a structure that is connected through the A translation initiation codon A T G.

Next, the above-mentioned 1. Okb I) NΔ fragment and synthetic oligonucleotide “CCT” produced by known methods
ACTTCG” and “AATTCGAAGTAGGT
GCA” was mixed and reacted with T4 DNA ligase, and then cut with EcoRl and old ndIIf to 0.65
The kb (EcoRI-HindIII) DNA fragment was isolated and purified. The DNA fragments obtained in this way are 5
V40 DNA-derived sequence and fL.

It has the entire leader sequence of the IL-2 gene and a base thread sequence encoding the four amino acids on the N-terminal side of the IL-2 protein molecule. On the other hand, the previously constructed pT B 406 was incubated with EcoRI-
) final, and contains the EGF gene, polyA addition site, and pBR322-derived sequence.
The NA fragment was separated and the 0.65 kb EcoRl
-11indlll DNA fragment and pTB
4jQ was constructed.

Furthermore, rl1LOTi35-8 [JP-A-60-1.1
No. 5528 Publication J was cut with PsLl and AIuI to obtain an O having the I L , -,, -, 2 gene r-leader sequence and the sequence that coats the 11 amino acids on the N-terminal side of the r L-2 molecule. , l6kb DNA fragment was isolated and EcoR1 linker-CGGAATTCCG was added to Alu
DNA fragments with blunt ends added by I cleavage were prepared. Cono o, 1.6kb DNA fragment, 1) TB406
The 2.8 kb DNA fragment obtained by cutting Ecoll 1-It ind ITI and p'r B
p'lI''B4] was ligated with a 0.52 kb D'NA fragment obtained by cleaving 410 with old nold IT-Ps tl by T4 DNA ligase reaction.

These EGF' expression vectors p'J' B 406.
410 and I)T''B41] is shown in FIG.

In addition, the construction (base sequence and predicted amino acid sequence) of the region containing the IL-2 leader sequence and the binding site of the HiI-EGF synthetic gene in these recombinants is shown in Figure 3. Ta.

The IL-2 gene-derived sequence of pTB406 has nucleotide numbers I to 123. pTB 410
is No, I~135°1) TB41] is No, ]-
1.58 (Figure 1).

(11) Plasmid pT I3503. p'T'
Construction of B504 and human I)'■゛B505 l\ctorρ for human f', GF expression containing the human +-II, -2 leader sequence obtained in Example I (1)
'FB406. pTB410 and rlTB411 were isolated from C1, which is present in the SV40 promoter, respectively.
al and 1lindI [1te cut, p7FB3]4
[Special application 1986- +334i+o (June 1, 1985)
80 Application) Specification (corresponding to JP-A-61-63282) Abelson murine leukemia virus (Δ-M u L, isolated and purified from Example 1 (iii) j).

V) 1. Containing LTR region. lkb C1aI-
pT+3 by inserting the tlindllD NΔ fragment.
503. pT r3504 and rl'FB505
was built. All of these recombinants are 5V407'
Compared to the case of promoter alone, high-efficiency expression of the target gene is expected, especially in mouse cells. pT
The construction of B503 is illustrated in FIG.

(iii) Transformation of animal cells Dulbecco's modified Eagle MEM (1) containing 10% tallow serum in a falcon petri dish (6 cm in diameter)
ME = 19~ M) medium, and mouse HP RT (hypoxan
thinphosphoribosyl trans
ferase) deficient cells (LA9 cells)
efield, J.W., Experimental Cell Research (EXp, Ce11. Res,) 3↓:1
．． 90-1.96 (1966)] was cultured overnight at 37°C. After culturing, plasmid p4aA8 (Hi1-1-TPRTc) was added to these cells (7x10 cells/Dingyu).
DNA-containing plasmids) [Jolly, DJ et al., Prosecuting National Academy of Science (
Proc, Natl, Acad.

3ci, USA) 80:477-481. (1,
983)] 0.5 μg and 1.0 μg pT B 50
5D NA and the method of Graham et al. [Pyrology (Vi
52-:456-467 (1973)
], the mixture was mixed, inoculated, and co-transformed. After incubating at 37°C for 4 hours, the medium was replaced with a new one, cultured overnight, and the next day, I-I A T medium (15μ
The culture was continued at 37°C.

Continue culturing by replacing the culture medium once every 3 to 4 days.
After about 2 to 3 weeks, the HP RT+ cells -20- proliferated to form colonies.

(1v) Cloning of transformants and quantification of EGF Cloning of the transformed cells obtained in Example 1 (iii) was carried out according to the limited diaryon method. After cloning, the cloned cells were cultured in DMEM medium containing 10% tallow serum.

The isolated cloned cells were sown on lymphoid dishes (Flow Inc.), and when the cells became approximately 80% confluent, the medium was replaced with fresh medium, and 2 days later, the EG in the culture-1 supernatant was inoculated.
The amount of F was measured. The amount of EGF was determined by radioreceptor assay (RRA method) [Cohen, S et al., Proceedings of the National Academy of Sciences (Proc., Natl. 8cad, Sci.
, USA).

Akira, '1.31.7-1.321 (1,975)] did not show the same activity and expressed the weight of a purified mouse EGF standard. Sunaichi, first human fetal foreskin cell Plo
w 7000 (Flosha.

(commercially available) was cultured in a lymphoid dish with a diameter of 1' and 6 cm using DMEM medium containing 10% tallow serum.

Discard this medium, wash the cells with DMEM medium containing 0.1% BSΔ, and add 0.2 ml of the same medium and chloramine']
Mouse ECP (Col
5 ng (commercially available from Laborativc Research, Inc.) and the culture supernatant of the transformed cell clone obtained above were added to a51, and cultured at 37° C. for 1 hour.

Next, after washing with the same medium, treated with 0.2N Na01 (
Transferred to Dupu, γ-ray counter, captured 12
5I was measured. Using the same operation, create a mouse with known weight E G
The human EGF finger in the culture supernatant was calculated from the calibration curve obtained from the competitive reaction with F.

Culture of the obtained LA9 cell transformant - 70% from the supernatant
EGF amounts of ~80 ng/ml were detected. Results first
Shown in the table.

Table 1 Culture - Transformed cell clone mouse T-A 9-IsIIEGP-2 in which l'l GF was detected in the supernatant
T P O50057), intracellular and culture NJ:
The amount of EGF produced during 2t'r was compared. ■7Δ
9-1sIIEGF-2 cells were sown in a falcon petri dish (6 cm in diameter), and just before the cells became confluent, the cells were prepared with 5 ml of culture medium.The next day, the culture-1 supernatant was sampled, and the cells were washed using a rubber policeman. It was peeled off and collected by centrifugation (2000 rpm for 5 minutes). The collected cells were then treated with 200μρ of 10mM Tris.
-11cI(1)H7,5) and sonication (5
Disrupt the cells by centrifugation (2000 (lrp
(m, 4°C, 30 minutes), the EGP activity in the obtained supernatant was measured according to the method described above.

In addition, the sampled culture - EGF in the second serum
The amount was determined similarly. As a result, mouse I7Δ9-IS
1. IEGl"2 cells contain approximately 400 ng of E G per 107 cells in the culture medium and approximately 75 ng in the cell extract.
It was found that F was produced.

Next, the animal cell transformant L obtained in Example 1 (iv)
Regarding A9-1sI IEGF-2, EGF levels in the culture supernatant were measured over time.

5XiQ' pieces of 1. A9-15LIEGF-2 cells were seeded and cultured in 2 ml of DMEM medium containing JO% tallow serum in a 37°C CO2 incubator. From day 3 after the start of culture, the number of cells and EGPM in the two culture supernatants were measured by the RΔΔ method (FIG. 5). EGP is produced and accumulated as cells proliferate, and even after cell proliferation has stopped, EGP remains
F production continued, with a maximum value of 680 ng/ml.

Example 2 Expression of H. and H.I for human IgE secretion
Animal cell transformant producing gE (i) Plasmid pTB541. pTB542.
pTB543 and pTB! Construction of M4 Plasmid pGETtrp302 [Kurokawa
a et al. 1 Nucleitsk Ass Research
icAcids Res, ), 11.3077-30
85 (1983) 1) was cut at C1al between the trp promoter and the translation initiation codon A'FG, and the sticky ends were blunted by S1 nuclease treatment to create Eco.
Rl linker-GGAATTCC was attached. EcoRl
and Pstl to separate a 1.2kb DNA fragment encoding IgE, and this 1.2kb E
coRl-Pstl DNA fragment and EcoRI-P
Plasmid pTB91 cut with st I. [Special application 1984. , No. 210502 (filed on October 9, 1988) (corresponding to JP-A-61-88881) Example 5(ii)], and by T4DNAΔ ligase reaction, l)T B 145 was constructed. pTB],
45 was cut with Pstl, the sticky ends were blunted with S, nuclease, and the Bamtll linker-CCGGATCCG
After coupling G, 1, . 2kh EcoRl-BamHID
A NΔ fragment was prepared. On the other hand, plasmid pTB505 (EGF secretion type expression vector) constructed in Example 1(ii) was cut with EcoRl-Bgln to remove most of the sequence encoding IICF', and then
A 1.2kb EcoR with a sequence that coats the polypeptide of the C7C4 region of IglE prepared from TB1-5.
l-BamHID NA fragment, 11TI3
543 was constructed. That is, pT+3543 is IL-
The sequence encoding the 1] amino acid on the N-terminal side of IL-2 and the sequence encoding IgE in alignment with this sequence combine, and upstream of these , a recombinant plasmid with A-Mul, VLTR and SV40 promoter regions as promoters for expression in animal cells (Figure 6).

Similarly, Example 1 (ii) (7) pT B503
Mari i:j: pT B 504 with EcoRI-Bg
pTB]45, cut with I
gE], 2kb Bam old-EcoRID
pT B 541 or pT B 5 from NA fragment
42 was constructed. pTB541 has an IgE-encoding sequence connected to the leader sequence of DingL-2, and p'r
B542 is a leader sequence with I>2 and I L-2
It has a structure in which a sequence encoding TgE is connected to a sequence encoding four N-terminal amino acids.

Also, Patent Application No. 176976 (August 13, 1985)
pT described in Specification Example 7 (1) (corresponding to E p c Patent Application Publication No. 177915)
The EGF'-encoding sequence of B506 was replaced with the rgE-encoding sequence derived from pT8145 in the same manner as above to create a pT8145-derived pT8145-derived rgE-encoding sequence, which does not have a leader sequence derived from the Ding L-2 gene.
TB544 was constructed.

(]1) Transformation of animal cells Falconnoyale (diameter 6 cm) t: , 1.0
Eagle's MEM medium containing % tallow serum was added, and mouse TK-deficient I cells were cultured overnight at 37°C. After culturing,
For this cell (7X]O'' cells/dinco), the plasmid p'l' contained 6] (3.5 kb Ba containing the TK gene of herpes symplex virus (HSV)).
The mHID NΔ fragment was cloned into r) Bn322 and the recombinant E. coli strain LE578 [None (G
ene), 7, 335-342 (1979); Dr.
A plasmid was isolated from a 2 kb PvulT fragment containing the TK gene [Pronouncing of the National Academy of Science (Pro, N.
a11. Acad, Sci, USA) %! ! ,
1441-1445 (1981)] recloned into pBR322) and 0.271 g respectively],
071g pT B 541. , ! ')TB S4
2. pT”B 543 and pTB!M4DN△ using the method “Virology” of Graham et al.
, 52.456~=27-. 167 (1973)]. After culturing at 37°C for 4 hours, replace with fresh medium and culture overnight.
The next day, HAT medium containing 10% tallow serum (1 g at 15°C)
/ml Hyboxanthin, ]tt g/ml Aminopterin, 57B/ml Demidine, 0.25 μg/ml
The culture was continued at 37° C. (MEM medium containing n+1 glycine).

If you continue culturing by replacing the culture medium once every 3 to 4 days,
After about 2 to 3 weeks, the TK+ cells proliferated and formed colonies.

(ili) Cloning of transformants and IgE
Quantification of Cloning of the transformed cells obtained in Example 2 (ii) was carried out according to the Limited Dairy Corn Yong method. After cloning, the cloned cells were cultured in Eagle's MEM medium containing 10% tallow serum. The isolated cloned cells were sown on a lymphoid plate, and the cells were reduced to about 80%.
When the wells reached confluence, 1 ml of new culture solution was added to each well, and the culture supernatant was sampled from the third white eye to measure the IgE content. At the same time, the cells were peeled off using a rubber policeman and centrifuged (2000 rpm
S minutes). Next, 03ml of IOmMTris-HCI (pH 7,5) was added to the collected cells, the cells were disrupted by sonication (5 seconds x 2), and centrifuged (20
]00 rpm, 4°C, 3 minutes), the amount of TgE in the obtained supernatant was measured.

TgE activity was measured using the RIST method [Radioi
mmuno 5orhent test1 immunology (1mmunology).

Shaku, 265 (1968)].

IgE expression vector pT B 541. having IL-2 leader sequence. , pT B 542 and p
Mouse 7 cells (TK") transformed with 'r B 543 were cultured at 500/ml or more in the culture medium.
Two IgE-detectable clones were obtained. Among them, clones of transformed cells using pT B 543 mouse L-13I1.1gE-9 (IF 050056
) produced and secreted 580 U/ml of IgE into the culture supernatant.

On the other hand, p'r B 5 without T L -2 leader sequence
From I7 cells transformed with 44, at most 1
Only clones were obtained in which 5 U/ml of IgE was detected in the two culture supernatants. Furthermore, the amount of IgE detected in the cell extract was higher than that of pTB54], pTB542 and pTB54], pTB542 and pTB54], pTB542 and pTB54],
In the case of cells transformed by TB543, approximately 10
70 U/ml, but a trace amount (less than 2 U/ml) of IgFl was detected from cells transformed with pT B 544.
or not detected. That is, transformed cells containing a TL-2 leader sequence for IgE expression are
The cells transformed with pT B 543 should produce and secrete at least 10 times as much JgE as those produced with a vector that does not have an IL-2 leader sequence. It turns out that it does. The results are shown in Table 2.

Table 2 Example 3 (1) Transformation of animal cells Mouse Bal'
b/3T3Δ31 cells International Journal of Cancer (Int, J, Cancer)
, 12', 463 (1,973)] and 10%
The cells were cultured overnight at 37°C in T) MEM medium containing calf serum. Plasmid pTB6 [Patent Application No. 1-133490 (filed June 18, 1985) Specification (Japanese Unexamined Patent Publication No. 6163-1983)
Corresponding to Publication No. 282) Example 1 (~!i)] 0.2 μg
and 1071 g of p obtained in Example 1, TB505DN
A and Graham et al.'s method
y) 52.456-467 (1973)], inoculation, and co-transformation. After culturing at 37°C for 4 hours, the medium was replaced with a new one, cultured overnight, and the next day 400 μg/
DM containing 041 g (Geneticin, manufactured by Gibco) of ml
The medium was changed to EM medium (containing 10% calf serum) and culture was continued at 37°C. When culturing was continued by replacing the culture medium once every 3 to 4 days, cells that had become resistant to (1,118) proliferated and formed colonies after about 2 to 3 weeks.

(11) Cloning of transformants and quantification of EGF Cloning of the transformed cells obtained in (i) above and culturing of the obtained clones - Quantification of the amount of EGF in the supernatant is the same as in Example 1 (iv) I went to 5-20'ng/ml of EGF from the culture supernatant of the obtained A31 cell transformant.
was detected.

(iii) Transformed cell clone in which EGF was detected in the culture supernatant of the A31 cell transformant grown in a low serum medium Mouse A31-1sIIEGF-2 (I F 05
0093), growth in low serum medium was performed using parental cell lines mouse Ba1b/3T3 A31 cells and mouse EG
F (Collaborative Re5earc
A comparative study was carried out with A31 cells supplemented with A.h., Inc.). 5XI in a falcon petri dish with a diameter of 3.5 cm.
O' cells were seeded in DMBM medium containing 10% calf serum, and after 8 hours, the culture medium was changed to DMEM medium containing 1% calf serum. Mouse EGF was used at a concentration of IOng/ml. The medium was replaced with fresh medium on the 3rd and 6th day after the start of culture, and the number of cells was measured over time. The results are shown in FIG. In DMEM medium containing 1% calf serum, the parent cell line A31 cells (not indicated by -0-) hardly proliferate, whereas the EGF-producing A31 cell transformant A
3l-IS], ]EGF-2 cells (indicated by -+-)
showed approximately 4-fold proliferation 7 to 9 days after the start of culture, which was comparable to when A31 cells were cultured in a medium supplemented with 10 ng/ml of mouse EGF (indicated by Kazuichi Issai). .

According to the present invention, glycosylated proteins can be advantageously produced as secreted proteins using transformants.

[Brief explanation of drawings]

FIG. 1 shows the base sequence of the leader sequence and structural gene portion of L-2 contained in plasmid pH, 0T135-8. Figure 2 shows plasmid pTB406. pT” B 4
10 and pTB41.1 are shown. FIG. 3 shows the detailed base sequence of the vector expressing EGF. Figure 4 shows the construction diagram of plasmid p'I''B503. Figure 5 shows the number of EGF-producing cells obtained in Example 1 and the amount of EGF in the culture supernatant. Figure 6 shows the plasmid A diagram of the construction of pT B 543 is shown. Figure 7 shows the growth of EGF-producing cells obtained in Example 3 in a low serum medium. In the figure, the abbreviations of restriction enzyme cleavage sites are indicated by "-". That's right. B: Bam1ll Bg: Bgl 汀 C: Co
al E: EcoRIH: l1indlTI
S: 5ail X: Xhol P: Pstl
Hg: 11g1AI Alu: Alul-35 = Figure 8 = Hokkoku 18 Kai 0U62-175182 (12)';1. ．． l
lipBR322ori pBR322or:

Claims (3)

[Claims] (1) An animal that has DNA in which a DNA sequence encoding a signal peptide of a different protein derived from animal cells is linked upstream of a DNA sequence encoding a protein, with the reading frame aligned, and DNA of an animal cell promoter. Expression vector for cells. (2) An animal that has DNA in which a DNA sequence encoding a signal peptide of a different protein derived from animal cells is linked upstream of a DNA sequence encoding a protein, with the reading frame aligned, and DNA of an animal cell promoter. Animal cells transformed with cell expression vectors. (3) An animal that has DNA in which a DNA sequence encoding a signal peptide of a different protein derived from animal cells is linked upstream of a DNA sequence encoding a protein, with the reading frame aligned, and DNA of an animal cell promoter. A method for producing a protein, which comprises culturing animal cells transformed with an expression vector for cells, secreting and accumulating protein in a medium, and collecting the protein.