JPS6232896A - Production of useful protein - Google Patents

Abstract

PURPOSE:To enable the mass-production of the objective useful protein utilizing a genetic engineering, by using an animal cell containing a vector integrated with a DNA coding the useful protein and proliferating and keeping the cell on a microcarrier. CONSTITUTION:An animal cell containing a vector integrated with a DNA coding a useful protein such as human interferon beta, human interferon gamma, human interleukin 2, etc., which can be produced by genetic engineering, is proliferated and maintained on a microcarrier. The microcarrier is generally a minute particle of a polysaccaride having positively charged chemical residue, or a minute particle of crosslinked dextran coated with collagen, etc. The necessary amount of bovine fetus serum to be added to the medium for maintaining the animal cell on the microcarrier is usually smaller than that for the proliferation medium.

Description

Detailed Description of the Invention (Industrial Application Field) The present invention provides a method for producing useful proteins by culturing and maintaining animal cells containing DNA encoding the useful proteins on microcarriers. Regarding.

(Prior art) In recent years, with the application of so-called genetic engineering technology, DNA corresponding to various useful proteins is introduced into prokaryotic cells such as E. coli or eukaryotic cells such as animal cells, and the information is expressed. It has become possible to produce this useful protein.

When comparing the protein productivity of systems using prokaryotic cells and systems using eukaryotic cells, it is currently generally believed that prokaryotic cell systems using E. coli are more advantageous. However, post-translat proteins such as glycoproteins
Some substances that require ionic modification cannot be produced in prokaryotic systems because they lack the mechanism. For these reasons, genetically engineered protein production threads that use animal cells as hosts are attracting attention. That is, by introducing DNA encoding the target protein into appropriate animal cells by some method, stably retaining it in the cells, amplifying the DNA as necessary, and expressing the DNA, the target protein can be obtained. Methods are being considered to produce proteins that The present invention relates to a method for producing useful proteins, which is characterized by culturing and proliferating animal cells into which a foreign gene has been introduced and amplified in this manner, and producing the protein encoded by the foreign gene. be.

There are many attempts to introduce foreign genes into animal cells and express them. (For example, proteins, nucleic acids, enzymes, special issue 19
83.12 vol 28 No 14, "Expression of recombinant genes into cells"). For example, mouse-derived C1271 cells are used as host cells, and bovine papillomavirus (
A method for sustainably producing human interferon α and γ using BPV) as a lid, (R, Fukuna
ga = tal, Proc, NAS81.5086
(1984) ), or 2. Method for sustainably producing human interferon T using Chinese hamster-derived CHO cells as a host and adenovirus as a vector (J, 1 Iaynes, C, Weissmann,
Nucl, 8cid, l1es, IL 687 (198
3)), etc.

However, since all of the animal cells listed as examples above are adhesion-dependent cells, suspension culture cannot be used when culturing genetically engineered animal cells. Therefore, in order to proliferate such adhesion-dependent cells, monolayer culture is generally performed. The monolayer culture method, which is still dominated by roller culture, has disadvantages such as being labor-intensive and susceptible to staining with bacteria and mold, and is often limited to a scale for experimental research. As mentioned above, to date, no technology has been established for culturing large amounts of genetically engineered animal cells.

(Problems to be Solved by the Invention) The purpose of the present invention is to cultivate a large amount of genetically engineered cultured animal cells, maintain them for a long period of time, and produce a large amount of desired useful proteins. do.

(Means for solving problems) The above object is achieved by the present invention as described below.

That is, the present invention is a method for producing a useful protein, which is characterized by growing and maintaining animal cells containing a vector incorporating DNA encoding the useful protein on a microcarrier.

The useful protein of the present invention is not particularly limited as long as it can be produced by genetic manipulation, and examples thereof include human interferon β, human interferon T, and human interleukin 2.

A vector incorporating a DNA encoding a useful protein has the property of being able to be amplified in animal cells, and preferably contains 1) a replication initiation site necessary for replication in E. coli, such as the replication origin of pBR322; Point, bovine papillomavirus type I (BPV-1) DNA Pvu II
A vector containing a truncated long-chain fragment, an Sv4° promoter (early or late), a DNA encoding a useful protein, and a polyrA addition signal, or
2) Contains the replication origin necessary for replication in E. coli, the SV4° early promoter, DNA encoding dihydrofolate reductase (DHFR) linked downstream thereof, DNA encoding a useful protein, and polyrΔ addition signal Vectors etc. are used. Animal cells have properties that allow the proliferation of vectors incorporating DNA encoding useful proteins, and are preferably mouse Cl
27-1 cells, Cll0 dhfr- (dihydrofolate reductase-deficient Chinese hamster Obari cells), etc. are used.

Usually, mouse C127-1 cells are used as the vector in 1), and C)10 dhfr- cells are used as the vector in 2).

The most common microcarriers are polysaccharide microparticles with positively charged chemical residues (e.g., diethylaminoethylated crosslinked dextran microparticles, dimethylaminopropylated polyacrylamide microparticles, etc.) or collagen-coated crosslinked dextran microparticles. Synthetic polymer microparticles (e.g. polystyrene microparticles)
, inorganic microparticles (for example, glass microparticles), or microparticles or microbodies made of other materials that have an affinity for cells.

A normal medium can be used for growing and maintaining animal cells containing a vector incorporating DNA encoding a useful protein on microcarriers, but Cl2
For 7-1 cells, fetal bovine serum (Fe
2) '2-10% added Darhenokou modified Eagle MEM
Medium, fetal bovine blood as maintenance medium? #0.05~2%
In the case of CtlOdhfr cells, MEM alpha medium (no nucleosides) supplemented with 2-10% fetal calf serum is used as the growth medium, and 0.1% fetal bovine serum is used as the maintenance medium.
MEM alpha medium supplemented with ~2% (no nucleosides added)
It is preferable to use

Here, the maintenance medium is a medium for maintaining proliferated cells, and usually requires only a small amount of fetal bovine serum added compared to the growth medium.

When producing useful proteins such as interferon and interleukin 2, this maintenance medium can be used as a medium for producing useful proteins.

〔Effect of the invention〕

The present invention involves culturing and maintaining animal cells on microcarriers containing a vector incorporating DNA encoding a useful protein and having the property of adhering to microcarriers, and culturing and maintaining the DNA on microcarriers.
The present invention relates to a method for producing the useful protein by expressing the protein. By the method of the present invention, it has become possible to mass culture animal cells of tube-grown eggplants that produce useful proteins, which was difficult in the past, and as a result, it has become possible to secrete and produce large amounts of useful proteins, which is also effective when considering commercial production. It is true. Furthermore, some examples of the present invention can be cultured for an unusually long period of time for conventional animal cell culture production systems, and moreover, produce useful proteins at high concentrations throughout almost the entire culture period. Continuing on, it has also been found that this method saves time and effort such as passage of cells, and that the amount of serum added, which generally accounts for a high cost in the composition of an animal cell culture solution, is kept to an extremely low level.

Below, mouse-derived C1271 cells are used as hosts, cells that express human interferon γ using BPV as a vector, Chinese hamster-derived CHO cells as hosts, and human interleukin 2 using adenovirus as a vector.
The present invention will be specifically explained with examples using cells expressing the following.

Needless to say, the present invention is not limited to the embodiments described below.

Example 1 (1) BPVT C127 :pSV
C127-■ cells (D, R, Lowy et al, J, Virol, 2
6,191.1978) clone No. 64-1 (Japanese Patent Application No. 170351/1982) was obtained. Hereinafter, these cloned cells will be referred to as BPVγ/C127 cells.

(2) Cultivation of seed cells: 7 x 10' BPVr/C127 cells were incubated with fetal bovine serum (
Dulbetsko-modified Eagle MEM medium (D-MEM) (manufactured by Hinaga Pharmaceutical Co., Ltd.) supplemented with 10% Fe2) and 1 g/l of glucose and QQed at 10 mA in a plastic flask (manufactured by Corning Co., Ltd., 25100. Cell adhesion area 25 cm). , and culture at 37°C. Confluency usually occurs in 3 to 4 days
Form an ent monolayer (6 to 8 x 106 cells/flask). 0.25% trypsin (Di
A cell suspension is prepared according to a conventional method using FCO (1:250).

BPVr/Cl27 cells were cultured in a plastic flask in the same manner as the seed cell culture, and the cells were cultured until confluent mon.
form an olayer. The culture supernatant of this monolayer is replaced with maintenance medium every 2 to 3 days. The cumulative titer of interferon thus obtained is shown in Table 1A.

Interferolysis-production by microcarrier culture: Seed cells cultured in plastic flasks were
- [[1 cell adhesion area approximately 150 cal)]. co
nfluent monolayer (3-4X I
After forming a cell suspension (O'/Roux bottle), trypsinize to prepare a cell suspension. Microcarrier “cytodex-1”
"(manufactured by Pharmacia) 0.6g FC55% and I
g//! Glucose added D-MEMl at 90 mA',
L4. Suspend the above cell suspension (2°4×10′ cells/
The cells were cultured with stirring in a 500rnff'gl spinner bottle with 10 mn FC (D-MEM supplemented with 35%). Every 2-3 days, 90% of the culture medium is replaced with fresh culture medium (FC3).
5% and glucose Ig/7! Add D-MEM). c on microcarriers on the 6th day after the start of normal culture.
Form an onfluent monolayer.

After this point, 90% of the medium is replaced with maintenance medium every 2 to 3 days to continue the culture. The cumulative titer of interferon thus obtained is shown in Table 1B.

Further, the progress of microcarrier culture and the cumulative interferon titer are shown in FIG.

A is growth medium (Darhenokow modified Eagle MEM medium (D-MEM) supplemented with FC35% and glucose 1g/!
Shows the number of cells when cultured (until 6th day after implantation).

△: Fe12.2% and glucose I as maintenance medium
The number of cells (from day 6 onwards) is shown when using D-MEM supplemented with g/β.

The system contained 10% Fe2 and glucose Ig as maintenance medium.
The number of cells (from day 6 onwards) is shown when D-MEM/Il added is used.

○ indicates Fe12.2% and glucose I as maintenance medium.
The cumulative interferon titer when using g/A-added D-MEM is shown.

・FC3IO% and glucose Ig as maintenance medium
The cumulative interferon titer when using /ff-added D-MEM is shown.

Interferon titration was performed by S. Rubinstei.
CP using FL cells and 5indbis Virus according to the method of N et al. (J. Virol F. 55. 1981).
This was carried out using the E50 method. The standard interferon is human interferon r8,3 derived from recombinant Escherichia coli that has been combined with the NIH standard human interferon T.
00 units/m/! was used. (Special application 1984-17Q35
1) Example 2 (1) CHO-interleukin 2 clone 1
Construction of 19: CHO-interleukin 2 clone 1-1 was constructed according to the following sequence. The basic genetic manipulation method is "Molecular cloning"
” (Maniatis et al. (1982) Col.
spring harbor 1 laboratory)
It depended on

i) Vector plL2 for cloning the human interleukin 2 gene of vector IL 2-28.3
-28.3 was produced as follows.

Lymphocytes were isolated from human tonsils, 2% fetal bovine serum, 1
0mM Hepes (N-2hydroxyet
hy I piperazine-N'-2-et
hanesulfonic acid) (p117.2
), 5X10-'M2-mercaptoethanol, 100
5X10 cells/m I in RPMl 164.0 medium containing beta penicillin, 100 μg/ml streptomycin! It was suspended so that Next, P HA (
phytohemagglutinin) (Difco
Laboratories) at 1%, TPA (12
-0-tetra decaoyl phorbor
-13-acetate) (Sigma) for 5 n
g/m It was added (Y, to, Yip
Infection and Immunity, 3
4.131 (1981), Hirano et al. Microb
Iology and Immunol, 27.87
(1983) ), this method allows IL2 and IFN-
Production of physiologically active substances such as r is induced. Cells were collected 16 to 24 hours after addition of PHA and TPA, and mRNA was prepared.

Preparation of mRNA, construction of cDNA, and cloning into plasmids were performed using known methods (Okayama et al.
r and Ce1lular Biology 3+
280 (1983)). Obtained cDNA
Known interleukin 2 cDNA from library
A (Noguchi et al. Na Lure, 302.305 (
1983) ) Complementary base sequence GAT GCT
TTG ACA AAA CGru by colony hybridization using GGT as a probe
nstern &:) Proc, Natl, 8cad.

Sci, 72.396H1975)) positive strain was selected and plasmid pIL2-28.3 was obtained. (Figure 2)
300 μg of pIL2-28.3 was digested with Bam III and subjected to agarose gel electrophoresis. A band of short chain fragments containing the human 1-interleukin 2 gene was excised, and the DNA fragments were recovered and purified from the gel by electroelution. This was further digested with Bgl I, and a long chain fragment containing the human interleukin-2 synthetic gene was separated and purified by agarose gel electrophoresis. Next, use T and DNA polymerase to make the ends of this DNA blunt ends, and add T to this end.
A BglII linker was added using 4 DNA ligase.

The fragment was further digested with B, l II and Rsa I, and a fragment containing the interleukin 2 structural gene was separated and purified by agarose gel electrophoresis. This is called fragment a.

On the other hand, pSV2 rabbitβglobin (R,C
, Mulligan et al., 5science 209.14
22 (1980)) with old ndIH1Bgl■, the longer of the two DNA fragments was 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' AGCTTTGCA88GATGT3 -
--・----d318ACGTTTCTACA5'
----------8 Next, 0.1 μg of fragment a, 0.1
．． ljg fragment b, 0°18μg oligomer d, 0.
09 μg of oligomer 〇 was mixed and reacted with T4 DNA ligase at 15°C for 18 hours to connect them, and to make E. coli MC1061 (M, J, Ca5a
daban et al. J, Mo1. Biol, +138, 1
7.9 (1980) was transformed.

Plasmid DNA was extracted from a strain showing resistance to ampicillin 100 μg/ml, and the base sequence around the ll1ndI[I cleavage site was determined using the Maxam G11bert method (Met
hEnzym, 65, 499 (1980)), and the desired vector 9-psVhlL2 was obtained. (Figure 3)
Creation of SV40 replication origin, E. coli plasmid pBR322
origin of replication and the tetracycline resistance gene, and DHF linked to the adenovirus major late promoter.
Known plasmid pAdD26SVA consisting of R gene
#3 (R, J, Kaufman et al.
cufar and Ce1lular Biolog
y 2, 1304 (1982)) was digested with EcoRI and treated with DNA polymerase to make the EcoRI cleavage site blunt-ended. 0.6 μg of this chain DNA ps
This was mixed with 0.6 μg of the Acc H-digested longest chain fragment of vh IL2 and ligated by reacting with T, DNA ligase at 15° C. for 18 hours. This reaction mixture 2
E. coli MCl061 was transformed using 0μ2, plasmid DNA was prepared from the obtained colonies exhibiting tetracycline resistance, and a restriction enzyme cleavage map was created to obtain the target plasmid pAdDh IL2. Since there are two orientations of interleukin 2 contained in this plasmid, use pAdD in which the direction in which the interleukin 2 gene is read is the same as the direction in which the DHFR gene is read.
hIL2T, and a different one was named pAdDhlL2C.

(Figure 4) iv) Transformation of dhfr-cells by hIL2 0 Human interleukin 2 expression vector pAdOhI
4 μg of L2C was converted to approximately 1×106 using the calcium phosphate method.
dhfr-Cll0 cells DXBII strain (G, LIr
laub et al. Proc, Natl, 8cad, Sci
US^-Jヱ, 4216 (1980)), and 2 days later, they were cultured in a 10 cm diameter dish with a selective medium consisting of nucleic acid-free alpha MEM (GIBCO) containing 10% dialyzed beef tallow serum (GIBCO). After re-seeding into 5 sheets and culturing for 10 days, about 1000 dhfr were grown.
” transformants were obtained. Of these, 12 clones were separated into a 24-well plate and confluen- ced.
The cells were cultured until reaching nt, and the interleukin (IL) 2 activity of the culture solution was measured (Table 2).

Table 2 CHOrL2-1 16 = 48 = 108 Cloned cell C from iv) above
110112-1 was grown on selective medium, and the diameter was 10 cm.
Approximately 2X10' cells per dish of 0.02 μM
The cells were cultured in a selective medium containing methotrexate.

After culturing for 14 days, 17 clones were separated into 24-well plates and cultured in a selective medium containing 0.02 μM methotrexate. The interleukin 2 activity of the culture supernatant was measured for five strains with fast growth rates. (Table 3) Table 3 cHOIL21* 50 As shown in Table 3, the clones selected in the medium containing bemethotrexate had 3 to 10 times higher interleukin 2 than CHOIL2-1 cultured in the medium without methotrexate. It showed activity.

Approximately 2×10′ CHO-IL2 produced by the above method
1-1 cells were placed in a Vetri dish (Cor
Nucleic acid component-free Alpha MEM (Alpha MEM) containing 0.05 μM methotrexate (MTX) and 10% dialyzed FC3 (GIBCO)
EM, GI BCO).

After culturing for 14 days, 10 clones were placed in a 24-well plate (
Corning, 25820). This was cultured in the above-mentioned medium, and clones with a fast growth rate were selected.

Among these clones, CHOIL2.1-1-9 (
The following experiment was conducted using the CHOIL2 119 (hereinafter referred to as CHOIL2 119).

(2) Cultivation of seed cells Clone 119 cells 1.5XIO6 cells at 0.05 μM
Alpha MEM medium 1 containing TX and dialyzed FC310%
Suspended in 0ml and 25CII! Plastic flask (C
orning 25100) at 37°C.

Confluent monolayer usually takes 3 to 4 days
r (6-7 cells/flask). 0
．． A cell suspension is prepared using 25% trypsin according to a conventional method.

(3) Interleukin 2 production by microcarrier culture Seed cells cultured in two plastic flasks are passaged into Louvre bottles. Confluent monolayer (
After forming 3XIO'/Ruby bottle, trypsinize to prepare a cell suspension. Microcarrier Cytode
x-3 (manufactured by Pharmacia) 0.8g to 0.05μM
Alpha MEM medium 1 containing MTX and 35% dialyzed FC
90 mjl! The above cell suspension (2,4X 1
0' cells/l Om j20.05MMTX, dialysis F
500 m with C35% supplemented alpha MEM medium)
Culture with stirring in a 1-volume spinner bottle. Every 2-3 days, 90% of the culture was replaced with fresh culture medium (0.05 μM MTX,
Replace with alpha MEM medium supplemented with 35% dialyzed FC.

Usually 6 to 7 days after the start of culture, C0nfl is placed on the microcarrier.
form a client monoloyer. After this point, 90% of the medium was replaced every 2 to 3 days with a medium for interleukin 2 production (alpha MEM medium supplemented with 0.05 μM MTX and 30° 2% dialysis FC), and the culture was continued. The cumulative titer of interleukin-2 obtained in this way was
Shown in the figure.

Interleukin-2 titer measurements were performed on CTLL-2 cells (S
, G1111g et al. J, Immnmol, 120
．． 2027 (1978)), T. Mosman
Method U of n, Immunological Meth
ods, 65.55 (1983)). The standard interleukin 2 is interleukin 2 prepared from human peripheral blood lymphocytes (5000 international units/
ml, whose titer had been adjusted to the international standard interleugin 2) was used.

[Brief explanation of the drawing]

FIG. 1 shows the progress of microcarrier culture of BPVr/C127 cells and the state of interferon γ production. Figure 2 shows the configuration of Hector p I L 2-28.3.
FIG. 3 shows the configuration of Hector psvhl L2. Figure 4 shows Hector pAdDhlL2c and pAdDh.
l The method for producing L2T is shown. FIG. 5 shows the progress of microcarrier culture of CHO-IL2clone19 cells and the state of interleukin 2 production.

Claims (1)

[Claims] (1) A method for producing a useful protein, which comprises growing and maintaining animal cells containing a vector incorporating a DNA encoding a useful protein on a microcarrier.