JPH04179485A - New vector and method for selecting highly expressing transformed cell using the same vector - Google Patents

Abstract

NEW MATERIAL:An exogenote expressing vector having a neomycin phosphotransferase gene with variation linked to the downstream side of a promoter having weak activity as a selective marker. USE:For screening in which transformed cells capable of highly expressing an optional exogenote are efficiently selected. PREPARATION:For example, a plasmid pMC1neo containing G418-tolerant gene (neomycin phosphotransferase II gene) normally used as a selective marker is digested with restriction enzymes EcoRI and BamHI to separate a DNA fragment of about 1 kb by agarose electrophoresis. An expression vector pAGS-3 is separately digested with a restriction enzyme Nde1 and dephosphorylated. The aforementioned separated DNA fragment is then liked to the dephosphorylated expression vector pAGS-3 and cyclized to afford the objective expression vector pAGneo.

Description

DETAILED DESCRIPTION OF THE INVENTION (Field of Industrial Application) The present invention relates to a novel expression vector and a method for efficiently selecting transformed cells highly expressing any foreign gene using the vector.

(Prior Art and Problems to be Solved by the Invention) With the advancement of genetic recombination technology, the production of useful substances using genetic recombination has been rapidly progressing in recent years. When expressing a foreign gene using genetic recombination technology, an appropriate host cell and an expression vector having an appropriate promoter for foreign gene expression are used.

Until now, easy-to-handle microorganisms such as E. coli and yeast have been widely studied as expression host cells, but it has been confirmed that these microorganisms have some limitations in expressing foreign genes, and in recent years In recent years, expression systems using cultured cells of higher animals as expression host cells have been actively studied. Until now, consideration has been given to using expression vectors with strong promoters as a method of highly expressing foreign genes using animal cells as hosts, and to increasing as much as possible the number of copies of the expression vector DNA that is introduced into the cells and maintained. I came. As an example of the latter, Chinese Muster ovary cells lacking the dihydrofolate reductase gene (dhfr) were treated with dhfr as a selectable marker.
Introduce an expression vector containing the gene.

Selection with progressively higher concentrations of mentrexate increased the copy number of the 2 expression vector DNA. There is a method to obtain transformed cells that highly express foreign genes. However, this method has problems in that it can only be effectively used with dhfr-deficient cells and that selection and cloning require a long period of time. Another method is to use an expression vector into which a self-replicating sequence derived from bovine papillomavirus has been introduced. In this method, the vector introduced into the cell is an episome,! : However, in reality, it is often integrated into the chromosome, and it is necessary to frequently clone transformed cell lines with high expression.

In order to commercialize the production of useful substances using genetic recombination technology, it is strongly desired to develop a technology that efficiently selects only transformed cells that highly express foreign genes from various cell lines. There is.

(Means for Solving the Invention) Under these circumstances, the present inventors used the G418 resistance gene (neomycin phosphotransferase gene), which has been commonly used as a selection marker, to inject 0418 at a high concentration. By selecting
We have devised a method for effectively selecting transformed cells into which an expression vector has been integrated in a high copy number, and have confirmed that the method is actually applicable, leading to the completion of the present invention. This method uses 0, which has been modified to show very weak activity.
The 418 resistance gene is inserted into an expression vector to create transformed cells.

Furthermore, by selecting at a high concentration of 6418, only cells that have integrated multiple copies of the expression vector are effectively left behind. + pSV2− as a selection marker
neo is known (JoMol, Appl.

Genet, 1.327 341.1982),
This confers very strong G418 resistance to the transformed cells, making it suitable for tree purposes. It has been reported that the neomycin phosphotransferase ■ gene produces an enzyme with very low activity due to mutations (Proc, Na
tl, Acad, Set, USA,, 87; 3
435-3439+1990). pMClneo has been reported as a plasmid that incorporates a gene with this mutation (Ce11).

51, 503-512.1987). In this plasmid, neomycin phosphotransferase■
The gene is integrated downstream of the herpes simplex virus thymidine kinase promoter linked to the polyoma-derived enhancer, and the poIJA signal derived from the SV 40 virus is linked to its 3' side. In the present invention, it is necessary that the promoter activity be weak, so it is used as a Neo cassette to be incorporated into a polyoma-derived lid (Fig. 1). Note that the promoter used for the Neo cassette may be any other promoter as long as it has weak activity. An example is the SV40 viral early gene promoter with the enhancer removed. Other genes may also be used as long as they have a mutation in the neomycin phosphotransferase gene that makes the activity weaker than that of the wild-type gene.

The basic structure of the foreign gene expression vector of the present invention is a strong expression vector that has already been reported, such as pAGS-3 (Gene, 79.269-277, 1
989) with the above-mentioned Neo cassette installed. The pAGneo 3-IL 2 plasmid shown in Example 1 further incorporates IL-2 cDNA (Fig. 2).

Furthermore, such expression vectors of the present invention.

Contains a gene derived from an E. coli plasmid to facilitate cloning in E. coli. As such a gene derived from an E. coli plasmid.

ori for replication in E. coli cells, and suitable genes that can serve as selection markers during cloning, such as drug resistance genes for ampicillin, tetracycline, and the like. In addition, a gene derived from plasmid pBR322 is often used as such a gene, but in this case, the pBR322 replication origin (or
i) is near. Toxic examples that inhibit replication in host cells (Nature +293, 79-81.1981
) is desirable to remove. Used in the examples described below.
The developed expression vector has this sequence removed.

Furthermore, by incorporating a self-replicating sequence derived from bovine papillomavirus into the above expression vector, a high concentration of 04
This makes it possible to more efficiently obtain highly expressing transformed cells after 18 selections.

An example of a preferred expression vector of the present invention comprised of the NA sequence as described above is pABneo3. This is pABne shown in Examples.

3- From the IL2 plasmid (Figure 3), IL-2c
It corresponds to something without DNA.

Using such an expression vector, a vector is constructed in which a foreign gene is introduced downstream of its promoter for foreign gene expression. The host cell into which the expression vector incorporating the foreign gene to be expressed may be introduced may be any host cell as long as the promoter for foreign gene expression functions well. Examples include mouse L cells, Chinese star ovary cells, human He1a cells, and the like.

Expression vectors can be introduced into host cells using known methods.

For example, the calcium phosphate method (Proc, Natl,
Acad, Sci.

USA, 76, 1373 1376, 1979),
DEAE-Dextran method (DNA clonin method)
g vol, n, IRL press, 0xfor
d, 143-190.1985), Electroporation Method (Proc.

Natl, Acad, Sci, USA, 81.7
161-7165.1984).

Lipofection method (Proc, Natl, Acad
, Sci., USA.

84, 7413-7417.1987). After that, C418 was added to 20011g/m
7 (titer) to obtain G418-resistant transformed cells. These cells were treated with high concentrations of 0418
The culture is continued for about 1 month in a medium containing (800 μg/mZ). During this process, only transformed cells into which multiple copies of the expression vector have been stably introduced remain. This was integrated into the Ne9 expression vector.

This was thought to be because the activity of the cassette was extremely weak, so that only transformed cells with multiple copies of vector DNA could proliferate under high concentrations of 0418. In fact, G418 is 2
80 μg/rnl compared to when the culture was continued at 00 μg/rnl.
When culture was continued at 0 μg/ml, the number of copies of the expression vector in the transformed cells increased by more than 5 times, reaching 200 to 200.
It has been confirmed by the Southern Pronoto method that the number of copies reaches 300 copies. Furthermore, this analysis confirmed that many of the introduced expression vectors were integrated into the chromosome in a row in the same direction. There was no significant change in the NA structure of the introduced expression vector. For vectors with autonomously replicating sequences derived from papillomaviruses, the expression vector introduced into cells is thought to be maintained in the nucleus of the cell as an episome;
Analysis using the Southern method showed that after 8 selections, multiple copies were finally integrated into the chromosome in a series.

In addition, N
When the same operation as described above is carried out using an expression vector having EO power, culture is performed under a high concentration of 0418. Selection of transformed cells that have integrated multiple copies of the expression vector is not permitted.

(Effects of the Invention) The novel foreign gene expression vector for animal cells having the Neo cassette of the present invention and the efficient selection method for highly expressing transformed cells using the same can be fully utilized in industrial-level production. It has become possible to easily obtain transformed cells using any cell line without cloning cells over a long period of time.In addition, the transformed cells obtained using the present invention can be stably obtained from foreign cells over a long period of time. Maintaining high gene expression.Since such an expression system was established for the first time by the present invention9, the technical benefits that the present invention brings to industry are evaluated to be extremely large.

(Example) Although transformed cells incorporating multiple copies of an expression vector can be efficiently obtained according to the present invention, the foreign gene is highly expressed in the actually obtained transformed cells. It has been confirmed that human IL-2 cDNA is present. The present invention will be described in detail using these as examples.

Plasmids, DNA, various enzymes, Escherichia coli,
For various operations involving cultured cells, etc., I referred to the following journals and publications.

(1) Genetic manipulation experiment method, Touka Takagi 1 editor (1980)
.

Kodansha (2) Gene manipulation manual, Toka Takagi 1 editor (198
2), Kodansha +3) MOLECULAR CLONING A L
ABORATORYMANUAL, TlMANIAT
Edited by IS et al. (1982), C0LDSPRING
HARBORLABORATORY.

(4) METHODS IN ENZYMOLOG
Y, Volume 65, L.

Edited by GRO8SMAN et al. (1980), ACADE
MICPRESSThe following abbreviations were used during implementation.

neo: neomycin phosphotransferase ■gene tk: monoM herpesvirus thymidine kinase gene B
PV: Bovine papillomavirus kb: 1000 base pairs CHO: Chinese hamster ovary cell Example 1: Construction of expression vector pABneo3-IL2 Plasmid p
Mc1neo (Cell, 51.503512゜1
987) with the restriction enzyme EcoRI (NEB#101),
After digestion with BamHI (NEB #136) and end repair with DNA polymerase Klenow fragment (NEB #210).

An approximately 1 kb Neo cassette DNA fragment consisting of the tk promoter, neo, and SV40 polyA signal was separated by agarose gel electrophoresis. GENEC this fragment
It was recovered from the gel and purified using LEAN (BIO101 Inc.). The structure of this Neo cassette is shown in FIG.

pAGS-3 (Gene, 79.26
9-277, 1989) with the restriction enzyme Ndel (NEB
#111), dephosphorylated by the action of alkaline phosphatase derived from calf small intestine, and the ends were repaired by DNA polymerase Klenow fragment.

By ligating and circularizing this using the aforementioned Neo-Kanachino) DNA fragment and 74 DNA ligase.

An expression vector pAGneo 3 was created. pZip provided by Professor Osaka Taniguchi, Cell Engineering Center, Osaka University
The SVIL2 plasmid was digested with the restriction enzyme BamHI (NEB
#136), subjected to agarose gel electrophoresis, and extracted from the gel approximately 0.5 kb of DNA containing human IL-2 cDNA.
Got a piece. This DNA fragment is a) IL-2 cDNA (
Nature, 302.305-310.1983)
The 41st base pair to the 542nd base pair are included. this DNA
The fragments were treated with DNA polymerase Klenow fragment and attached to BitXI adapters (Invitr
(genN408-18) was ligated with T4 DNA ligase.

The pAGneo 3 plasmid described above was transformed into BstXI (N
After digestion with EB#113) and dephosphorylation with alkaline phosphatase derived from calf small intestine, the human IL-2c
The DNA fragment was ligated and circularized using T4DNAIJ gauze.

In this way, human IL-2 expression plasmid pAGn
eo3-IL2 was produced. This structure is shown in FIG.

Example 2: Production of pABneo3-IL2 pdBP
V-1 plasmid (Proc, Natl, Acad,
Sci.

USA, 79.7147-7151.1982) was digested with the restriction enzyme Hindm (NEB#]04), treated with DNA polymerase Klenow fragment, and BamHI linker (
NEB #1021) was combined.

This was digested with Bam old, and a so-called 69% fragment of BPV with a length of 5.4 kb was recovered by agarose gel electrophoresis.

pAGneo 3-IL 2 constructed in Example 1 was
Digested with amHI, treated with alkaline phosphatase derived from calf small intestine, and combined with the above-mentioned BPV 69% fragment and T.
4 DNA was ligated and circularized using nogase. In this way.

pABneo 3-I L2 was constructed. This structure is shown in FIG.

Example 3: Human) Production of IL-2 in Mouse L Cells I x 10' mouse L cells were removed into a Falcon 6cm dish, and the next day, 5 μg of IL-2 was expressed using ribofectin reagent (BRL) according to the attached protocol. Plasmid pAGneo3-IL2. pABneo3
- Transfected with IL2. After 2 days, titer 2
Culture was continued for 18 days in a medium containing 00 μg/anl of G418.

As a result, pAGneo 3-I L2. pAB
Approximately 200 colonies were observed in both neo3-I L2 transfected deinters. These colonies were detached from desauches using trypsin-EDTA solution, divided into four 25 cm flasks, and each flask was incubated for 1 month in a medium containing 0418 at 200°400 and 600.8004 g/mZ (titer). Subculture was continued. The L cells maintained at each concentration were removed into a 6-well plate for Falcon cell culture at a rate of 1XIO3 cells/well, and G41
The cells were cultured in a medium containing no 8. After 48 hours, the culture supernatant was collected.

Human IL-2 measurement kit Quantikin@(R&D
Systems.

#D2000) was used to measure IL-2 activity. As a result, as shown in FIG. 4, when cells transformed with pABneo3-IL2 were selected at 600.800 μg/mt, the number of cells transformed with pABneo3-IL2 was 6 times higher than when selected at 400 μg/ml.

showed an 8-fold higher expression of IL-2. The effect of such high-concentration 0418 selection was clearly observed even when the BPV-derived sequence was not included, but the effect was small.

In addition, if a similar selection was made using a highly active (・Neo cassette) containing Enno-cer, a high concentration of G4
Almost no effect of 18 selections was observed.

Example 4: Production of human l-2 in Chinese Hamster Ovary (CHO) cells 2×10 5 CHO cells were plated in Falcon 6 cm Danish cells, followed by transfection and selection with G418 as in Example 3.

The activity of IL-2 produced in the same manner as in Example 3 was measured. CHO cells transformed with pABneo IL2 have a concentration of 800 μg/m! as in the case of L cells! G of
418 selection compared to the 200 μg/rnl selection.

Approximately 6 times more IL-2 production was obtained. This high production
Furthermore, there was no change at all after culturing in the presence of 800 μg/rnl G418 for 3 months. pAGneo3-
In the case of CHO cells transformed with IL2, 80
The transformed cells selected at 0 μg/ff1t were 200
pABneo 3-IL-2 showed higher expression of IL-2 than that selected at μg/ml;
It was less than in the case of

[Brief explanation of drawings]

FIG. 1 shows the structure of the Neo cassette constructed in Example 1. Due to the mutation, the 185th amino acid residue has been changed from Glu in the wild type to Asp. Figure 2 shows the expression vector pAGne constructed in Example 1.
The structure of o3-IL2 is shown. Figure 3 shows the expression vector pABne constructed in Example 2.
o 3-I Shows the structure of L2. Figure 4 shows pAGneo3-IL2 and pABn
After transfecting mouse L cells with eo3-IL2 and selecting transformed cells with 200 μg/m, 1 G418 for 18 days, further 200.400.600;
0/jg/m117) Human IL-2 activity secreted into the culture medium at 348 hours per 10' transformed cells obtained by single-sided selection in the presence of G418.
The results of rvi are shown. -4-, pABneo3-LL
2; -■-, pAGneo3-IL2 Figure 5 is
pAGneo3-IL2 and pABneo3-IL2
was transfected into Chinese hamster ovary (CHO) cells and treated with 200 μg/mt of 0418 for 20 min.
days. After selecting transformed cells, further 200.400.800
This figure shows the results of measuring IL-2 activity, which was distributed into the culture medium after 48 hours, per 106 transformed cells obtained by continuous selection for one month in the presence of μg/ml G418. −・−, pABneo3-IL2 knee■−, pAG
neo3-L2

Claims (5)

[Claims] (1) A vector for expressing a foreign gene that has a selection marker in which a mutated neomycin phosphotransferase gene is linked downstream of a weakly active promoter. (2) Claim (1) wherein the weakly active promoter is a thymidine kinase promoter derived from herpes simplex virus.
) A vector for expressing a foreign gene. (3) Claim (1) containing a self-replicating sequence that works in animal cells
) A vector for expressing a foreign gene. (4) The vector for foreign gene expression according to claim (3), wherein the self-replicating sequence is a gene sequence derived from papillomavirus. (5) A foreign gene is incorporated into the expression vector according to any one of claims (1), (2), (3), or (4), and cells transformed thereby are transformed at a concentration of 400 μg/ml.
A method for selecting transformed cells that highly express a foreign gene by selecting in a culture solution containing G418 at the above concentration.