JPH05503015A - mammalian expression vector - Google Patents

Abstract

(57) [Summary] This bulletin contains application data before electronic filing, so abstract data is not recorded.

Description

[Detailed description of the invention] Specification Much of the work in the field of recombinant DNA to date has focused on recombinant polypeptides. Bacterial hosts such as Escherichia coli (E. coli) to produce bacteria and proteins. We have focused on using cells. However, the use of bacterial cells has left many wanting It has certain aspects. For example, most proteins and molecules produced within E. coli Ripeptides accumulate in the cytoplasm or periplasmic space. The times of these gene products extraction requires cell disruption and is often accompanied by detergents, or urea or salts. Requires extraction with chaotropic agents such as acid guanidine. Therefore, times The recovered proteins often have abnormally folded structures, and there are numerous Difficult to purify as it must be isolated from other E. coli cell components . Additionally, it is possible to carry out the glycosylation necessary to complete the synthesis of many interesting gene products. and the correct conformation and biological activity of many eukaryotic proteins. The requisite, specific disulfide bonds often fail to form.

To overcome these shortcomings of bacterial expression systems, genetic engineering interests have turned to eukaryotic host cells. There is a rapid shift towards usage. Cells such as yeast and mammalian cells contain the desired gene. The product can be secreted into the culture medium, as is the essential glycosylation process. can be done. However, recombinant DNA cloning and expression The use of animal cells also presents many technical obstacles to overcome. for example , endogenous plasmids that have been shown to be extremely useful in bacteria are usually Not replicated in eukaryotic cells. As a result, another approach must be taken.

One method is to use E. coli, which can be cultivated and manipulated as easily as E. coli. The yeast, Saccharomyces cerevisiae, is a lower eukaryote. ces cerevisiae). Yeast cloning system is available possible, and such a system could be used to develop human interfaces in yeast. Efficient expression of the feron gene 17 (1981)]. However, yeast cells at least one heterologous mammalian gene, including the rabbit β-globin Another method that does not properly transcribe the bin gene is that the foreign gene is has been inserted into mammalian cells. For example, this is a cloned gene This was carried out by a calcium phosphate co-precipitation method with 2% of cells can be induced to take up DNA. However, such a low At this level, only a small amount of the desired gene product can be expressed. Chimidinki If mammalian cells lacking the enzyme gene (tk-cells) can be found, Co-transformation gives better results. Select HAT TK- cells that cannot grow in poxanthine-aminotherine-thymidine (poxanthine-aminotherine-thymidine) medium are Containing the tk gene (herpes simplex virus DN By incorporating foreign DNA (such as A), this defective enzyme activity can be restored again. Obtainable. tk DNA covalently linked to other DNA, or is simply that other DNA mixed with tk DNA is also taken up by the cell and will often be co-expressed [Scangos et al., Gene 14: 1 (198 1)].

Many researchers have developed expression vectors for use in eukaryotic cells. for example , Okayama et al. [Mo1. Ce11. Biol, 3:280 (1983) , opened the “pcD” vector system containing the simian virus 40 (SV40) regulatory region. uttered. Kaufman (U.S. Pat. No. 4,740,461) described S A eukaryotic vector containing a V40 region and a selectable marker is described. these The vector is said to be useful for the production of tPA in CHO cells, for example. ing. Cho et al. [DNA 5:529 (1986)] Developed two recombinant vectors for direct expression and amplification of cDNA in did.

Fahr et al. [DNA 4, 461 (1985)] developed pDsPl, a “modular” vector containing a transcription cassette for .

A variety of genetic engineering techniques are used to construct such recombinant vectors. general The method described by Cohen et al. (U.S. Pat. No. 4,237,224) and Collins et al. National Patent No. 4.304.863) and Maniais et al. Laboratory Manual, 1982, Cold Spring Harbor Laboratory). Panayotatos [Gene] 31:291 (1984)] following cleavage of palindromic restriction enzyme sites. In order to create new sites during ligation, restriction enzyme sites can be modified. Complete smoothing of the exit part is disclosed. Coach [Nuc, Ac1ds fragment Manipulating restriction enzyme sites, including partially filling in the ends to allow ligation It describes how to do this.

Summary of the invention The present invention involves cloning heterologous genes in E. coli and transferring those genes to various mammalian species. A recombinant plant that can be cloned and expressed in animal cells. Provide sumid. These plasmids contain strategically placed genetic elements. They are characterized by a single restriction enzyme site and a single restriction enzyme site. facilitates extraction and/or substitution and restoration of the original restriction enzyme site.

The recombinant plasmid of the present invention has in the direction of transcription: (a) Xba I and Bg±I The β-lactamase gene and pBR322 replication vector bounded by I restriction enzyme site. Starting point, (b) 5V-4 bounded by Dankotan dlII and xhdanI restriction enzyme sites. 0 or SRα promoter, (C) one or more bounded by Pstr and EcoRI restriction enzyme sites; a polylinker containing a single restriction enzyme site above, and (d) EcoRI and Xb a polyadenylation signal sequence bounded by an aI restriction enzyme site; It consists of a DNA sequence containing.

The polyadenylation signal sequence is human β-globin or 5V-40 early or a late region polyadenylation signal sequence.

Some of the recombinant plasmids of the present invention are transcriptionally oriented in mouse mammary tumor tumors. rus long terminal repeat (MMTv-LTR) promoter and DHFR cDNA1 A dihydrohydride consisting of a splined nognal sequence and a polyadenylation signal sequence. It further includes a lofolate reductase (DHFR) transcription unit. splicing signal arrangement The column preferably contains a small antigen intron, and also contains a polyadenylation signal sequence. Preferably, the sequence is a 5V-40 early region polyadenylation signal sequence.

When in a plasmid, the DHFRHF index is linked to the β-lactamase gene and pB DNA sequence containing the R322 origin of replication and one 5V-40 or SRα promoter In another embodiment of the invention, the recombinant plasmid is β-galactonase (β-gal) or chloramphenicol acetate It consists of a DNA sequence containing transferase (CAT). If it exists, this These sequences are also bounded by Bg±II and Dan-Thu-Dan dIII restriction enzyme sites.

In yet another embodiment, the splice song signal sequence is a 5V-40 or SRα protein. exists between the DNA sequence containing the promoter and the DNA sequence containing the polylinker. , bounded by the xhdanI and PstI restriction enzyme sites, according to the direction of transcription.

This splicing signal sequence is preferably a 16S/19S intron.

Brief description of the drawing The present invention can be more easily understood by reference to the accompanying drawings, which illustrate and: Figure 1 shows the recombinant plasmid pD showing the single restriction enzyme site and elements. It is a schematic diagram of SVS.

Panels A-H of FIG. 2 depict plasmid pDSVS from various starting plasmids. It is a schematic diagram showing the construction.

FIG. 3 is a schematic diagram of plasmid pDSR5. This plasmid is pDSVS except that the 5V-40 promoter of is replaced with the SRα promoter. , similar to pDSVS.

FIG. 4 is a schematic diagram of pSR5. This plasmid is pSR3 with DHFRHF Similar to pDSRS except that the index position has been removed.

Panels A and B of Figure 5 are schematic illustrations of the construction of plasmids pDSRG and pSRG. Ru. These plasmids include plasmids pDSRG and pSRG after 5V-40. polyadenylation signal sequence of human β-globin instead of the phase domain polyadenylation signal sequence. They are similar to pDSRS and pSR3, respectively, except that they contain a conjugation signal sequence. Similar.

Panels A-C of Figure 6 show recombinant human interleukins in mammalian cells. Schematic diagram of the construction of plasmid pGSRG-hIL5, which enables the production of Ru.

Description of the invention All references cited herein are incorporated herein by reference in their entirety. be absorbed.

As used herein, the term "transcription" refers to transcription of transcription from a DNA template. refers to the synthesis of mRNA (mRNA). “Promoter” means RN A DNA sequence that binds polymerase and thereby "facilitates" transcription. Ru. “In the direction of transfer” means from upstream to downstream (i.e. from 5° to 3′) or means the direction in which RNA polymerase moves when transcribing DNA. be. The SRα promoter used in the present invention was developed by Takebe et al. [Mo1. Ce It is stated by I1゜.

An “enhancer” is a term that enhances the transcription of a certain gene, regardless of the nature of that gene. This is a possible DNA sequence. A promoter is located upstream of a gene to be expressed. However, the location of the enhancer is not critical. They are , upstream or downstream of a gene, and whether they are reversed with respect to another DNA sequence. It can function even when facing 3° to 5'.

“Splicing signal” refers to a gene that encodes a certain protein. It is a DNA sequence that can also enhance transcription. The “origin of replication” is the origin of replication in the host cell. This is the point on the plasmid at which replication of the plasmid begins.

A “polyadenylation signal sequence” is a sequence in which an adenine ribonucleotide is Translation of a gene added to form a polyadenylated end at the 3° end This is the DNA sequence that exists downstream of the region.

``Polylinker'' is a DN containing one or more single restriction enzyme sites. This is A array. The polylinker is the linker for the difference to be expressed in the plasmid of the invention. Facilitate the insertion of seed genes (i.e. genes not normally present in the host cell) . As used herein, the term heterologous “gene” refers to an isolated genetic Contains both offspring and cDNA prepared from mRNA.

A "restriction enzyme site" is a DNA sequence that defines the cutting point of a restriction enzyme. certain characteristics A site is “single” if there is only one such site on the plasmid. be. The plasmid of the present invention contains a single XbalSEcoRI, SmaI, Ba Contains mHI, and II, Dan1ku■, Kkudan■, and Dantudand111 sites. Good too.

In its most basic embodiment, the plasmid of the invention has in the direction of transcription: (a) β-lactamase gene and pBR bounded by restriction enzyme sites I and II. 322 origin of replication, (b) bordered by the Dantutan dllll and Xhol restriction enzyme sites. 5V-43 or SRα promoter, (C) one or more bounded by Danand1■ and Dandandan R1 restriction enzyme site; a polylinker containing a single restriction enzyme site above, and (d) Dandandan RI and Xb Polyadenylation signal sequence bounded by the aI restriction enzyme site Contains.

With the heterologous gene inserted into the polylinker, elements (b)-(d) are contains the transcription unit of a heterologous gene.

The β-lactamase gene encodes an enzyme that breaks down the antibiotic ampicillin. are doing. Because of this activity, the presence of this β-lactamase gene within the host cell is essential. The cells then become resistant to ampicillin in the culture medium. As a result, the present invention Host cells carrying the plasmid can be selected by growth in such a medium. , other cells lacking such resistance would not survive. In the attached drawing Therefore, the presence of the β-lactamase gene is determined by “AMP” or “AMP’”. Displayed.

A recombinant plasmid containing only the aforementioned elements is inserted into a polylinker. It can be used for the temporary expression of heterologous genes. Add to polylinker can be easily qualified using standard methods for matching them. can be done.

The polylinker does not need to contain all the restriction enzyme sites mentioned above, so it may not be necessary. Areas that are damaged can also be removed using standard methods. Conversely, if you need a single site, and cutting with the corresponding restriction enzyme will not cut the plasmid at that site. You can also add another cutting site unless you want to.

In another embodiment of the invention that can be used for transient expression, β-gal or CA The T transcription unit is a DN containing the β-lactamase gene and the origin of replication of pBR322. Inserted between the A sequence and the DNA sequence containing the 5V-40 or SRα promoter be done. Both the β-gal gene and the CAT gene are well known to those skilled in the art, :1044 (1982)]. present in plasmids If these genes are present in the transformant/transfectant ctant) provides useful marker activity for screening.

Although all of the aforementioned plasmids are useful for transient expression in mammalian cells, Long-term expression cannot be achieved with these plasmids. It is the host cell This is because there is no selective pressure to maintain this plasmid in the cell. Over time, they is lost. For longer term expression, the plasmid of the invention can be used with β-gal or Preferably, a DHFRHF site is included instead of a CAT transcription unit. DHFRHF The leading element is based on Lee et al. [Nature λ94: 228 (1981 ) is stated by Ko.

The presence of DHFRHF sites in the plasmid provides two advantages. Firstly, Insertion into host cells lacking hydrofolate reductase (e.g., into CHO-dhfr- cells) If entered, this unit provides selection means. Cells with plasmids are It can be easily selected from other cells in culture medium lacking xanthine and thymidine.

Second, cells containing high levels of dhfr cDNA are capable of de novo purine biosynthesis. Can survive in culture medium containing high levels of the inhibitor methotrexate; Cells expressing this cDNA at low levels will not survive. Therefore, this These conditions select for cells with increased or amplified copies of the dhfr gene. It would be possible. − When the heterologous gene to be expressed is present on the plasmid together with the dhfr gene , expression of the heterologous gene occurs as the host cell makes multiple copies of the dhfr gene. will be co-amplified. In the following examples, DH in the coexistence of methotrexate By culturing cells carrying a plasmid containing FRHF, dihydrofolate reductase and increased interleukin-5 (IL-5) production.

In the following examples, the 5V-40 late region [Okayama et al., Mo1. Cel 1. It was used downstream of the B car. These arrays are interchangeable and 5V-40 Instead of the initial region array (Lee et al., supra), it can also be used in the DHFRHF index. can be done. This 5V-40 early region sequence is also used downstream of the polylinker. You will be able to do it. The only requirement for such substitution is if polyadenylation If it is desired to later remove the signal sequence, such a The sequences used in place must be terminated by the restriction enzyme sites shown in Figure 1. This cannot happen. Since the nucleotide sequences of all three are known, For such uses, they can be chemically synthesized and/or applied using conventional methods. can be used.

The plasmid of the present invention can optionally contain an enhancer region. useful engine Hansa is 5V-40, polyoma virus, false papilloma virus, and rhinoceros. Is it an animal virus such as tomegalovirus, retrovirus or adenovirus? obtained from Ideally, the enhancer used is a virus that is tolerated by the host cell. (i.e., from a virus that normally infects cells of the host type). Ru.

Enhancer regions of numerous viruses are known [Lunyu et al., Ce1133 :705 (1983)]. Announcements have been made regarding the selected viruses. Excise these regions based on the restriction enzyme map and, if necessary, For example, the recombinant plasmid henin enhancer can be spliced into its Modifying the terminus is a conventional matter. Alternatively, the published sequence It is also possible to synthesize enhancers from data.

The only requirement for the selected enhancer is that the restriction enzyme site described above be unique. It is preferable that it does not contain any restriction enzyme sites that would cause damage. Those skilled in the art will understand the published This can be easily determined from the sequence data and known restriction enzyme site sequences. will be possible. Although many virus-derived enhancers can be used, the present invention 5V-40 (Takebe et al., supra) is preferred for use in. en in the plasmid The location of the hanger is not critical, but once the ±ndIII restriction enzyme site is inserted It is a convenient point (part).

blood clotting or fibrinolytic enzymes, or various lymphokines, or hormones. growth factors, oncogene products, and soluble or cell surface antigens and receptors. A wide range of variants including, but not limited to, genes encoding regulatory proteins. Seed genes can be used for insertion into plasmids. The plasmid of the present invention The first is to express the gene encoding the soluble gamma interferon receptor. It is used to In the following example, the gene encoding IL-5 is expressed. The use of different plasmids for expression has been described.

Such genes often contain pre-pro-polypeptides (e.g. secretory league- ), which contains the wild-type translated sequence encoding the mature protein structure, This may be desirable for stability and/or secretion. Such polypeptides It is removed during post-translational modification by the cell. Alternatively, these blemish blows The sequence can also be removed by known methods before inserting the gene into the plasmid. .

The gene can also contain 5' and/or 3' non-coding sequences.

The 5V-40 or SRα promoter used has an essential transcription initiation sequence. The gene or cDNA to be expressed is responsible for translation initiation and Must include a stop codon.

The preferred order of the elements in the plasmid is as described above, except for β-ga 1 or a protein for a heterologous gene such as the CAT transcription unit or the DHFRHF sequence. Any element inserted upstream of the promoter will It can be inserted downstream of a transcription unit. This is done by using the Xbal restriction enzyme site. It can be done easily.

The placement of the genetic elements and single restriction enzyme site in the plasmid is a separate plus. Substitution of elements or elements for use in mids or vectors promote the removal of This also replaces the original restriction enzyme site after the cleavage has taken place. Allows for easy recovery.

For example, the DHFRHF index is easily removed. Following BgllI/Klenow , by performing dI/exoVI I treatment, is played. Following Dan 1st I I I/Klenow, Dan gl I I/exoV II treatment with Leno polymerase and religation. The splicing signal of the 21■ fragment is removed by live.

The plasmids of the invention can be used in a number of mammalian host cells, including initial transplantation from a variety of tissues. Can be used in cells. However, established and/or transformed It is preferred to use cell lines that have been tested. Available cell lines include African Green Monkey kidney (CO5), Chinese hamster ovary (CHO), N5-1 ．． 5P210. NIH3T3, NIH3T6, C127, CV-1, He These include, but are not limited to, the La, murine L and Bowes cell lines. DHF If co-amplification using RHF rates is desired, CHO-dhfr-cells [U. Liaube et al., Proc, Natl, Acad, Sci, USA 4216 (1980), dihydrofolate reductase-deficient cells are preferred.

Plasmid DNA can be introduced into mammalian cells by many techniques. C.O. Temporal expression studies in mammalian cells, such as 8 cells, have shown that chloroquine boosts It can be carried out by the DEAE-dextran method using [Yokota et al., Pr.

c, Natl, Acad, Sci, USA Dan 2: 68 (1985).

Transfection of suspension cells (e.g. N5-1) is performed by electroporation. [Botter et al., Proc. Natl. Acad. 5ciUSA 81: 7161 (1984)] o CHO thin Fi etc. 0) Fibrosperm FJt, which can be transfected by lJ7 acid Cal/Um precipitation method [Graham et al., Vir.

1ogy 52, 456 (1973)]. All three methods are detailed below It is stated in

Identification of cells harboring plasmids can be performed using standard methods. For example, The genetic material of principal cells can be determined by Southern blot analysis. desired Heterologous gene expression can be determined using standard immunological or enzymological assays or bioassays. can be detected by assay.

kankoshi man In the following examples, mixing solids into solids, mixing liquids into liquids, mixing liquids into Unless otherwise specified, the ratio for mixing solids is wt/wt, vo]/ It is given based on vol and wt/voI. Under sterile conditions, cell culture maintained during cultivation.

General method Small-scale isolation of plasmid DNA from saturated overnight cultures was described by Birnboim et al. According to the method of [Nuc, Ac1ds Res, E: 1513 (1979)] It was conducted. This method is characterized by the isolation of small amounts of DNA for analysis from bacterial cultures. [ J. Bacteriol. 110:1135 (1972)]. .

Specific restriction enzyme fragments derived from cutting the plasmid DNA were prepared in an agarose gel. isolated by preparative electrophoresis followed by electroelution (Manniais et al., supra). Out, p. 164). Gels of 9 x 5 1/2 cm were prepared in Tris-acetate buffer (Mania (Nice et al., supra, p. 454) for 1 hour at 5QmA, then the DNA was allowed to run. For visualization, it was stained with lug/ml ethidium bromide. suitable gel part was cut out and melted at 65°C for 10 minutes, then 5 ml of 0.2M NaCl, 20 Low salt buffer containing mM Tris-HCl (pH 7.4) and 1 mM EDTA diluted with liquid. The Ltip-D column (Schlei Concentrate the DNA using a 100% DNA solution (Hier & Schnill, Köhne, NH) and g yeast tRNA carrier (Bethesda Research Laboratories, Bethesda , MD) at -20°C.

Restriction enzymes, DNA polymerase I (Klenow fragment) and T4 DNA ligase are products of Niney England Biolabs, Beverly, MA, and these The method and conditions for using the enzyme were basically in accordance with the manufacturer's instructions. T4 DNA ligase Ligacon was prepared using 50mM Tris-HCl, pH 7, 8, 10mM gCl2.20mM dithiothreitol, 1mM ATP and 50mg/ml The test was carried out at 4°C for 16 hours in a buffer containing bovine serum albumin. to Clenow To blunt the ends of single-stranded DNA, use 1mM dGTP, dATP, dCT. It was carried out in a restriction enzyme buffer adjusted to contain P and TTP.

chemical synthesis The oligonucleotides used as linkers were Available from Orabo, Beverly, MA or Model 380A DNA Synthesizer (Pride Biosystems, Foster City, CA) using standard Prepared by method.

Transformation and transfection E. coli cultures were transformed essentially according to Maninais et al., supra.

For transient expression in CO8 cells, plasmid DNA was introduced by DEAE-dextran treatment with boost [Yokota et al. Proc, Natl, Acad, Sci, USA 82:68 (1985)] .

Briefly, 10 ml of CO8 cell suspension was supplemented with 10% fetal bovine serum ()\ Iclone, Logan, UT), DME medium, penicillin/strep Tomycin (pen/5trep) and 2mM glutamine (Gibco, Grand - Island, NY) in a 100 mm culture dish containing approximately 10' cells/ml. It was written. After the cells reach approximately 50% confluence, the culture medium is removed and the cells are Washed with m1 serum-free DME.

The plasmid DNA-DME-DEAE-dextran mixture was prepared as follows. It was prepared by For each plate, 50mM Tris-HCl, pH 7 , 4, glutamine and the above-mentioned antibiotics (4.2 ml), DEAE-dextran (20 mg/ml in sterile distilled water: Macia, Bistowey, NJ). Plasmid DNA (10μg/plasmid rate) to 4.2 ml of DME-EDAE-dextran mixture, and The mixture was added to the washed cells. Each 100mm culture plate for 4 hours. Incubate the plate and aspirate the DNA solution from the plate. ml of serum-free DME. 5ml chloroquine solution (7% bovine) After adding fetal serum (150 μM chloroquine DME solution), cells were incubated for 3 hours. Incubated. After this incubation con, inhaling chloroquine The plate was washed with 5 ml of DME and aspirated. Next, each play Add 10 ml of DME (as described above, 10% 0% run serum, antibiotics and (containing glutamine) was added and incubated for about 72 hours. bioapp Cells were harvested or conditioned media was collected for analysis.

For stable transfection of myeloma cells, plasmid DNA is Introduced by lectroporation [Botter et al., Proc, Nat 1. Ac ad, Sci, USA 81 Near 161 (1984). To put it simply, about Myeloma cultured in suspension in 10 ml of complete DME medium added as above. Cells (5X10' cells/ml) were centrifuged and resuspended in 1 ml complete DME medium. .

Add plasmid DNA (40 μg) to 1 ml of suspension and mix cells/DNA. The liquid was divided into 0.25ml aliquots. Electrolyte each aliquot of cells. Gene Pulsar (Bio-Rad, Rockville Center, NY), 300 volts, 960 micro Fd electrical Shocked with a capacitive electrical pulse. Cells were left undisturbed for 10 min at room temperature, then 4 min. A plastic T-shirt containing complete DME with a total volume of 10 ml including the book aliquot. 75 tissue culture flasks. After incubation for 3 days, cells were concentrated for final concentration. mycophenolic acid (BRL/Life Chinosis, Gaisburg, MD), 100 μg/ml xanthine (Sigma, St. Louis , MO), 15 μg/ml hypoxanthine (Sigma), 10% fetal bovine serum and Diluted with 880 μg/ml glutamine selection medium. These cells were placed in a set of 96 wells. The cells were diluted to approximately 1,000 cells per 0.2 ml in tissue culture plates.

Colony formation of transfected cells could usually be observed in 7-14 days. Up Cells were harvested or conditioned media was harvested for assays similar to those described above.

Calcium phosphate precipitation method stably transfects fibroblasts with plasmid DNA. It was used for the purpose of Mammalian Transfection Kit (Stratagi) The protocol contained in the University of Pennsylvania, La Jolla, CA) was followed. Briefly stated, the above and 1x non-essential amino acids (100x non-essential amino acids available from Gibco). solution) and 4 μg/ml thymidine and 15 μg/ml hypoxanthine (both complete DME medium (contained in the 100X preservation solution sold by Gibco) In the ground, fibroblasts were placed in 100 mm tissue culture dishes at 5 x 10' cells/dish. Sowed. After 24 hours incubation, add 10 μg per plate. Mid DNA (as a calcium phosphate precipitate) was added. Incubate cells for 24 hours. plates were washed with serum-free DME medium and aspirated.

The cells were incubated for 24 hours, divided into 1/10 portions, and supplemented with selective medium. The cells were allowed to grow for 24 hours before addition. The selective medium contains DME, glutamine and the non-essentials mentioned above. Contains amino acids, 5% dialyzed fetal bovine serum (Gibco), and no antibiotics. Excluded. After colonies begin to form, grow in methotrexate at the 1 μM level. Medium containing methotrexate was added stepwise until viable cells were obtained.

This amplified the inserted site [Kaufman et al., J. Mo1. Bi.

1, 159+601 (1982)]. For assays similar to those described above, cells were The collected or conditioned medium was collected.

cell culture E. coli strain C600 (ATCC23724) was used for plasmid construction.

The cells were grown in 20:10:5 TYE medium (20g per liter). Lipton, Log yeast extract, 5g NaCI).

COS cells (ATCC CRL 1650) were treated with antibiotics, 2mM as described above. Glutamine (Gibco) and 10% fetal bovine serum (Iklon, Logan, UT) in DME medium (Gibco, Grand Island, NY). Leta. N5-1g cells (ATCCTIB 18) are DM similar to cO5[1tfi It was cultured in E culture solution. CHOdhfr- cells (clone DXB-11) were Obtained from Dr. Chasin, London University, NY. These cells are conventional 1x non-essential amino acids, thymidine and hyboxanthin (sold by Gibco) The cells were maintained in complete DME medium supplemented with 100X).

Construction of plasmid pDSVS The starting material, the plasmid, and some of the elements it contains are The single danglII site of lasmid pMTV-dhfr was cut at dangen±II; Subsequently, the 5' overhanging ends were blunted with DNA polymerase Klenow fragment, and the protruding ends were blunted. A lasmid pMTV-DHFR was created. Murine dihydrofolate reductase CD The DNA fragment containing the NA and 5V-40 early region polyadenylation signals was (containing the SV-40 polyadenylation signal and dhfr cDNA) ) The Tan-Do-tan dIII/Dan-and-R1 fragment of pL23 is the Tan-Do-tan RI/Tan-1-dII fragment. It is inserted into the cut plasmid pMT11s to create plasmid pL12R-A. Served. To create plasmid pL13R-B, plasmid pL12R -The single HindI11 site in A is a 5-truncation resulting from Dan-Satudan dIII cleavage. The protruding end was blunted with Klenow polymerase and then added with a linker (CAGA). TCTG: No. 1036, New England Biolabs) First, the single BamHI site of plasmid pL13R-B was first cut with amHI. After blunting the resulting 5' protruding end with Klenow polymerase, HindII I linker (CA, AGCTTG; No. 1022, New England version) iolapoz) ligation, Hind111 cleavage, ligation and colon The ndI11 site was converted by transformation of strain C600. It was.

5V-40 splicing signal sequence from pcD and murine gamma I The fragment containing the interferon cDNA was isolated from the plasmid pMg by cutting with XhoI. amma18 and this plasmid backbone creates plasmid pL27B. It was re-licensed in order to 5V-40 early protein of plasmid pL27H The motor and 5V-40 late region polyadenylation signals were restricted with 8ccI. The 5゛ protruding end was blunted with Klenow polymerase, and Ps±■ linker (GCTGCAGC; No. 1024, Niney England Biolabs) By ligation and simultaneous cleavage of Pst I/Hind I removed. The resulting Dankodan dlII/P containing the above-mentioned 5V-40 DNA The s±1 fragment was digested with Dankodan dIII/Ps±I to create pL15R-B. ) was ligated with L14R. A single strain of pL15R-B The DanamHI restriction enzyme site immediately adjacent to the I11 site is the part created by Dandan mHI. It was removed by cutting, yielding linear DNA. Following this, plasmid pL60 2, the 5° overhang was blunted with Klenow polymerase and This plasmid was religated.

The small fragment of Dandandan RI/Dandoku of pL602 was obtained by cleavage of Dandandan R1/dan mutual ±I and However, this is because complete recognition sequence reconstruction does not occur and both sites are lost. This is the result. In addition, this synthetic NA contains a recognition sequence for XbaI. It was. The nucleotide sequences of this synthetic NA, AB176 and AB177 are shown in the figure. 20. The resulting plasmid pL603 is an Ec of pL602. It lacked the oRI/Ps±1 fragment and instead contained a single Xba1 site.

To create plasmid pKMP-1, the 5V-40 late region polyadenylacetate Blunt the 5″ overhang with Renaud polymerase and religate. removed by.

DN phase containing the 5V-40 early promoter and intron from plasmid pL1 The DNA fragment containing the promoter and intron was obtained from plasmid pUcL1-E. In order to fabricate, dankutan dIII/danba ± II cutting and dankudan dIII/dan and m Removed from plasmid pKML-1 by insertion into HI cut pUC13 It was done. This The DNA fragments are annealed. However, the entire recognition sequence for both enzymes is lost. Both parts were destroyed in order to

Destroyed by -7 Yon. To create plasmid pcDL-4, 1) TanindIII/XbaI fragment 1 containing 5V-40 of UCL2-A ; k, inserted into HindIII/XbaI-cut plasmid pKMP-2 . To create plasmid pL64G, the production of plasmid pcDL-4±I The I and Xba1 sites were modified by Bg±II/XbaI restriction enzyme cleavage followed by 5 ° The overhanging ends were cut with exonuclease VII and religated. plastic To create Sumid pT443-2, a plasmid containing the 5V-40 intron was The X Watadan■/Tanmi mHI fragment of pL64G was cut with X Watadan I/Tanmi mHI. Inserted into L603.

To create plasmid pT512-2, a single clone of plasmid pCDV-1 The Nde1 site was cut with NdeI and a 5° overhang was created using Klenow polymerase. Smooth the ends and add Xbal linker (CTCTAGAG: No, 1032, -'- Ligation with N.England Biorapoz) and cleavage with XbaI. The XbaI site was converted by religation. Plasmid pT51 4-2, the 5V-4Q polyadenylation of plasmid pT443-2 The DNA fragment containing the conjugation signal was cleaved with BamHI/Xbal and ligated. The DNA fragment containing the 5V-4Q polyadenylation signal of pT512-2 was Replaced with piece. To create plasmid pT519-1, BamHI, S Synthetic NA fragment containing recognition sites for maI and EcoRI (pT514-2 The original BamHIa position of plasmid p was lost during cloning). T514-2+7) was inserted between BamHI and Sa Dodobu site. DN used The nucleotide sequences of A, namely AB199 and AB200, are shown in Figure 2G. Ru. To create plasmid pLT7-A, two H The indI11 site was first cut with ±ndIII, and the 5' overhanging ends of both DNA fragments were Smoothen with Klenow and insert ±II linker (CAGATCTG; No, 1036 , New England Biolabs), and was ligated with Dansei↓II The DNA mixture was cut and transferred to the BglII site by ligation. was replaced.

Finally, to create pDSVS, a general eukaryotic expression vector, The DNA fragment containing the MMTV-LTR promoter was removed by cutting with Bg±II. It was inserted into the single Bg±II site of plasmid pT5191.

The 5V-40 bromotor of pDSVS was linked to the SRα promoter [Takabe et al., Mol.

Ce11. Biol, 8:466 (1988)] to replace SRα Promotion 9-f;! , HidandIII and Ps±■ cleavage to pcDL-5R Cut out from α296. To create pDSR3, the SRα promoter The DNA fragment containing HidandIII/PΣ± was electroeluted from the agarose gel and ■ It was ligated with the cut pDSVS. This construction method is schematically shown in Figure 3. The synthetic oligonucleotides AB361 and AB362 shown (Figure 4) are as described above. When annealed, these oligonucleotides attach to one end. Generate double-stranded DNA with one dlII overhang on the top and a dlII overhang on the other. It was.

Synthetic NA is prepared by heating to 95°C in a volume of 20 microliters. Annealed and left until it reaches room temperature! It was done.

The general expression plasmid pDSVS is cut with danl±II and dansutanddllll. , electroeluted from an agarose gel. The annealed oligonucleotide is To create the lasmid psvs, Bg±II/HKDIII-cleaved pD Ligated with SVS. As a result, the dhfr transcription unit is removed and both The restriction enzyme site has been regenerated. Next, the SRα promoter is the SV40 promoter of pSVS. Replaced with motor. The SRα promoter is derived from plasmid pcDL-3Rα2 96 was isolated by cutting with 1ndlII and oI on X. plasmid p To generate SR3, this promoter element was transformed into HindllI/Xh It was ligated with pSVS cut with oI.

Construction of plasmids pDSRG and SRG Synthetic oligonucleotide AB523 and AB524 (Figure 5) were synthesized as described above. When annealed, these The oligonucleotides present in pUc19 are R1 and DIII. Between parts 1:, X mutually I, tan! mHI, Danseiko II and 5alIa restriction enzyme sites Introduced. AB523 and AB524 synthetic oligonucleotides were added to plasmid p 679-2, obtained by amHI cleavage as described above, To create plasmid p658J, p679 cut on Bg II and ligate was applied. This plasmid is inserted into an expression vector containing the SRα promoter. used as the human beta-globin polyadenylation signal in It was. Plasmid p658J was cut with HindII and DNA Klenow The ends were blunted with the fragment, Klenow was inactivated, and the plasmid was Cleaved with coRI. This fragment was cut with XbaI, treated with Klenow, and then EcoRI-cleaved pDSR5 to create pDSRG and pSRG, respectively. and ligated with pSR3.

Construction of plasmid pGSRG-h IL5 Human IL-5 cDNA was PCR chain reaction (PCR) [Saiki et al., 5science 23rd: 487 (19 [88) ] from the plasmid pcD-3Rα-hlL. Same direction Primer AB790 (Fig. 6) was used to obtain the optimal Kozak conformation [Kozak, J. Ce11. Biol, Kodandan: 229 (1989)]. It was designed to introduce a SaI restriction enzyme site upstream of this region. Reverse plastic The imer, AB791 (Figure 6), is immediately downstream of the stop codon for human IL-5. Designed to introduce a coRI restriction enzyme site. The direction of the primer is It is indicated by an arrow in FIG. 30 cycles of PCR (95°C 2 min, 40°C After 2 minutes at 72°C and 2 minutes at 72°C, the amplified DNA fragment was transferred to the expression vector pSR. To create 3-hIL5, it was cleaved with 5alI/dIIl, and then I/dankodandIII was ligated into pSR3. SR Alpha hIL-5 cDNA up to the promoter, splice site and EcoR1 site. Expression cassettes for human IL-5 containing pDSRG-hIL5 and To create pSRG-hIL5, pSRG-hIL5 was cleaved by HidandllI/dandandanRI. was removed from plasmid pSR3-hIL5 and digested with Dankodan dIII/EcoRI. ligated with pDSRG and pSRG. Next, the dominant marker Escherichia coli xanthine-guanine phosphotransferase (gpt) was introduced into the L-5 expression plasmid pSRG-hlL5. E. coli gpt transcriptional monomer position, part of the key variable region (Vhl) rearranged immunoglobulin, The region containing the marker and the E. coli plasmid replication origin contains Xhol and 1 11 cleavage, blunting of ends with Klenow polymerase, inactivation of Klenow enzyme HindIII cleavage to generate plasmid D by cleavage and Xbal cleavage. and inserted into Klenow-treated and Xbal-cleaved psRG-hIL5. child The human IL-5 expression plasmid contains the SRα promoter, the SV40 splice /Gnar and human beta globin 3 untranslated region to induce human IL-5. It will manifest.

The murine BCL, subculture strain (ATCCTlB197) described in However, the erythroleukemic human cell line TF-1 is available from T Kitamura Hiroshi. Obtained from Mr. (University of Tokyo). TF-1 cells were incubated with 10% fetal Uno serum, lng/ m1 Granulocyte-macrophage colony stimulating factor (GM-C5F) and penicilli in RPM 11640 medium (Gibco) containing streptomynon/streptomynon. cultured. For bioassays, cells were placed in phosphate-buffered saline (PBS, Gibco) and plated 4x10 in a 96-well plate containing RPM11640. Divided into aliquots at 4 cells/well. RP! vL11640 and 1 A solution containing the test sample in 0% fetal serum was added to the cells. 3 cells Incubate for 48 hours at 7°C. 10 μg of 3E4. dissolved in PBS. D-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT, Sigma) was added to each well and the cells were incubated for 6 hours. I took Kyubenodine.

Add 11 dilutions in each well to acidic isopropanol (0.1M hydrochloric acid). -95% isopropanol) and the contents of the wells were thoroughly mixed. . Mosman colorimetric determination [J, Immunol, Methods 65:55 (1 983)] was measured using a v-max spectrophotometer (Molecular Detailer) set at 570 nm. Vices, Palo Aldo, CA). Control absorbance at 630nm The yield was subtracted from the measurement at 570 nm. The activity (expressed in units/ml) is , the dilution of sample required to give half-maximal mitotic induction when incubated for 48 hours. defined as the reciprocal of the

Use of pDSVS derivatives to generate recombinant human rL-5 Human I as described above The L-5 expression plasmid was transfected into three different cell lines and the culture The conditioned medium derived from human L-5 was tested for human L-5 bioactivity using a TF-1 cell bioassay. was assayed for. Conditioned medium from CO8 cells was prepared as described above. . For growth in unamplified or 1 μM methotrexate The selected recombinant CH○ cells were cultured in 5 ml of DME medium (without antibiotics, 10% Uno fetal dish serum supplemented with 1x non-essential amino acids and 880ag/ml glutamine. 5×10 5 cells/ml and cultured for 72 hours.

The cell culture conditioned medium was harvested and placed in a 0°22μ nalu solution for bioassay preparation. Gen Low Protein Binding Syringe Filter. As mentioned above about CH○ cells, Similarly, recombinant myeloma cells (NS-1, ATCCTl818) Also, 5 ml of HBIOI medium (Hana Ba) supplemented with 800 ag/ml glutamine (Iological, Alameda, CA) at 5 x 105 cells/ml, It was cultured for 2 hours. After incubation, the conditioned medium is harvested for bioassay. The results are shown in Table 1.

Table 1 pSR5-hrL5 2.00On, d, n, d, n, dpDSRG-hIL5 n, d, <2.00020.00On, dpSRG-h IL, 5 2.0 00 n, d, n, d, n, dpGSRG-hII, 5 n, d, n, d, n, d, 12.000a plasmid was not amplified.

b Plasmids were amplified in the presence of 1 μM methotrexate.

n, d, = not measured The data in Table 1 show that human L-5 was effectively produced in three cell types. It shows things. The biological activity of IL-5 in conditioned medium was 2°0 in CO8 cells. 00 units/m1 (transient expression) to 20.000 units/m1 in CHO cells It spanned 1. Production in CHO cells was determined using methotrexate, It increased more than 10 times. Unamplified production of IL-5 in NS-1 cells Increased production in CH○ cells in view of the variability of the bioassay used It was almost the same.

Plasmid deposit Plasmids pDSVS, pDSR5SpDSRGSpSR5 and pSRG are Under the provisions of the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purposes of Administrative Procedures, Lycan Type Culture Collection, Rockville, MD, 1990. It was deposited on February 21st. They have accession numbers Nos, ATCC68, respectively. Assigned 231, 68232, 68233, 68234 and 68235 .

Many modifications and variations of this invention will be apparent to those skilled in the art, as will be apparent to those skilled in the art. This will be done without departing from the Certain aspects described herein are examples. The invention is limited only by the scope of the appended claims.

Figure 2A Figure 4 Figure 6A abstract The present invention provides methods for cloning a heterologous gene in E. coli and for cloning the gene in mammals. Provides novel recombinant plasmids that can be used for expression in plant cells . These plasmids contain deletions or substitutions of genetic elements within the plasmid. A large number of single restriction enzymes that can be used to facilitate conversion and insertion of heterologous genes Including parts. The nature and arrangement of this single restriction enzyme site! is the section following restriction enzyme cleavage. regeneration can be easily accomplished with minimal operational effort. This recombination Host cells containing the body plasmids are also provided by the present invention.

August 28, 1992

Claims (15)

[Claims] 1. In the transfer direction: (a) β-lactamase residue bounded by XbaI and BgIII restriction enzyme sites. Gene and pBR322 replication origin, (b) HindIII and XhoI restriction enzyme region SV-40 or SRα promoter, bounded by (c) one or more bounded by PstI and EcoRI restriction enzyme sites; a polylinker containing a single restriction enzyme site above, and (d) Polyadenylated sig bounded by EcoRI and XbaI restriction enzyme sites. null array, A recombinant plasmid consisting of a DNA sequence containing. 2. In the direction of transcription, the MMTV-LTR promoter, DHFR cDNA, and DHFR transcription consisting of ising signal sequence and polyadenylation signal sequence further comprising a transcription unit which contains the β-lactamase gene and the pBR322 duplex. It exists between the production start point and the SV-40 or SRα promoter, and BgIII and and HindIII restriction enzyme sites. Lasmid. 3. β-lactamase gene and pBR322 origin of replication and SV-40 or S It further contains a β-gal transcription unit between the Rα promoter and this β-gal transcription Claims wherein the unit is bounded by BgIII and HindIII restriction enzyme sites. Recombinant plasmid of Section 1. 4. β-lactamase gene and pBR322 origin of replication and SV-40 or S It further includes a CAT transcription unit between it and the Rα promoter, and this CAT transcription unit is of claim 1, bounded by BgIII and HindIII restriction enzyme sites. Recombinant plasmid. 5. There is a splice between the SV-40 or SRα promoter and the polylinker. further comprising a splicing signal sequence, the splicing signal sequence being and any one of claims 1 to 4, bounded by the PstI restriction enzyme site. A recombinant plasmid. 6. Any of claims 1 to 5, further comprising a viral enhancer. A recombinant plasmid. 7. The recombinant according to claim 1, wherein the recombinant plasmid is pDSVS. Plasmid. 8. The recombinant according to claim 1, wherein the recombinant plasmid is pDSRS. Plasmid. 9. The recombinant plasmid of claim 1, wherein the recombinant plasmid is pSRS. Lasmid. 10. The recombinant according to claim 1, wherein the recombinant plasmid is pDSRG. body plasmid. 11. The recombinant according to claim 1, wherein the recombinant plasmid is pSRG. Plasmid. 12. Claim 1, wherein the recombinant plasmid is pGSRG-hIL5. recombinant plasmid. 13. Claim 1, wherein the recombinant plasmid is pDSRG-hIL5. recombinant plasmid. 14. Having a recombinant plasmid according to any one of claims 1 to 13 E. coli. 15. Having a recombinant plasmid according to any one of claims 1 to 14 mammalian cells.