KR100450266B1 - Bicistronic vector for gene-expression by animal cell, the cell line thereof, and the preparation method for antibody peptide thereof - Google Patents

Abstract

The present invention is an animal cell that expresses a desired protein at high concentration by efficiently amplifying a target gene when selecting as a selection marker gene or amplifying an amplification gene using a bicistronic expression vector in an animal cell. The present invention relates to a dragon expression vector, an animal cell line including the same, and a method for producing a protein using the same, and can be used to efficiently express various genes including an antibody gene.

Description

Bicistronic vector for gene-expression by animal cell, the cell line, and the preparation method for antibody peptide

The present invention uses a bicystronic expression vector, which can be useful for preparing cells which continuously and stably express genes in mammalian cells, to amplify a gene for selection or amplification as a selection marker gene. The present invention relates to an expression vector for an animal cell expressing a desired protein at a high concentration by efficiently amplifying a target gene, an animal cell line including the same, and a method of producing a protein using the same.

Although various cellular systems are used to make genetically engineered proteins, many proteins derived from human genes are known to be active only when expressed in animal cells (Wasley et al., Blood (1991) 77, 2624). . This is because, in most cases, when mammalian cell proteins are expressed by E. coli, the tertiary structure is formed incorrectly or inefficiently, resulting in lack or lack of protein activity. In addition, most mammalian proteins are synthesized to form biologically active proteins through a process of glycosylation, phosphorylation, oligomerization and cleavage of proteins at specific sites.

Antibody genes are composed of heavy chain and light chain genes, and heavy and light chain proteins made from these genes bind to the Golgi of eukaryotic cells and are fixed to the cell wall or secreted out of the cell. In the case of immunoglobulin G (IgG), which is widely used as a therapeutic antibody, the heavy and light chains are bound by disulfide bonds in Golgi, and a molecular weight of about 150 kDa in which the heavy and light chains are again bonded to each other. The antibody molecule which has a secretion is extracellularly.

Genetically engineered Chinese Hamster Ovary (CHO, CHO) cells are used to express therapeutic antibody proteins in animal cells (Kaufman et al., Mol. Cell. Biol. (1985) 5, 1750), the cells can be suspended and cultured in large quantities, and are approved by the US Food and Drug Administration (FDA) for the manufacture of pharmaceuticals.

Briefly summarizing the process for preparing cells expressing foreign genes (Kaufman, Methods in Enz. (1990) 185, 537), CHO cells are transformed into foreign genes and then chemicals are used to recombine the external genes to chromosomes. Cells are isolated by selective culture so that only the cells can be grown. In addition, increased amounts of chemicals are added to the medium to induce more protein to induce amplification of foreign genes recombined into the chromosome.

This process enables the production of cell lines with about 1000 or more amplified foreign genes. At this time, the dihydrofolate Reductase (DHFR) gene and its inhibitor, Methotrexate (hereinafter referred to as 'MTX'), are mainly used as the selection gene and the selection agent. .

In general, to prepare CHO cells that continuously express antibody genes, two DNAs are co-transfected with a plasmid vector DNA containing a heavy and light chain gene to be expressed and a vector DNA having a DHFR gene. At the same time, cell lines integrated on the chromosome are isolated via selective culture (Kaufman, Methods in Enz. (1990) 185, 487).

However, the disadvantage of this co-transformation system is that in the amplification process using drugs, only the DHFR gene, which is a selection gene, is selectively amplified and the external gene to be expressed is often eliminated. There was a problem of leanness (Kaufman, Nucl. Acids Res. (1991) 19. 4485).

In order to solve the above problems, the present invention designs and manufactures a vector such that the heavy and light chain genes of the antibody and the selectable genes, the dihydroxyfolate reductase (DHFR) and the NEO gene, are expressed as a single gene. It is an object to provide an expression vector that can be amplified.

1 is a process diagram illustrating a cloning process of pLCD-MoK-Mok, a bicystronic expression vector for animal cell expression.

2 is a map showing the detailed structure of the expression vector pLCD-MoK-Mok.

3 is a process diagram illustrating a cloning process of pLCN-MoH, a bicystronic expression vector for animal cell expression.

4 is a map showing the detailed structure of the expression vector pLCN-MoH.

5 is a flowchart illustrating the cloning process of pANTIBODY.

6 is a map showing the detailed structure of the expression vector pANTIBODY.

In order to achieve the above object, the present invention is (1) a bicistronic expression vector (bicistronic expression vector), the portion required for transcription RNA production is cytomegalovirus (CMV) promoter, the light chain of the antibody as a foreign gene It provides a biscistronic expression vector comprising at least one foreign gene, an IRES sequence, a selection gene and a poly (A) sequence selected from among genes or heavy chain genes.

In another aspect, the present invention (2) provides the bicystronic expression vector, characterized in that the selection gene is DHFR (dihydroxyfolate reductase) or NEO ^R (neomycine resistance gene).

The present invention also provides a transgenic animal cell, characterized in that (3) the bicistronic expression vector is produced by transforming the animal cell.

In another aspect, the present invention (4) provides a method for producing a protein using animal cells expressing a large amount of foreign genes using the transformed animal cells.

In addition, the present invention (5) the part required for transcription RNA production is characterized in that the cytomegalovirus (CMV) promoter, a foreign gene, an IRES sequence, a selection gene consisting of the sequence of DHFR and poly (A) sequence It provides a bicistronic expression vector.

In addition, the present invention (6) bistronic expression vector pLCE-MoK (KCTC 10151BP), characterized in that the RNA is transcribed in the sequence of the sequence of 'light chain gene-IRES-DHFR-poly (A) of the antibody' To provide.

In another aspect, the present invention (7) provides a CHO cell line transformed with the expression vector of (6).

In addition, the present invention (8) bicistronic expression vector pLCN-MoH (KCTC 10150BP), characterized in that the RNA is transcribed in the order of the sequence of 'heavy chain gene-IRES-NEO-poly (A) of the antibody' To provide.

In another aspect, the present invention (9) provides a CHO cell line transformed with the expression vector of (8).

In addition, the present invention is characterized in that (10) the RNA is transcribed in the order of the sequence of 'light chain gene-IRES-DHFR-poly (A) -antibody heavy chain gene-IRES-NEO-poly (A) of the antibody' It provides a bicistronic expression vector pANTIBODY (KTCC 10152BP).

In another aspect, the present invention (11) provides a CHO cell line transformed with the expression vector of (10).

In general, in prokaryotic cells such as Escherichia coli, ribosomes can be attached to various sites on one mRNA, and a starting codon AUG can be found nearby, resulting in the synthesis of multiple polypeptides from one mRNA. This is called a polycistronic system.

In eukaryotic cells, on the other hand, when translation of a protein from mRNA, ribosomes are attached to the 5 'end of the mRNA, and then a scanning method is used to find the starting codon AUG, and the synthesis of protein from the AUG codon begins. do. Thus, one polypeptide is necessarily formed from one mRNA. Such a system is called a monocistronic system.

However, in case of poliovirus or EMC virus (Encephalomyocarditis virus), it has an RNA sequence with a special structure called Internal Ribosome Entry Site (IRS). Like E. coli , ribosomes can also synthesize polypeptides inside mRNA (Jang et al., J. Virol. (1989) 63, 1651).

In the present invention, Cytomegalovirus (CMV), one of the most powerful animal cell promoters known to date; Bohart et al., Cell (1985) 41, 521; Thomen et al., Proc. Natl. Acad. Promoter of Sci. USA (1984) 81, 659).

This promoter's enhancer is also active in a wide range of hosts, as well as the early CMV promoter known as one of the powerful enhancers in the transient expression system (Foecking et al., Gene (1986) 45, 101). Induce high expression of the light and heavy chain genes of antibodies, and in particular, the open reading frame (ORF) of the DHFR gene, a selection gene, is placed under the light chain gene in order to efficiently amplify the light chain gene. The expression vector was designed so that the IRES sequence was directly above the start codon.

Thus, the RNA transcribed by the CMV early promoter will include the light chain and the DHFR gene. This means that when MTX is used to amplify the DHFR gene, the portion encoding the heavy chain is also amplified. For the heavy chain gene, the expression vector was designed to be arranged in the order of CMV promoter-heavy chain gene-IRES-NEO gene in the same manner as for the light chain gene.

The gene of the antibody used in the present invention used a humanized antibody gene for 4-1BB (or CD137), which is induced to express in activated T-immune cells (Korean Patent Application Publication No. 2000-034847). The antibody expression vector of the antibody-producing cell line prepared using the antibody expression vector (pANTIBODY, KCTC 10152BP) prepared in the present invention was compared with each other. The present invention has been completed by revealing that the productivity is improved at least five times more.

Hereinafter, the present invention will be described in more detail with reference to Examples. The following examples merely illustrate the invention and do not limit the scope of the invention.

Example 1 Preparation of Light Chain Expression Vector pLCD-MoK-Mok

HinDIII, XbaI of pcDNA3 (manufactured by Invitrogen, 3985 B Sorrento Valley Blvd. San Diego, Ca92121, USA) with multicloning site, CMV promoter, SV40 intron and poly A signal sequence Two DNA fragments (DNA fragments 1 and 2) were conjugated to the restriction enzyme recognition site to prepare a light chain expression vector pLCD-MoK-Mok.

DNA fragment 1 was prepared by PCR (Polymerase chain reaction, hereinafter 'PCR') using primers Mok H3 and primers Mok Sal in plasmid pRc-HzB4-Mok-gs with light chain gene (Korean Patent Application Publication No. 2000-034847). Amplification).

The base sequences of all primers used in the present invention are shown in Table 1 below.

The amplified DNA fragments were purified by cleavage with restriction enzymes HinDIII and SalI, and the pure purification of all DNA fragments was performed by NucleoTrap DNA Extraction Kit (Clontech laboratories, Inc.). Product). Meanwhile, DNA fragment 2 containing IRES and DHFR genes of EMC virus (Encephalomyocarditis virus) was purified by cutting pLCED4 gDs (Korean Patent Publication No. 1997-006485) with XhoI and XbaI.

In addition, a conjugation reaction vector and a DNA fragment to be inserted were used about 100 ng, and 2 μl of 10-fold conjugation reaction solution (50 mM Tris-HCl), pH 7.8, 10 mM magnesium chloride (MgCl ₂ ), 10 mM dithiothreitol, 1 mM ATP, 25 μg / ml bovine serum albumin (BSA) and 10 units of T4 DNA ligase were added, and distilled water was added to a total volume of 20 μl, followed by reaction at 16 ° C. for 12 hours. After the reaction was completed, E. coli HB101 (ATCC 33694) was transformed and selected from the plate containing the empicillin and cleaved with an appropriate restriction enzyme to obtain a plasmid containing the desired DNA fragments, finally confirmed the DNA sequence.

PCR was performed using a temperature circulator (DNA Thermocycler, manufactured by Perkin-elmer, USA). The circulation program was 94 ° C. for 30 seconds; 45 ° C., 30 seconds; The cycle of 72 ° C. for 1 minute was repeated 25 times and finally further reacted at 72 ° C. for 10 minutes. Restriction enzyme cleavage and conjugation were performed using restriction enzymes and conjugate enzymes purchased from New England Biolabs, USA, and the reaction conditions were followed by the manufacturer's recommended method. Cloning of the expression vector is shown in FIG. 1 and a detailed map of the vector is shown in FIG.

Primer names and their sequences used for PCR and mutagenesis Primer name Primer Sequence Mok H3 5'-gctagtaagcttagagcttcatcagacaggca-3 ' Mok sal 5'-gctagtgtcgacgaatgagagaaacca-3 ' MoH H3 5'-gctcagaagcttgccgccaccatgggatggag-3 ' MoH Sal 5'-gctcaggtcgacctgacccgtggaaag-3 ' NEO Cla1 5'-atggtaatcgatatgattgaacaagatgga-3 ' NEO Xba1 5'-gggccctctagatcagaagaactcgtcaag-3 ' MoH Xho 5'-gtgagctaacctcgagtcacattaat-3 ' Mok xho 5'-tctgaggcggaactcgagagaaccag-3 '

Example 2 Preparation of Heavy Chain Expression Vector pLCN-MoH

The heavy chain expression vector pLCN-MoH was prepared by conjugating three DNA fragments (DNA fragments 3, 4 and 5) to pcDNA3 cleaved by HinDIII and XbaI restriction enzymes obtained in Example 1 to prepare a heavy chain expression vector pLCN-MoH. It was.

DNA fragment 3 was amplified by PCR (Polymerase chain reaction) using primers MoH H3 and primers MoH Sal in plasmid pCI-HzB4-MoH (Korean Patent Application No. 99-16750) containing a heavy chain gene. Amplified DNA fragments were purified by digestion with restriction enzymes HinDIII and SalI.

DNA fragment 4 contains the IRES gene of EMC and was purified purely by cutting pLCED4 gDs (Korean Patent Application No. 1995-282864) with XhoI, ClaI. DNA fragment 5 was a fragment containing a neomycin gene (NEO gene) and amplified by PCR using primers NEO Cla1 and primers NEO Xba1 in pcDNA3. The amplified DNA fragments were purified by cleavage with restriction enzymes Cla1 and XbaI.

PCR, restriction enzyme cleavage, conjugation, and separation and purification of DNA fragments related to DNA manipulation were performed in the same manner as in Example 1. The cloning process of the expression vector is shown in FIG. 3 and a detailed map of the vector is shown in FIG. 4.

<Example 3> Preparation of the light chain / heavy chain expression vector pANTIBODY

Light chain expression vector pLCD-MoK between SV40 poly (A) of the heavy chain expression vector pLCN-MoH and the plasmid replication origin (ColE1 origin) to create an expression vector system capable of simultaneously expressing the light and heavy chains of an antibody in one plasmid. PANTIBODY was prepared by inserting DNA fragments containing the CMV promoter-light chain gene-EMC IRES-DHFR gene of -Mok.

First, to facilitate cloning, the XhoI restriction enzyme recognition sites were introduced into the pLCN-MoH and pLCD-MoK-Mok using site-directed mutagenesis method as follows. Primer MoH Xho was used to introduce XhoI restriction enzyme recognition sites into pLCN-MoH, and primer Mok Xho was used to introduce XhoI restriction enzyme recognition sites into pLCD-MoK-Mok, respectively.

Site-directed mutagenesis is a U.S.-based Promega company (2800 Woods Hollow Road, Medison, WI, USA) under the trade name GeneEditor ^TM in vitro Site-Directed Mutagenesis Kit. Catalog number Q9280) was used, which followed the method recommended by the manufacturer.

As a result, the modified plasmids were named pLCN-MoH (xho) and pLCD-MoK-Mok (xho), respectively.

After pLCN-MoH (xho) was completely cleaved with restriction enzyme XhoI and partially cleaved with restriction enzyme SalI, about 8.1 kb fragment was isolated and purified, which was named 'DNA fragment 6'. Meanwhile, pLCD-MoK-Mok (xho) was completely cleaved with restriction enzyme XhoI and restriction enzyme SalI, and separated and purified about 3.1 kb fragment, which was named 'DNA fragment 7'.

Then, DNA fragment 6 and DNA fragment 7 were conjugated to obtain pANTIBODY having a size of 11.16 kb. Cloning of the expression vector is shown in FIG. 5 and a detailed map of the vector is shown in FIG. 6.

<Example 4> Expression test of the light chain / heavy chain expression vector pANTIBODY.

(Step 1) Transformation

To develop a cell line stably expressing the antibody protein, DHFR deficient CHO cells (ATCC CRL 9096) were first diluted 12-fold in 36 mm plates the day before the transfection experiment, and then cultured in a 37 ° C. carbon dioxide incubator.

After confirming that the cells had grown to about 70 to 80% on the plate, the plasmid pANTIBODY DNA was transfected into the cells with Lipofectamine plus reagent (Cab. No. 10964-013 / 11514-015 manufactured by Gibco BRL). At this time, IMDM medium (Iscove's modified Dulbecco's media, manufactured by Gibco BRL, catalog No. 12200-069) containing 10 μg / ml of 10% calf serum, hypoxanthine and thymidine was used. Used.

Two days later, a complete alpha medium containing 10% of hypoxanthine and thymidine and dialyzed calf serum and Geneticin (Geneticin, Gibco BRL, Cat. No. 11811-031) at a concentration of 800 μg / ml; It was replaced with a minimum essential medium (MEM), manufactured by Gibco BRL, catalog # 12000-022) and cultured so that only transformed CHO cells could grow.

Colonies of surviving cells began to form after 10-14 days from the start of selection culture. Cells with good growth rate were cultured until 10 ⁷ or more per 100 mm diameter dish, and some were frozen and some were subjected to gene amplification.

(Step 2) Antibody Gene Amplification of Transformed CHO Cells

Amplification of the transformed gene was performed by adding methotrexate (MTX), an inhibitor of DHFR, to the selection medium, and inducing amplification of the antibody gene along with the DHFR gene by gradually increasing its concentration while culturing the cells.

Initially, the concentration of MTX starts at 30 nM and increases by about 3 times. When the MTX concentration shows normal CHO cell growth and growth rate, at least 2 to 3 times of cultivation at each concentration of MTX, the concentration of MTX is the next level. Increased.

In particular, the expression rate of antibody protein was divided into 40 cell groups with good growth rate at 1 μM MTX concentration, and the enzyme-linked immunosorbent assay (ELISA) using a plate coated with GST-human 4-1BB was referred to as 'ELISA'. It was quantified by the method). ELISA was performed by adding 100 μl of 2 μg / ml GST-human 4-1BB protein per well of a 96-well plate and incubating at 37 ° C. for 4 hours. After blocking with 1% bovine serum albumin (BSA) containing 0.05% thimerosol, 100 μl of the primary antibody was added and reacted at 37 ° C. for 1 hour, followed by 1 × PBS, 0.05% Tween 20 Washed five times with (tween20).

Subsequently, 100 μl of the secondary antibody was added thereto, followed by reaction for 1 hour at 37 ° C., followed by washing five times with 1 × PBS and 0.05% tween 20. After developing with o-phenylenediamine (OPD; o-Phenylenediamine), the color development was measured by ELISA reader (ELISA READER, Molecular Devices, product name SPECTRA MAX340) at 490 nm. Since then, the productivity of the antibody was measured by culturing clones showing good growth rate and high expression rate (see Example 5).

Example 5 Comparison of Antibody Productivity of Cell Lines Transformed by pANTIBODY and Cell Lines Transformed by Existing Antibody Expression Vectors

(Step 1) The cells transformed with pANTIBODY obtained in Example 4 were cultured in flask T175 with serum-free medium (CHO-S-SFMII, manufactured by Gibco) for 3 days while maintaining 37 ° C. and 5% CO 2 conditions. The amount of expressed antibody contained in this culture was quantified by the same ELISA method as described above. The results of comparing the antibody productivity with the cell line SB500-23 (KCTC 0541BP) transformed by the existing antibody expression vector are as follows.

Antibody Expression Vectors Cell line Expression amount (mg / L) pANTIBODY HB02-164 6.9 pCI-HzB4-MoH / pRc-HzB4-Mok-gs SB500-23 0.13

As shown in Table 2, the amount of the antibody expressed using the vector according to the present invention was found to be increased by about 530% or more compared to the amount of the antibody expressed in cells transformed by the existing antibody expression vector. .

As described above, the present invention can efficiently express a desired protein by efficiently amplifying a target gene when selecting it as a selection marker gene or amplifying an amplification gene using a bicistronic vector system in an animal cell. It is a useful invention to provide an expression vector for an animal cell, an animal cell line comprising the same, and a method for producing a protein using the same.

Claims (8)

In the bicistronic anti-peptide vector, The antibody expression vector comprises a sequence of 'promoter-antibody heavy chain gene-IRES (internal ribosome entry site) -selection gene' sequence and 'promoter-antibody light chain gene-IRES sequence-DHFR (dihydroxyfolate reductase) gene' sequence Bicistronic expression vector comprising a. The method of claim 1, The selection gene is a bistronic expression vector, characterized in that NEO ^R (neomycine resistance gene). The method of claim 1, The promoter is a biscistronic antibody expression vector, characterized in that the cytomegalovirus (CMV) promoter. The method of claim 1, The antibody expression vector is an antibody light chain gene-IRES (internal ribosome entry site) -DHFR (dihydroxyfolate reductase) -poly (A) -antibody heavy chain gene-IRES (internal ribosome entry site) -NEO ^R- poly (A) Bistronic antibody expression vector pANTIBODY (accession number KCTC 10152BP), characterized in that the sequence of the sequence of '. The transgenic animal cell of claim 1, wherein the bistronic antibody expression vector is transformed into Chinese hamster ovary cells (CHO). In the method of producing protein by the proliferation of animal cells, Inoculating the transformed animal cell of claim 5 in the medium; Culturing the inoculated medium; And Purifying the antibody in the cultured medium; production method of the antibody comprising the. delete delete