KR20210081989A - Expression vector for animal cell - Google Patents

Abstract

The present invention relates to an animal cell expression vector, which includes a promoter for regulating expression of a target gene, a promoter for regulating expression of a selectable mark gene and a factor for increasing gene expression, and specifically, provides the animal cell expression vector capable of increasing efficiency of recombinant cell selection and stabilizing expression of a target gene for a long period of time. Furthermore, it is possible to provide a recombinant animal cell transformed with the animal cell expression vector having high expression efficiency.

Description

Animal cell expression vector {EXPRESSION VECTOR FOR ANIMAL CELL}

The present invention relates to an animal cell expression vector comprising a promoter for controlling the expression of a target gene, a promoter for controlling the expression of a selection mark gene, and a gene expression increasing factor.

Various expression systems such as microorganisms, plants, yeast, insect cells, and animal cells are used to express and obtain a target protein in large quantities and use it for pharmaceutical, industrial, and the like purposes.

Among them, although the animal cell expression system is the most suitable system for expressing animal proteins, the production cost is high due to the low expression efficiency of the recombinant protein compared to the microbial expression system, and the manipulation process of animal cells is complicated. Currently used industrial animal cell lines include CHO (Chinese Hamster Ovary), BHK (Baby Hamster Kidney), myeloma, and the like. make it manifest

In animal cells, various protein modification mechanisms including glycosylation are maintained, and when the protein is secreted into the culture medium, the protein is more easily obtained and purified. Whereas most animal cells necessarily require complex additives such as serum proteins in the culturing process, CHO cells can be cultured in a medium free of serum and protein, and thus are used as the most suitable host for recombinant protein expression. In addition, the characteristics of CHO cells are well known as many studies have preceded them, and they have the advantage of fast growth rate and suspension culture for mass culture.

In general, when a target gene is to be expressed in an animal cell, a vector having the target gene and a selection mark gene is simultaneously transfected (transfected). Transformed cells are selected by culturing in a selective medium. However, their expression frequency is very low in most cases. One of the reasons is that, unlike microbial systems, in animal cells, these target genes must be inserted into the chromosomes of the host cell. Also, even if stable transfectants are selected in which the target gene is stably inserted into the chromosome of the host cell, the expression level is difficult to predict. This is because the insertion site of the gene is different for each cell, and the expression pattern is different depending on the insertion site. Therefore, there is no clear correlation between the number of target genes inserted into animal cells and the gene expression level (Grindley et al., 1987, Trends Genet. 3, 16-22; Kucherlapati et al., 1984, Crit. Rev. Biochem. 16, 349-381; Palmiter et al., 1986, Annu. Rev. Genet. 20, 465-499) In most cases, gene expression in animal cells is suppressed by the nucleotides surrounding the inserted nucleotides, and the purpose of stably inserted Even stably integrated transgenes are often expressed at very low levels (Eissenberg et al., 1991, Trends Genet. 7, 335-340; Palmiter et al., 1986, Annu. Rev. Genet. 20, 465-499).

The possibility of using a nucleic acid factor to protect the expression of a target gene from these gene location-specific effects has been reported in several systems. The above-mentioned nucleic acid factors include an insulator factor and a nuclear lattice structure binding base (Nuclear Matrix Attachment Region: hereinafter referred to as "MAR"), a scaffold attachment region (hereinafter referred to as "SAR"), or a chromatin insulator (hereinafter referred to as "SAR"), or a chromatin insulator ( Chromatin Insulator: hereinafter referred to as “insulator”), etc. may be utilized. Although their mechanism of action has not been elucidated, when they are included in a transgene construct, they induce gene expression irrespective of the insertion site, and thus the expression level is determined according to the copy number of the gene. (McKnight, RAet) al., 1992, Proc. Natl. Acad. USA.89, 6943 - 6947)

Also, even in a transformant in which the target gene is stably inserted into the chromosome of the host cell, the efficiency of expression of the recombinant protein from the target gene inserted into the chromosome of the host cell may be affected by various factors. In particular, the most direct influence is determined by the promoter that regulates the expression of the target gene, and in general, viral promoters such as CMV or SV40, or the promoter of the EF1-alpha gene are used.

In addition, in the case of a target gene randomly inserted into the host cell chromosome, the protein expression efficiency may be affected depending on the insertion site, and when inserted into an inactivated location of the host cell chromosome, expression may be inhibited. Therefore, among recombinant cells into which the expression vector containing the target gene and the selection mark gene has been stably inserted, the expression vector is inserted into the activated position of the host cell chromosome to selectively distinguish only the cell group that does not inhibit the expression of the target gene. If the target gene expression efficiency of the stabilizing cell group obtained after the selection process can be increased, the expression efficiency of the cell line selected using the stabilizing cell group can be greatly increased.

Carlos et al. combined the MAR factor of the human apolipoprotein B gene into a minimal promoter transgene construct and inducing gene expression in animal cells to increase the expression of the transcript by about 200 times. (Kaloset al., 1995, Mol. Cell. Biol. 15, 198-207) Similarly, in vertebrate cells, the MAR factor of the chicken lysozyme A gene and human interferon β SAR factors of genes have also been reported to have the property of increasing target gene expression regardless of the insertion position in the host cell chromosome. (Eissenberg et al., 1991, Trends Genet. 7, 335-340; Klehr et al., 1991, Biochemistry30 , 1264-1270) However, there are still no attempts to substantially increase protein production in CHO cell lines by applying a promoter that controls the expression level of the selection mark gene together with these MAR/SAR or insulator factors or cases where industrial profitability has been verified. has not been reported

Under this background, the present inventors increase the efficiency of recombinant cell selection by using a gene expression increasing factor such as MAR, SAR, or insulator, in parallel with a method for minimizing the expression of a selectable mark gene, while at the same time stabilizing the expression of a target gene for a long time. The present invention was completed.

Korean Patent Publication No. 10-2012-7006308 Korean Patent Publication No. 10-2012-7012896

Accordingly, it is an object of the present invention to provide an animal cell expression vector capable of increasing the efficiency of recombinant cell selection and stabilizing the expression of a target gene for a long period of time.

Another object of the present invention is to provide a recombinant animal cell transformed with an animal cell expression vector having high expression efficiency.

In order to achieve the object of the present invention as described above, the present invention provides a promoter for regulating the expression of a target gene; promoters regulating select mark gene expression; Provided is an animal cell expression vector comprising one or more gene expression increasing factors selected from the group consisting of MAR factors, SAR factors and insulator factors as gene expression increasing factors.

In one embodiment of the present invention, the promoter controlling the expression of the selection mark gene may be derived from the SV40 promoter.

In one embodiment of the present invention, the promoter controlling the expression of the selection mark gene can minimize the expression of the selection mark gene.

In one embodiment of the present invention, the MAR factor may be beta-globin MAR or chicken lysozyme MAR.

In one embodiment of the present invention, the SAR factor may be CSP-B SAR or interferon beta SAR.

In one embodiment of the present invention, the insulator factor may be a chicken lysozyme insulator (chicken lysozyme insulator).

In one embodiment of the present invention, the gene expression increasing factor is the 5' end of the promoter controlling the expression of the target gene, the 3' end of the transcription termination factor, or the 5' end of the promoter controlling the expression of the target gene and on both sides of the 3' end of the transcription termination factor, it may be located in the forward or reverse direction.

In one embodiment of the present invention, 2 copies or more copies of the MAR, SAR or insulator factor may be located at the 5' end or 3' end of the promoter controlling the expression of the target gene. In addition, the MAR factor and the SAR factor may be positioned by one copy each at the 5' end or 3' end of the promoter that regulates the expression of the target gene.

In one embodiment of the present invention, the promoter for regulating the expression of a target gene may be a CMV, SV40 promoter or EF1-alpha.

In one embodiment of the present invention, the promoter controlling the expression of the selection mark gene may be a puromycin gene, a neomycin gene, a blasticidin gene, a hygromycin gene, a zeocin gene, a glutamine synthetase gene, or a DFHFR gene.

In addition, the present invention provides a promoter for regulating the expression of a target gene; promoters regulating select mark gene expression; Provided is a recombinant animal cell transformed from an animal cell expression vector comprising one or more gene expression increasing factors selected from the group consisting of MAR factors, SAR factors and insulator factors as gene expression increasing factors.

In one embodiment of the present invention, the recombinant animal cells are CHO cells, Hela cells, BHK cells, NIH/3T3 cells, COS-1 cells, COS-7 cells, CHO-K1 cells, DG44 cells, NS0 cells or HEK293 cells. It may be a cell.

The animal cell expression vector of the present invention increases the efficiency of the expression of the recombinant protein of the target gene compared to the existing expression vector, the stability of the expression of the recombinant protein from the gene of interest introduced into the chromosome of the host cell is high, and as a result, the production of the recombinant protein is increased. It has its usefulness in significantly improving the performance of a stable cell pool or a stable cell line prepared for this purpose.

1 is a schematic diagram showing the configuration of an animal cell expression vector according to an embodiment of the present invention. The CMV promoter is used as a promoter for controlling the expression of a target gene, the SV40 promoter is used as a promoter for controlling the expression of a selection mark gene, and the MAR factor is This is a picture of an expression vector containing 1 copy.
2 is a schematic diagram showing the configuration of an animal cell expression vector according to an embodiment of the present invention, and is a diagram of an expression vector including one copy of each of the SAR factor and the MAR factor.
3 is a schematic diagram showing the construction of an animal cell expression vector according to an embodiment of the present invention, using the CMV enhancer Early Promoter for expression of a target gene, and using a variant of the SV40 promoter for expression of a selectable mark gene; It is a picture that shows that
4 is a graph measuring the expression level of a target gene according to an embodiment of the present invention.
5 is a diagram illustrating purification and analysis of a target protein produced according to an embodiment of the present invention.
6 is a graph including information on cell line selection according to an embodiment of the present invention.
7 is a diagram comparing productivity values with and without MAR factors according to an embodiment of the present invention.
8 is a diagram comparing the specific productivity rate in the case of including and not including the MAR factor according to an embodiment of the present invention.
9 is a diagram showing a result of selecting a high-efficiency cell line according to an embodiment of the present invention.
10 is a diagram showing the results of a productivity experiment of a stable cell pool prepared according to an embodiment of the present invention.
11 is a diagram showing conditions for a culture experiment of an optimized cell line prepared according to an embodiment of the present invention and results thereof.

The present invention relates to an animal cell expression vector comprising a promoter for controlling the expression of a target gene, a promoter for controlling the expression of a selection mark gene, a gene expression increasing factor and a transcription termination factor.

In the case where it is stated throughout this specification that a component "includes" another component, it does not exclude any other component, but further includes any other component unless otherwise stated. It could mean that you can.

Furthermore, when it is stated that a component is "connected", "installed" or "installed" of another component, the component may be directly connected to or installed in contact with another component, It may be installed spaced apart by a certain distance, and in the case of being installed spaced apart by a certain distance, a third component or means for fixing or connecting the corresponding component to another component may exist, and this It should be noted that the description of the third component or means may be omitted.

Similarly, other expressions describing the relationship between each component, that is, “at the end of”, “on”, etc., should be construed as having the same meaning.

In addition, in the present specification, terms such as "one side", "other side", "one side", "other side", "end", "first", "second", etc., if used, are this for one component. It should be understood that one component is used to be clearly distinguished from another component, and the meaning of the component is not limitedly used by such terms.

In addition, in the present specification, terms related to positions such as "upper", "lower", "left", "right", and "end", if used, indicate a relative position in the drawing with respect to the corresponding component. It should be understood that, unless absolute positions are specified for their positions, these position-related terms should not be construed as referring to absolute positions.

In the drawings attached to this specification, the size, position, coupling relationship, etc. of each component constituting the present invention are partially exaggerated, reduced, or omitted for convenience of explanation or in order to sufficiently clearly convey the spirit of the present invention. may be described, and therefore the proportion or scale may not be exact.

Hereinafter, preferred embodiments of the present invention will be described in detail as follows.

The present invention provides a promoter for regulating the expression of a target gene; promoters regulating select mark gene expression; It provides an animal cell expression vector comprising one or more gene expression increasing factors selected from the group consisting of MAR factors, SAR factors and insulator factors as gene expression increasing factors.

In one embodiment of the present invention, the promoter controlling the expression of the selection mark gene may be derived from the SV40 promoter.

In one embodiment of the present invention, the promoter controlling the expression of the selection mark gene can minimize the expression of the selection mark gene.

In one embodiment of the present invention, the MAR factor may be beta-globin MAR or chicken lysozyme MAR.

In one embodiment of the present invention, the SAR factor may be CSP-B SAR or interferon beta SAR.

In one embodiment of the present invention, the insulator factor may be a chicken lysozyme insulator (chicken lysozyme insulator).

In one embodiment of the present invention, the gene expression increasing factor is the 5' end, the 3' end of the promoter controlling the expression of the target gene, or both the 5' end and the 3' end of the promoter controlling the expression of the target gene In, it can be located in the forward or reverse direction.

In one embodiment of the present invention, two copies of the MAR factor may be located at the 5' end or 3' end of the promoter controlling the expression of the target gene. In addition, two copies of the SAR factor may be located at the 5' end or 3' end of the promoter that regulates the expression of the target gene. In addition, the MAR factor, the SAR factor, or the insulator factor may be located one copy each at the 5' end or 3' end of the promoter that regulates the expression of the target gene.

In one embodiment of the present invention, the promoter for regulating the expression of a target gene may be a CMV, SV40 promoter or EF1-alpha.

In one embodiment of the present invention, the promoter controlling the expression of the selection mark gene may be a puromycin gene, a neomycin gene, a blasticidin gene, a hygromycin gene, a zeocin gene, a glutamine synthetase gene, or a DFHFR gene.

In addition, the present invention provides a promoter for regulating the expression of a target gene; promoters regulating select mark gene expression; Provided is a recombinant animal cell transformed from an animal cell expression vector comprising one or more gene expression increasing factors selected from the group consisting of MAR factors, SAR factors and insulator factors as gene expression increasing factors.

In one embodiment of the present invention, the recombinant animal cells are CHO cells, Hela cells, BHK cells, NIH/3T3 cells, COS-1 cells, COS-7 cells, CHO-K1 cells, DG44 cells, NS0 cells or HEK293 cells. It may be a cell.

Hereinafter, the present invention will be described in more detail through examples. These examples are for explaining the present invention in more detail, and the scope of the present invention is not limited to these examples.

Expression vector preparation method

1, 2 or 3, the expression vector of the present invention utilized the CMV enhancer Early Promoter for expression of a target gene (target gene), and a selection mark gene as a variant of the SV40 promoter for expression of the selection mark gene. A method was introduced to minimize the expression of

In addition, in order to increase the homeostasis of target gene expression, the MAR factor was inserted in the 5' direction of the target gene.

Expression of target gene

As shown in FIG. 4, after transient transduction of 20 μg or 40 μg of CMC-A vector into CHO-k1 cells cultured as floating cells, the cell growth curve and expression level (ug/ml) of the target gene were measured for 7 days. It was confirmed through the ELISA method, and it was confirmed that the target gene was well expressed.

Protein A column purification assay

As shown in FIG. 5 , it was confirmed that the target protein produced through transient expression was purified by protein-A column and then expressed through SDS-PAGE.

cell line selection

CHO-k1 cells and CHO-k1 transformed cells, V23 cells, were transduced with a vector containing no MAR factor and a vector containing MAR factor, followed by antibiotic selection for up to 19 days to create a stable cell pool. was manufactured (see FIG. 6).

Productivity check according to the introduction of MAR factor

CHO-k1 cells or CHO-KS1 cells were transduced with pCMC-A vector to construct a stable cell pool, and then CDM4CHO (CDM4) or Hycell CHO (Hycell) culture medium was used to produce a stable cell pool (specific productivity rate, picogram per cell per). day), and when comparing the case where the expression vector did not contain the MAR factor (A) and the case where the MAR factor was included (MA), it was confirmed that the productivity was more excellent in the case where the MAR factor was included (A). (See Fig. 7).

In addition, as shown in FIG. 8, productivity was confirmed using the Specific Productivity Rate analysis method. Specifically, the stably transduced CHO-k1 cell pool was compared in the condition without the MAR factor (A) and the condition including the MAR factor, and the productivity of 9.027 pg/cell/day in the condition including the MAR factor was compared. It was confirmed that the effect of 83.4% productivity improvement compared to the case where the MAR factor was not included in the expression vector was confirmed.

Selection of high-efficiency cell lines and confirmation of productivity

In order to select high-efficiency cell lines and check their productivity, a stable cell pool prepared through a selection process with puromycin after transducing a vector containing the MAR factor was used to select high-efficiency cell lines using the Clonepix-2 (Molecular Devices) cell line selection device. was selected. It was confirmed that the high-efficiency cell line selected in this way exhibited a productivity of up to 19.84 pg/cell/day (see FIG. 9 ).

Selection of optimal cell lines and confirmation of productivity

In order to select an optimized cell line, a stable cell pool was prepared using puromycin (Puro-r) or glutamine synthetase (GS) as a selection marker using CHO-k1 or CHO-ke1 cells, and then a productivity test was conducted. As a result, as shown in FIG. 10, it was confirmed that CHO-ke1 cells exhibited a specific productivity rate of 4.37 pg/cell/day.

In addition, as shown in Table 1 below, a stable cell pool showing a specific productivity rate of 4.37 pg/cell/day in CHO-ke1 cells was selected into three cell groups using MoFlo-XDP, a FACS high-speed flow cytometer, The bulk sort 1 cell group recorded a production phase of 5.2 p/c/d, confirming that the productivity was improved by about 19% compared to the productivity before selection (4.37 p/c/d). Cell selection was classified according to the degree of fluorescence using an RPE-conjugated antibody with binding affinity to the target protein known as the cold capture method.

Classification Initial Count Final Count Sample ug/ml cells/ml cells cells/ml cells ICA SPR Bulk sort 1 3.68 50700 152100 617000 1851000 2124523 5.20 Bulk sort 2 0.06 10700 32100 21500 64500 145097 1.32 Bulk sort 3 1.85 39200 117600 368000 1104000 1376481 4.02

Then, after recovering the cells of Bulk sort 1 through culturing for about 2 days, the cells were reselected as single cells in a 96-well plate using MoFlo. As a result, 4 single cells with a productivity of 13 p/c/d or more were recovered. Cell lines were isolated, and the results are shown in Table 2 below.

TSB ug/ml (l) Cells (F) Cells SPR Day SPR DT 2 14.28 1.40 21.40 2.917 13.4 17.8 17 28.20 2.74 35.00 2.917 15.3 19.0 21 16.23 2.24 5.69 1.875 23.4 33.5 46 15.66 11.70 171.60 3.958 19.9 30.7

Additionally, cell line 46 in Table 2 was used to maximize IgG productivity, maximize the main population of charge variants that occur in connection with additional protein structural changes such as glycosylation, and culture conditions to minimize the population of basic proteins ( In order to achieve optimization (pH, culture temperature, type of medium), the experiment was conducted under conditions of BD1+FD3, pH 6.97, and culture temperature of 32-34° C. Accordingly, the results of the fed-batch culture experiment for up to 17 days are shown in Table 3 below.

Day Productivity (g/L) Cell viability (%) 10 days 1.57 98.3 11 days 2.08 98.4 12 days 2.51 98.1 14 days 3.37 97.4 15th 3.94 97.6 17 days 5.09 94.0

As can be seen in Table 3, cell line 46 recorded a maximum productivity of 5.09 g per volume and 94.0% viable cell density for 17 days under the same conditions. Through these results, it can be predicted that if the culture is continued for 2-3 days additionally, the productivity of up to 6 g/L can be achieved while the cell viability is maintained at 80% or more, which is a separate process for gene amplification. On the other hand, as a result of using a fusion protein, which is well known to be very difficult to achieve high productivity, as a target gene, the productivity of the animal cell expression vector derived through the present invention exceeds the average value of the existing industry to a high level. could check

So far, with respect to the present invention, the preferred embodiments have been looked at. Those of ordinary skill in the art to which the present invention pertains will understand that the present invention can be implemented in a modified form without departing from the essential characteristics of the present invention. Therefore, the disclosed embodiments are to be considered in an illustrative rather than a restrictive sense. The scope of the present invention is indicated in the claims rather than the foregoing description, and all differences within the scope equivalent thereto should be construed as being included in the present invention.

Claims (12)

An animal cell expression vector comprising:
a promoter regulating the expression of a target gene;
promoters regulating select mark gene expression;
One or more gene expression increasing factors selected from the group consisting of MAR factors, SAR factors and insulator factors. According to claim 1,
The promoter for controlling the expression of the selection mark gene is an animal cell expression vector, characterized in that it is derived from the SV40 promoter. According to claim 1,
An animal cell expression vector, characterized in that the promoter controlling the expression of the selection mark gene minimizes the expression of the selection mark gene. According to claim 1,
The MAR factor is an animal cell expression vector, characterized in that beta-globin MAR or chicken lysozyme MAR. According to claim 1,
The SAR factor is an animal cell expression vector, characterized in that it is CSP-B SAR or interferon beta SAR. According to claim 1,
The insulator factor is an animal cell expression vector, characterized in that the chicken lysozyme insulator (chicken lysozyme insulator). According to claim 1,
The gene expression increasing factor is located at either the 5' end, the 3' end of the promoter for controlling the expression of the target gene, or on both the 5' and 3' ends of the promoter for controlling the expression of the target gene, in the forward or reverse direction. Characterized, animal cell expression vector. 8. The method of claim 7,
Two copies of the MAR factor are located at the 5' end of the promoter regulating the expression of the target gene, or 2 copies (copies) of the SAR factor at the 5' or 3' end of the promoter controlling the expression of the target gene ( copies), or wherein the MAR factor and the SAR factor are located at the 5' end or 3' end of the promoter controlling the expression of the target gene, characterized in that each 1 copy (copy) is located, animal cell expression vector. According to claim 1,
The promoter controlling the expression of the target gene is an animal cell expression vector, characterized in that it is a CMV, SV40 promoter or EF1-alpha. According to claim 1,
The promoter controlling the expression of the selection mark gene is a puromycin gene, a neomycin gene, a blasticidin gene, a hygromycin gene, a zeocin gene, a glutamine synthetase gene, or a DFHFR gene, an animal cell expression vector. A recombinant animal cell transformed with the animal cell expression vector according to any one of claims 1 to 10. 12. The method of claim 11,
The recombinant animal cells are CHO cells, Hela cells, BHK cells, NIH/3T3 cells, COS-1 cells, COS-7 cells, CHO-K1 cells, DG44 cells, NS0 cells or HEK293 cells. cell.

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