KR101629564B1 - Recombinant vector for enhancing target gene-expression and use thereof - Google Patents

Abstract

The present invention relates to a vector comprising a gene fragment derived from CHO (Chinese Hamster Ovary) cells for promoting the expression of a gene of interest in animal cells and the use thereof, and a vector containing the gene fragment of the present invention can be used to produce Since the expression of the target protein is confirmed to be enhanced, it can be usefully used for the production of biological drugs such as therapeutic antibodies.

Description

Recombinant vector for enhancing the expression of a target gene and its use {Recombinant vector for enhancing target gene-expression and use thereof}

The present invention relates to a vector comprising a gene fragment derived from CHO (Chinese Hamster Ovary) cells for promoting recombinant protein expression in animal cells, and the use thereof.

The synthesis of proteins is initiated through a transcription process in which genomic information encoded in DNA is copied into RNA. The transcription starts by binding RNA polymerase to a promoter located upstream of the gene. The nucleotide sequences of all promoters are not completely identical, but they have a common form. This nucleotide sequence is called a consensus sequence. Most promoters differ in the actual base sequence of the common base sequence and the distance from the transcription initiation point. It is recognized that this diversity determines the frequency at which transcription begins in a particular promoter, i. E., The promoter strength.

This promoter is one of the important factors determining the efficiency of recombinant protein production. Therefore, techniques for searching strong and specific promoters from various genes have been developed. One of the most commonly used methods of such techniques is the use of a reporter vector. The reporter vector includes a gene encoding a reporter protein that can easily confirm a reaction product by a biochemical reaction. Since the gene contains only the region encoding the protein and does not contain the promoter required for transcription of the gene, the researcher inserts the DNA fragment to be investigated upstream of the reporter gene and then transcribed and translated from the reporter gene By measuring the amount of the protein, the activity of the promoter can be measured.

Reporter genes include green fluorescent protein (GFP), luciferase, secreted embryonic alkaline phosphatase (SEAP), b-galactosidase, and chloroamphenicol acetyltransferase (CAT). However, when an external gene is inserted into a chromosome, various factors may change the activity of the promoter, or even inactivate it. The GFP gene expressing green fluorescence is widely used as a reporter for the expression of a gene in a living cell, the position and the movement path of the expressed protein. That is, the intensity of the promoter can be compared in the cell into which the vector DNA of the present invention is introduced by observing the intensity of the fluorescent protein using a flow cytometer or fluorescence microscope in a living state without preparing the cells as an extract.

The Flp-In cell line (Invitrogen) has a nucleotide sequence that can be recognized by a recombinase (flp recombinase) inserted into the chromosome and has the advantage of recombining a single gene. Therefore, by using the Flp-In system, the activity of the promoter can be measured under stable expression conditions.

Thus, the present inventors have completed the present invention by finding a gene fragment that increases the expression of a recombinant protein by measuring the activity of the promoter using the reporter vector and the Flp-In system.

KR Patent Registration No. 0783491 (2007.12.07)

It is an object of the present invention to provide a recombinant vector for increasing the expression of a target gene into which a gene fragment comprising a nucleotide sequence represented by any one of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 and SEQ ID NO:

It is still another object of the present invention to provide a method for producing a target protein, which comprises culturing an animal cell transformed with an expression-increasing recombinant vector to obtain a target protein.

The present invention provides a recombinant vector for increasing the expression of a target gene comprising a gene fragment comprising a nucleotide sequence represented by any one of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 and SEQ ID NO:

For purposes of the present invention, a "vector" refers to a DNA construct containing an exogenous DNA sequence operably linked to a suitable regulatory sequence capable of expressing DNA within the appropriate host. The vector of the present invention can typically be constructed as a vector for expression. Preferably, the vector of the present invention is a vector for expressing a recombinant peptide or protein. In addition, the vector of the present invention can be constructed by using prokaryotic cells or eukaryotic cells as host cells. The recombinant expression vector of the present invention may be, for example, a bacteriophage vector, a cosmid vector, a yeast artificial chromosome (YAC) vector, or the like. For the purpose of the present invention, it is preferable to use a plasmid vector. Typical plasmid vectors that can be used for such purposes include (a) a cloning start point that allows replication to be efficiently made to include several hundred plasmid vectors per host cell, (b) a host cell transformed with the plasmid vector And (c) a restriction enzyme cleavage site into which the foreign DNA fragment can be inserted. Even if an appropriate restriction enzyme cleavage site is not present, the vector and the foreign DNA can be easily ligated using a synthetic oligonucleotide adapter or a linker according to a conventional method. The vectors used in the present invention can be constructed through various methods known in the art, and specific methods for this can be found in Sambrook et al. Molecular Cloning. A Laboratory Manual. Cold Spring Harbor Laboratory Press (2001).

In the present invention, the "target gene" is a gene encoding a foreign product to be expressed, and is a gene encoding all kinds of proteins capable of being expressed as a recombinant protein. Typically, insulin, cytokine (interleukin, tumor necrosis factor, interferon, colony stimulating factor, chemokine and the like), erythropoietin and the like are included. Such a target gene is a restriction enzyme for insertion into the vector, Quot; cloning site "

In the present invention, "promoter" means a nucleic acid sequence that is upstream of the coding region and contains a binding site for the polymerase and has a transcription initiation activity of mRNA of the promoter subgenus. In the present invention, the promoter is preferably a virus-derived or mammal-derived promoter, more preferably a cytomegalovirus (CMV) promoter, but is not limited thereto.

The gene fragment is preferably derived from CHO (Chinese Hamster Ovary) -K1 cells, but is not limited thereto.

The gene fragment is preferably inserted in front of the target gene, but is not limited thereto.

In addition,

1) preparing a recombinant vector for increasing expression according to the first aspect;

2) transforming an animal cell with a recombinant vector for increasing the expression of step 1); And

3) culturing the transformed animal cells of step 2) to obtain a target protein.

The animal cell is preferably a CHO (Chinese Hamster Ovary) cell, but is not limited thereto.

According to the present invention, it is possible to search for a gene fragment that promotes the activity of the promoter in the genomic DNA derived from CHO-K1, and when the vector containing the retrieved gene fragment is transformed into CHO (Chinese Hamster Ovary) Can be produced in large amounts. This may be useful for production required for the production of biologics such as therapeutic antibodies.

1 is a schematic diagram of a GFP reporter vector used for measuring the activity of a promoter in the present invention.
FIG. 2 is a schematic diagram of a vector for searching a CHO-K1 genomic DNA fragment for promoting the activity of a promoter in the present invention. FIG.
FIG. 3 is a graph showing the fluorescence intensity of green protein fluorescence obtained by fluorescence-activated cell sorter (FACS) by obtaining a cell population expressing various green fluorescent proteins by transfecting the DNA pool prepared in Example 1 into Flp-In CHO-K1 cell line .
FIG. 4 is a graph showing the intensity of a green protein fluorescence in a single cell line isolated from the Pool 2 cell group obtained in Example 2. FIG.
FIG. 5 is a graph showing the intensity of green protein fluorescence by transforming animal cells with a vector having the E1, E2, E3, and E4 gene fragment inserted therein according to the present invention.

It will be apparent to those skilled in the art that various modifications and variations can be made in the present invention without departing from the spirit or scope of the invention as defined by the appended claims. It will be obvious to you.

Example 1: Preparation of a vector library into which a CHO cell gene fragment was inserted

1- (1): Preparation of green fluorescent protein expression vector (pcDNA5 / FRT / EGFP vector)

The multiple cloning site of the pcDNA5 / FRT vector (Invitrogen) was digested with NheI and XhoI restriction enzymes and EGFP cDNA was inserted to construct a green fluorescent protein expression vector. The thus prepared vector was named pcDNA5 / FRT / EGFP vector (Fig. 1).

1- (2): Production of protein expression enhancing gene screening vector (pmCMV / FRT / EGFP vector)

In the pcDNA5 / FRT / EGFP vector prepared in Example 1- (1), the CMV promoter / enhancer site was cleaved with BamHI and NheI restriction enzymes and the enhancer site was removed. The thus prepared vector was named as pmCMV / FRT / EGFP vector (FIG. 2) and used as a vector for screening the gene promoting protein expression.

1- (3): Preparation of CHO-K1 genomic library

A library was obtained by inserting a gene fragment of CHO-K1 cell line (ATCC®CCL-61 ^™ ) into the pmCMV / FRT / EGFP vector prepared in Example 1- (2). First, genomic DNA of CHO-K1 cell line was obtained from CHO-K1 cells using DNeasy blood & tissue kit (Qiagen). Extracted genomic DNA was sectioned by treatment with restriction enzyme Sau3AI (NEB). DNA was reacted at 37 ° C with 2 units of Sau3AI restriction enzyme per 1 μg of DNA, and the reaction-terminated DNA was electrophoresed on agarose gel. DNAs of 1 Kb or less were extracted from the gel electrophoresis using gel extraction kit (Qiagen) and used as gene fragments to be inserted into the vector.

The pmCMV / FRT / EGFP vector prepared in Example 1- (2) was treated with BamHI restriction enzyme and CIAP, and the prepared gene fragment was ligated and introduced into Escherichia coli DH5a. The vector was introduced into a solid culture plate containing ampicillin and cultured overnight at 37 ° C. The number of colonies grown on the plate was checked and the colonies were collected in an LB medium flask. Colonies collected in one flask were incubated for 3 hours, and then a vector was obtained using a plasmid midi kit (Qiagen). The obtained sample was named DNA pool and used as a gene fragment library.

Example  2: Performing transduction and cell sorting

The DNA pool prepared in Example 1 was transfected into the Flp-In CHO-K1 cell line, and cell populations expressing various green fluorescent proteins were obtained through selective culture, and a fluorescence activated cell sorter (FACS) The intensity was compared with the control group.

Specifically, the Flp-In CHO-K1 cell line was cultured in RPMI1640 (Gibco) medium containing 10% fetal bovine serum (Gibco), and the introduction of the recombinant vector was carried out using Lipofectamine 2000 (Invitrogen) Respectively. After introducing the recombinant vector, the cells were removed by trypsinization after 48 hours, and the cells were transferred to a selective medium containing hygromycin (Invitrogen) at 10% v / v for 2 weeks. Cells transfected with the pmCMV / FRT / EGFP vector without the inserted gene fragment were also generated in the same manner and used as a control group for the cells of the high incidence group.

As a result of measuring the average value of green fluorescence expressed by each cell group using a fluorescence activated cell sorter (FACS Aria, BD), it was found that the average value of green fluorescence was increased in the Pool 2 cell group as compared with the control group ).

Example  3: Single cell line formation and gene fragment analysis

The pool 2 cell group obtained in Example 2 was cultured as a single cell line and the gene fragment having the vector introduced into each cell was identified.

Specifically, to obtain single cell line, sorted cells were plated at 1 cell / well in a 96-well plate, cultured for 2-3 weeks, and fluorescence of green protein was measured with a microplate reader (Bioteck). Single cell lines showing high fluorescence were selected and cultured until reaching T25 flask. Then, each cell was separated from the flask, and the average value of green fluorescence was obtained using a flow cytometer, thereby comparing the expression of each cell line (FIG. 4).

As shown in FIG. 4, the fluorescence intensities of the 2C5, 2H5, 3B5, 3G11, 4B12, and 5D3 cell lines were more than twice as high as those of the control group. In particular, Which is more than three times. A representative histogram of the cells is shown in Fig.

Genomic DNA was extracted from each cell and used as a template to perform PCR using the primer shown in Table 1 to confirm which gene fragment contains the 1G8 cell line having a high expression level. The primers were prepared by sequencing the BamHI restriction enzyme in front of the CMV promoter of the vector. (SEQ ID NO: 5) and gDNA-R primer (SEQ ID NO: 6) for 1 minute at 95 DEG C, 1 minute at 60 DEG C and 4 minutes at 72 DEG C to confirm the inserted gene sequence PCR amplification was performed 30 times in total.

SEQ ID NO: Name of sequence order 5 gDNA-F GGG CCA GAT ATA CGC GGA TC 6 gDNA-R ATA ATC AAT GTC AAC GGA TC

Sequence analysis of the PCR products revealed that four gene fragments were obtained and sequencing results were analyzed using NCBI and CHOgenome database (www.chogenome.org). As a result of the analysis, E1 has the sequence of SEQ ID NO: 1 (160 bp, NW_003614399.1), E2 has the sequence of SEQ ID NO: 2 (55 bp, NW_003614015.1), E3 has the sequence number 3 (77 bp, NW_003614727.1) 413 bp, NW_003613602.1).

Example  4: Promotion of protein expression enhancement by gene fragmentation

E1 (SEQ ID NO: 1), E2 (SEQ ID NO: 2), E3 (SEQ ID NO: 3) and E4 (SEQ ID NO: 4) gene fragments were used to confirm whether the gene fragment obtained in Example 3 promoted protein expression The inserted vector was constructed and introduced into animal cells to measure the fluorescence of the green protein.

Specifically, the pmCMV / FRT / EGFP vector prepared in Example 1- (2) was treated with BamHI restriction enzyme and CIAP, and the prepared gene fragment was ligated to obtain a vector using plasmid midi kit (Qiagen). The prepared DNA vector was transfected into the Flp-In CHO-K1 cell line. After 48 hours, the cell population expressing the green fluorescent protein was measured for fluorescence intensity through a fluorescence activated cell sorter (FACS) (FIG. 5).

As shown in FIG. 5, it was confirmed that the fluorescence intensities of the vectors inserted with the E1 to E4 gene fragments were higher than those of the control group. The E3 gene fragment inserted vector was increased by about 86% when compared with the control group, and about 108% when the E4 gene inserted vector was increased. Therefore, it was confirmed that the gene fragment of E1 to E4 promoted protein expression.

<110> Korea Research Institute of Bioscience and Biotechnology <120> Recombinant vector for enhancing target gene-expression and use          the <130> P14R16D0767 <160> 6 <170> Kopatentin 2.0 <210> 1 <211> 160 <212> DNA <213> Cricetulus griseus <400> 1 gatctatccc cttcatattt ctcattagaa aacaaacaat cttctaaggg attttaatat 60 ataatataat acaatacaat acaatataat atacactgat aatgatttcc cttccctcta 120 ctccttccag gtcctcccta catcccctcc caccttgatc 160 <210> 2 <211> 55 <212> DNA <213> Cricetulus griseus <400> 2 gatccagagg caatggaatg ttatccaggt aactgaggcc tgtgcagagg gatcc 55 <210> 3 <211> 77 <212> DNA <213> Cricetulus griseus <400> 3 gatcctaata ttagaagggg cggggtagcc agccaatggt ggtgctcacc tttaatccca 60 gcagaggcag gcggatc 77 <210> 4 <211> 413 <212> DNA <213> Cricetulus griseus <400> 4 gatctgtctg agcattttcc tggtgttcac tgggataact tctacttttt agcatggaaa 60 ctgttaactc tgaaaacttt gttctaatag ttacccttag caacagtcac tctgtagatg 120 tcattggcca tagttgacac atcacttcat tgactactca cagtaactat aggtcaggta 180 ttatgaatga gagagtcatt caagttcaaa aagttctttt agaaactact cttaaagcca 240 agataccagc ccaggtgtgt gtctgaggtc cacactcctc tctggaggtc cctagagttc 300 agatttctgg tttctgcctg gacagttgct tccctccgcc ccagtattta ttcacttcta 360 tttgggacct gagagaagcc ggagggccta atgagaccca catgctgagg atc 413 <210> 5 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> gDNA-F <400> 5 gggccagata tacgcggatc 20 <210> 6 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> gDNA-R <400> 6 ataatcaatg tcaacggatc 20

Claims (4)

1. A recombinant vector for increasing expression of a target gene comprising a gene fragment comprising a nucleotide sequence represented by any one of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 and SEQ ID NO: 4,
Wherein the cell whose expression of the target gene is increased is a rodent cell selected from the group consisting of a hamster and a mouse.
2. The recombinant vector according to claim 1, wherein the gene fragment is inserted before the target gene.
1) preparing a recombinant vector for increasing expression according to the first aspect;
2) transforming an animal cell with a recombinant vector for increasing the expression of step 1); And
3) culturing the transformed animal cell of step 2) to obtain a target protein,
Wherein the animal cell is a rodent cell selected from the group consisting of a hamster and a mouse.
[4] The method of claim 3, wherein the animal cell is a CHO (Chinese Hamster Ovary) cell.

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