KR100529281B1 - Expression vector for animal cell containing nuclear matrix attachment region of interferon beta - Google Patents

Abstract

The present invention relates to an animal cell expression vector comprising an interferon beta MAR factor, and includes pPGM-1, pGM-2, and pPGM-3 including a Nuclear Matrix Attachment Region (MAR) factor of human interferon beta genes. It is about. The animal cell expression vector of the present invention increases foreign gene expression and efficiently expresses recombinant protein regardless of the insertion position in the host cell chromosome.

Description

Animal cell expression vector containing interferon beta MAR factor {EXPRESSION VECTOR FOR ANIMAL CELL CONTAINING NUCLEAR MATRIX ATTACHMENT REGION OF INTERFERON BETA}

The present invention relates to an animal cell expression vector comprising an interferon beta MAR factor, and more particularly, pPGM-1 and pPGM- containing a MAR factor of the interferon beta gene (hereinafter referred to as 'MAR'). 2 and pPGM-3.

Various expression systems, such as microorganisms, plants, yeasts, insect cells, animal cells, etc., have been used in order to express and obtain a large amount of the desired protein for use in medicine, industry, and the like. Among them, microorganisms are the most easy to use systems, and have been developed and commercialized as expression systems suitable for various applications.

However, microbial expression systems have some limitations. The biggest limiting factor is that the protein expression and modification mechanisms of microorganisms (glycosylation, phosphorylation, and amidation) are different from those of animal cells. It is not. Therefore, the recombinant protein generation using the microbial expression system is very often inactivated due to little modification after synthesis, but even if there is no significant difference in function, there are many cases of expressing a protein having a partial difference in the modification or structure. In addition, the production process of recombinant proteins using a microbial expression system is cumbersome to be accompanied by the removal of secondary contaminants due to microbial contamination, microbial endotoxin contamination.

On the other hand, although the animal cell expression system is the most suitable system for expressing animal proteins, the expression efficiency of recombinant proteins is lower than that of microbial expression systems, resulting in high production costs, and the difficulty of manipulating animal cells. Currently used animal cell lines include Chinese Hamster Ovary (CHO), Baby Hamster Kidney (BHK), and myeloma (myeloma) .The expression vector containing the gene is transduced into these animal cell lines to target foreign proteins of interest. Expression.

Animal cells maintain various protein modification mechanisms, including glycosylation, and are easier to obtain and purify proteins when secreting proteins into culture medium. While most animal cells require complex additives such as serum proteins during the culturing process, CHO cells can be cultured in a medium without serum and protein, and thus are used as the most suitable host for recombinant protein expression. In addition, CHO cells have been well-characterized by many studies, and have the advantage of fast growth rate and suspension culture for mass culture.

In general, when a foreign gene is to be expressed in an animal cell, the foreign gene and a vector having a marker are simultaneously transfected. Transformed cells are selected by culturing in selective medium. However, their expression frequency is very low in most cases. One reason for this is that in animal cells, unlike microbial systems, these foreign genes must be inserted into chromosomes in the host cell. In addition, even if the foreign gene selects stable transfectants that are stably inserted into the chromosome of the host cell, the expression level is difficult to predict. This is because the insertion position of the gene is different for each cell, and the expression pattern is different according to the insertion position. Therefore, there is no clear correlation between the number of foreign genes inserted in animal cells and gene expression (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 is inhibited by gene expression of the inserted nucleic acid base in the vicinity of the animal cells, stably Even highly 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 availability of nucleic acid factors to protect the expression of foreign genes from such gene location specific effects has been reported in various systems. As the nucleic acid factor, buffer factor and nuclear lattice binding base (MAR) or scaffold attachment region (SAR) are used. Can be. Their mechanism of action has not been elucidated, but when they are included in the transgene construct, they induce gene expression irrespective of the insertion position, and the amount of expression is determined by the number of copies of the gene (McKnight, RA et. al. , 1992, Proc. Natl. Acad. USA.89, 6943-6947).

Carlos et al. Combine the MAR factor of the human apolipoprotein B gene with a minimal promoter transgene construct and induce gene expression in animal cells to increase the expression of the transcript by about 200-fold. (Kalos et al ., 1995, Mol. Cell. Biol . 15, 198-207) Similarly, MAR factor of chicken lysozyme A gene and human interferon beta in vertebrate cells. SAR factors of genes have also been reported to have the property of increasing foreign gene expression regardless of the insertion site in the host cell chromosome (Eissenberg et al. , 1991, Trends Genet . 7, 335-340; Klehr et al. , 1991, Biochemistry 30, 1264-1270) However, these MAR / SAR factors apply to the industrial profitability or practical to try to increase the protein production verification cases are still reported in CHO cell bars The.

Therefore, an object of the present invention is to provide a vector for increasing the expression efficiency of foreign proteins in animal cells.

It is also an object of the present invention to provide a vector expressing a large amount of foreign protein irrespective of the insertion chromosome position of the host cell.

It is also an object of the present invention to provide a vector that increases the frequency and expression of foreign protein expression in animal cells, including the interferon beta MAR factor.

It is also an object of the present invention to provide a vector that is easy for cloning a foreign gene, including a plurality of cloning sites.

It is another object of the present invention to provide a vector capable of expressing a foreign protein in an animal cell.

In order to achieve the above object, the present invention provides an animal cell expression vector comprising a MAR factor (Nuclear matrix attachment region) and promoter of the interferon beta gene.

In another aspect, the present invention provides a cell transformed with the animal cell expression vector introduced with the target gene.

In another aspect, the present invention provides a method for producing a protein expressed from the foreign gene by introducing a foreign gene into the animal cell expression vector, introduced into the animal cell.

Hereinafter, the present invention will be described in detail.

The present inventors have devised an optimal expression vector for overcoming position-specific expression effects and improving gene expression in expression of foreign genes in animal expression systems.

Animal cell expression vector of the present invention is a further addition of the nucleotide sequence useful in the existing expression vector. The above useful sequences protect the expression of foreign genes from the site-specific inhibitory effects of host cell chromosomes in CHO (Chinese hamster ovary) or BHK (Baby hamster kidney) cells, which are commonly used as commercial animal cells. To increase expression. Preferred useful base sequences are Nuclear Matrix Attachment Regions (hereinafter referred to as "MAR") or Scaffold Attachment Regions (hereinafter referred to as "SAR").

The MAR / SAR factor is chicken lysozyme 5 'MAR (Phi-Van, L. and Stratling, WH, 1996, Biochemistry 35, 10735-10742, Gene bank #: X98408), chicken phi alpha globin 5' MAR (Kraevskii , VA et al. , 1992, Mol. Biol . 26, 672-678, Gene bank #: X64113), CHO DHFR intron MAR (Kas, E. and Chasin, LA, 1987, J. Mol. Biol . 198, 677 692, Gene bank #: X06654), human HPRT intron MAR (Sykes, RC et al. , 1988, Mol. Gen. Genet . 212, 301-309, Gene bank #: X07690), human CSP-B gene flanking (flanking) SAR (Hanson, RD and Ley, TJ, Gene bank #: M62716), and human interferon beta gene flanking SAR (Mielke, C. et al. , 1990, Biochemistry 29, 7475-7485, Gene bank #: It is preferable to select from the group which consists of M83137).

The effects of MAR / SAR factors on foreign gene expression by inserting six MAR / SAR factors into pSV-β-gal / ver I or pSV-β-gal / ver II were investigated. Analyzed.

The test vector was completed by inserting six MAR / SAR factors located above the promoter of pSV-β-gal / ver I or pSV-β-gal / ver II, respectively. A transformant was prepared by introducing a test vector into CHO DG44 cells, and the β-gal expression frequency and expression amount were measured.

Recombinant vector pSVβ / I containing interferon beta MAR factor was compared with control pSV-β-gal. The number of positive cell lines increased 6-fold as the number of colonies in G418 selective medium increased by 71% and the incidence of positive cell lines expressing β-gal protein increased to 71% compared to 35% of the control group.

In addition, when the pSVβ / I transformants were selected in DHFR selection medium, 10 times more positive colonies were generated in the initial selection medium without nucleosides. When the MTX was added to the culture medium to induce the amplification of foreign genes, the ratio of β-gal positive colonies of pSVβ / I transformants was 90% or more of the total colonies, and the number of positive cells was about 7 than that of the control group pSV-β-gal. It increased fold (Figure 3).

Therefore, the interferon beta MAR factor of the present invention enhances the expression efficiency of foreign and select genes, greatly enhances the growth of transformed cell lines in the selection medium, and shortens the time required for selection of the transformed cell lines in the selection medium.

The present invention also provides animal cell expression vectors comprising the interferon beta MAR factor on the promoter 5 '. The animal cell expression vector is preferably pPGM-1, pPGM-2 and pPGM-3. The promoter is preferably selected from the group consisting of an SV40 promoter, a cytomegalovirus (CMV) promoter and a Mouse Mammary Tumor Virus (MMTV) promoter.

The pPGM-1 vector is a 5409 bp vector including the MAR factor, the SV40 virus promoter, and the multiple cloning site (MCS) of the human interferon beta gene, and can express various genes by inserting a foreign gene into the multiple cloning site. Many cloning sites include HindIII, NheI, NotI and XhoI cleavage sites. The base sequence of the pPGM-1 vector was prepared as SEQ ID NO: 1 in the Sequence Listing Builder, and the major factors and positions thereof included in the vector are shown in Table 1 and FIG. The pPGM-1 vector was deposited with the Korean Culture Collection of Microorganisms and its accession number is KCCM 10232.

SEQ ID NO: function   1-413 Early promoter and enhancer of SV40 virus 414-448 Multiple Cloning Sites (MCS) 449-584 Small T antigen of SV40 virus 1194-2054 Beta-lactamase (β-lactamase: Amp ^R ) gene 3225-5397 MAR factor of the interferon beta gene

The pPGM-2 vector is a modification of the multiple cloning site of the pPGM-1 vector and includes the interferon beta MAR factor, the SV40 promoter, and the multiple cloning site. The base sequence of the pPGM-2 vector was prepared in SEQ ID NO: 2, and the structure of the vector is shown in FIG. 8 and Table 2. Multiple cloning sites of the pPGM-2 vector include NheI, PmeI, AflII, BamHI, BstXI, EcoRV, BstXI, NotI, XhoI, XbaI, ApaI, PmeI, and MluI cleavage sites. The pPGM-2 vector was deposited with the Korean Culture Collection of Microorganisms and its accession number is KCCM 10338.

SEQ ID NO: function 1-413 SV40 promoter 426-445 T7 promoter 446-579 Multiple Cloning Sites (MCS) 1331-2191 Beta-lactamase (β-lactamase: Amp ^R ) gene 3361-5534 MAR factor of the interferon beta gene

The pPGM-3 vector is composed of 5601bp in which the SV40 promoter of the pPGM-1 vector is replaced with a promoter derived from CMV (Cytomegalovirus). The base sequence is shown in SEQ ID NO: 3, the structure is shown in Figure 9 and Table 3. The pPGM-3 vector was deposited with the Korean Culture Collection of Microorganisms and was assigned accession number KCCM 10339.

SEQ ID NO: function 1-611 SV40 promoter 612-640 Multiple Cloning Sites (MCS) 1396-2246 Beta-lactamase (β-lactamase: Amp ^R ) gene 3417-5589 MAR factor of the interferon beta gene

The pPGM-1 vector, pPGM-2 vector, and pPGM-3 vector of the present invention are all excellent in the number, frequency, and expression of positive cells expressing foreign proteins as compared with the conventional vector. Therefore, the pPGM-1 vector, pPGM-2 vector and pPGM-3 vector of the present invention can be used for expressing and producing foreign proteins in animal cells, and can produce recombinant proteins such as enzymes and cytokines.

In another aspect, the present invention provides a transformant prepared by introducing a foreign gene into a vector containing an interferon beta MAR factor, and introduced into the animal cell. The transformant was selected from the medium to which the selection marker or MTX was added. Foreign genes are genes that encode all kinds of proteins that can be expressed as recombinant proteins. Representative examples include insulin, cytokines (interleukin, tumor necrosis factor, interferon, colony stimulator, chemokine, etc.), erythropoietin, and the like. Transformants can be carried out by conventional methods.

In another aspect, the present invention provides a method for introducing a foreign gene into a vector containing an interferon beta MAR factor, and introducing it into an animal cell to produce a protein expressed from the foreign gene. The vector is preferably selected from the group consisting of pPGM-1 (KCCM 10232), pPGM-2 (KCCM 10338) and pPGM-1 (KCCM 10339). The animal cells refer to all kinds of cells derived from animals, most preferably Chinese hamster ovary (CHO).

Hereinafter, preferred examples are provided to aid in understanding the present invention. However, the following examples are provided only to more easily understand the present invention, and the present invention is not limited to the following examples.

Example 1: Cloning of MAR or SAR Factors

Genomic DNA was isolated from human G-2 cells using a DNA Genomic DNA purification kit (Promega USA). 10 u g of DNA was digested with Cla I, Sma I, Xba I, Xho I restriction enzymes, and then used for PCR. In addition, CHO cell lines, chicken embryos (chickem embryos) were purified through DNA similar to those described above for PCR.

200 ng of the isolated genomic nucleic acid was used as a template, and each polymerase chain reaction (PCR) was added by adding 25 pmole of each primer, 0.5 mM dNTP, and ExTaq DNA polymerase (Takara Shuzo Co., Japan). Was carried out. The base sequence of the primers used for each MAR / SAR factor and the size of the DNA fragments obtained by PCR are shown in Table 4 below. Polymerase chain reaction was performed using GeneAmp PCR system 9600 (Perkin-Elmer Corp, USA), and PCR conditions are shown in Table 5 below.

 MSR / SAR Factor primer SEQ ID NO:  size Chicken lysozyme 5 'MAR 5'- GGA TCC ATA ATA TAA CTG TA -3'5'- AAG CTT AAA AGA TTG AAG CA -3 ' 4 1668 bp 5 Chicken phi alpha globin 5 'MAR 5'- AAG CTT TTA ACC AAC AAA AA -3 '5'- CTG CAG ACC TAA CCT GTC AC -3' 6 619 bp 7 CHO DHFR Intron MAR 5'- TAT ACG TGA ATA GTT TTT CT -3'5'- GAG TTG GAA CTG AGA AGT TC -3 ' 8 549 bp 9 Human HPRT Intron MAR 5'- AAG CTT GGT CAA GAA TGC TG -3'5'- GCT GGG CGT GGT GGT GCC TG -3 ' 10 580 bp 11 Human CSP-B Gene SAR 5'- GGA TCC CAT TCT CCT TGA TG -3 '5'- GAA TTC AAA CAA CTC AAT AG -3' 12 1233 bp 13 Human Interferon Beta SAR 5'- GAA TTC AGC AAG GTC GCC AC -3'5'- TTG TAT CAA CTT TCT ACA AT -3 ' 14 2174 bp 15

step Condition Number of cycles One 94 ^o C, 2 minutes One 2 94 ^° C., 40 seconds; 65 ^° C., 40 seconds; 72 ^o C, 40 seconds 2-31 3 72 ^o C, 10 minutes 32

Chicken liasozyme 5 'MAR (Gene bank #: X98408, hereinafter referred to as "lyso MAR"), chicken phi alpha globin 5' MAR (Gene bank #: X64113, hereinafter referred to as "phi-a MAR") ), CHO DHFR intron MAR (Gene bank #: X06654, hereinafter referred to as "DHFR MAR"), human HPRT intron MAR (Gene bank #: X07690, hereinafter referred to as "HPRT MAR"), human CSP-B gene flanking SAR (Gene bank #: M62716, hereinafter referred to as "CSP-B MAR"), and human interferon beta gene flanking SAR (Gene bank #: M83137, hereinafter referred to as "interferon beta MAR") gene fragments were isolated.

PCR products were subcloned into either pT7blue (R) (Novagene, USA) or pCR 2.1 (Invitorgen, USA) vectors. HPRT MAR, DHFR MAR and lyso MAR were subcloned in pT7blue (R) vector, phi-a MAR, CSP-B MAR and interferon beta MAR were subcloned in pCR 2.1.

Example 2: Preparation of pSV-β-gal / ver I and pSV-β-gal / ver II Vectors

The vector pSV- modified to efficiently clone a number of MAR / SAR factors subcloned into pT7blue (R) or pCR2.1 vector above the SV40 promoter of the pSV-β-gal (referred to as 'pSVβ') vector. β-gal / ver I and pSV-β-gal / ver II were prepared (FIG. 1).

(1) pSV-β-gal / verI preparation

PCR was carried out using primers of SEQ ID NO: 16 and SEQ ID NO: 17 with pSVβ as a template, to obtain a fragment containing the SV40 promoter. The fragments were treated with the Spe I and Hin dIII enzymes, and then 443 bp DNA fragments containing the SV40 promoter were purified with a JinClean III kit (Bio 101, USA). PBS / SV40 I was prepared by subcloning it into pBluescript SK (+) vector (Stratagene, USA) which was ring-opened with Spe I and Hin dIII enzymes.

pBS / SV40 I was digested with Sca I and Hin dIII enzymes to isolate DNA fragments of 1240 bp and subcloned into pSVβ treated with the same enzyme to complete the pSV-β-gal / verI vector.

(2) Preparation of pSV-β-gal / verII Vector

pSVβ was treated with Eco RI and Hin dIII to recover the 420 bp fragment in the same manner as above, and then pBS / SV40 II was prepared by inserting and connecting to the pBluescript SK (+) vector opened with the same enzyme.

pBS / SV40 II was treated with restriction enzymes Sca I and Hin dIII to insert a fragment containing the SV40 promoter into the pSVβ vector treated with the same restriction enzyme to prepare a pSV-β-gal / verII vector.

Example 3 Preparation of a Vector Containing MAR Factor and β-gal Gene

MAR factors in pT7blue (R) / HPRT MAR, pT7blue (R) / DHFR MAR, pT7blue (R) / lyso MAR, pCR 2.1 / phi-a MAR, pCR 2.1 / CSP-B MAR, and pCR 2.1 / interferon beta MAR It was isolated and cloned into pSVβ / I or pSVβ / II.

phi-a MAR and HPRT MAR (human HPRT intron MAR) for pSVβ / I were subcloned using Spe I / Sma I, and interferon beta MAR and lyso MAR using Apa I / Spe I. The vectors produced were pSV-β-gal / phi-a MAR, pSV-β-gal / HPRT MAR, pSV-β-gal / interferon beta MAR (hereinafter referred to as 'pSVβI') and pSV-β-gal / lyso MAR to be.

In addition, pSV-β-gal / verII DHFR MAR and CSP-B MAR were subcloned using Bam HI / Xba I. The produced vectors are pSV-β-gal / DHFR MAR and pSV-β-gal / CSP-B MAR, respectively.

Example 4: Preparation of pCMVβ and pCMVβ / I Vectors

Spe I- Xho I fragments containing MAR factors were removed from the pSVβ / I vector and self-ligation again. After treatment with Apa I and Hin dIII, a CMV promoter fragment was inserted to prepare a pCMVβ vector. The CMV promoter fragment was PCR by primers SEQ ID NO: 18 and SEQ ID NO: 19 in the pcDNA3.1 vector. SEQ ID NOs: 18 and 19 include Apa I or Hin dIII recognition sequences.

In addition, pSVβ / I vector was treated with Apa I and Hin dIII enzyme, and a CMV promoter fragment was inserted therein to prepare a pCMVβ / I vector.

That is, pCMVβ is a vector without MAR factor, and pCMVβ / I is a vector with MAR factor.

Example 5 Measurement of β-gal Gene Expression of pSVβ and pSVβ / I

(1) transfection

CHO cell line DG44 lacking the DHFR gene was cultured in MEM-α medium (α-Minimum Essential Medium, Gibco BRL) to which 10% serum (fetal bovine serum) was added. 2 × 10 ⁵ cells were inoculated in 60 mm plates with 3 ml of medium and incubated overnight at 37 ° C., 5% CO ₂ incubator until transduction.

Transduction was performed according to the liposome method. PSV2Neo or pDCH1P vector having the selected gene was co-transduced with each test vector in a molar ratio of 100: 1, and the same experiment was performed using the pSVβ vector as a control. 2 u g of each vector was mixed with DOSPER (Boehringer Mannheim, Germany), a serum-free MEM-α medium, and reacted at room temperature for 45 minutes, and then incubated for 5 hours by adding the medium to the washed cells. The reaction solution was removed. Transformants using Neo genes were cultured in MEM-α medium containing 10% FBS and G418 (500 u g / ml) for 2 weeks, and MEM-α without nucleosides was selected for DHFR gene selection. 2 weeks of culture in the medium was analyzed for β-gal expression.

(2) Frequency measurement of β-gal expressing positive cell line

Positive cells expressing β-gal were selected.

Cells incubated in a 100 mm plate were treated with fixative (2% formaldehyde and 0.2% glutaraldehyde) at 4 ° C. for 10 minutes. Washed twice with 1 × PBS and reacted at 37 ° C. with the addition of X-gal (1 mg / ml).

β-gal expressing cells can be easily identified since they break down X-gal and become blue. After separating positive cells with Trypsin-EDTA solution, the number of cells was measured by a hematocytometer.

Figure 2 is a photograph of X-gal treatment to identify cells expressing β-gal after transfection with pSVβ or pSVβ / I. (a) is a cell transfected with pSVβ and (b) is a cell transfected with pSVβ / I. In the upper plate photograph, blue positive cell line was observed in pSVβ / I transformant.

In addition, after transfection, transformants were selected using Neo gene or DHFR gene, and positive cells expressing β-gal were measured.

Table 6 shows the number of β-gal positive cells of DG44 / pSVβ or DG44 / pSVβ / I selected using Neo selectors. Table 7 shows the number of β-gal positive cells of DG44 / pSVβ or DG44 / pSVβ / I selected using the DHFR selector.

Total cell count Positive cell count Negative cell count Positive cell frequency (%) DG44 / pSVβ 222 145 77 34.68 DG44 / pSVβ / I 659 191 468 71.02

Total cell count Positive cell count Negative cell count Positive cell frequency (%) DG44 / pSVβ 120 105 15 12.50 DG44 / pSVβ / I 350 200 150 42.86

When selected as a selection factor, the total number of colonies and the frequency of positive colonies increased simultaneously, especially the number of positive colonies significantly increased.

Transformants (DG44 / pSVβ, DG44 / pSVβ / I) were also adapted to medium containing MTX to induce gene amplification. Β-gal expression frequency of transformed strains adapted to MTX 10 nM and 50 nM was measured. 3 is a graph measuring β-gal activity of transformed strains adapted to MTX. More than 90% of the DG44 / pSVβ / I transformants selected from MTX expressed β-gal, producing 7-fold more positive colonies than the control (pSVβ).

Therefore, the interferon beta MAR factor further enhanced the expression activity of the SV40 promoter and increased the frequency of positive cell lines 6-10 times. Therefore, transgenic cell lines have to be selected from the selection medium for a very long time in a conventional transgenic line production process. However, the use of the MAR factor is expected to significantly reduce the time required for the development of transformed cell lines. In addition, the MAR factor simultaneously affects the expression of not only the target gene but also the select gene, resulting in greatly enhanced growth of recombinant cell lines in the selection medium.

(3) β-gal activity of DG44 / pSVβ and DG44 / pSVβ / I

Β-gal activity of the same cells used in measuring the frequency of the β-gal expressing positive cell line was measured.

Transformants were incubated for 48 hours and washed twice with 1 × PBS. 1 ml of STE (0.1 M NaCl, 10 mM Tris-Cl pH 8.0, 1 mM EDTA pH 8.0) solution was added and placed on ice. The cells were scraped off with a scraper, transferred to an Eppendorf tube, centrifuged at 4 ° C., 14000 rpm, for 40 seconds, the supernatant was removed, and 100 ul of 0.25 M Tris-Cl (pH 7.5) was added and mixed with the cells. It was frozen five times in liquid nitrogen and immediately dissolved at 37 ° C. The pulverized cells were centrifuged at 12000 rpm for 4 minutes for 10 minutes and the supernatant was transferred to a new tube for analysis.

Cell extract 30 ul, 100 × magnesium solution (0.1 M MgCl ₂ , 4.5 M β-mercaptoethanol) 3 ul, 1 × ONPG (4 ml / ml in 0.1 M sodium phospate (pH 7.5)) 66 ul , 0.1 M sodium phosphate pH 7.5) After mixing 201 ul well, the reaction was continued until it turned yellow at 37 ° C. 500 ul 1 M Na ₂ CO ₃ After addition, absorbance was measured at 420 nm.

The amount of protein in the solution was measured according to the bicinchoninic acid (BCA) method. (Smith et al. , 1985, Anal. Biochem. 150, 76-85)

4 is a graph measuring β-gal activity of DG44 / pSVβ or DG44 / pSVβ / I screened using Neo selector. 5 is a graph measuring the β-gal activity of DG44 / pSVβ or DG44 / pSVβ / I selected using DHFR selector.

Β-gal activity was increased by 7.5-fold (FIG. 4) and 32-fold (FIG. 5), respectively, when MAR factor was present in the expression vector. This is a significant increase in β-gal protein activity expressed per positive cell considering the frequency of positive cells.

Example 6: Measurement of β-gal Expression of pCMVβ and pCMVβ / I

PCMVβ and pCMVβ / I were transfected into DG44 in the same manner as in Example 5, and then β-gal positive cell lines and activities were measured.

6 is a graph showing β-gal activity of DG44 / pCMVβ and DG44 / pCMVβ / I, and Table 8 shows the β-gal positive cell numbers of DG44 / pCMVβ and DG44 / pCMVβ / I.

Total cell count Positive cell count Negative cell count Positive cell frequency (%) DG44 / pCMVβ 180 147 33 18.33 DG44 / pCMVβ / I 326 79 247 75.77

The MAR factor increased the frequency of positive cell lines from 18.33% to 75.77% even under the CMV promoter. The total colony count increased by 1.8 times and positive colony count increased by 7.5 times. Thus, the effect of the interferon beta MAR factor is not limited to the use of the SV40 promoter, it indicates that the MAR factor of the present invention can be used in various combinations with the SV40, CMV promoter and other such promoters.

Example 7: Preparation of pPGM-1 Vector

A pPGM-1 vector having the genetic map of FIG. 7 was prepared by removing the β-gal gene region from the pSVβ / I vector and introducing a multiple cloning region (MCS).

The Hin dIII- Bam HI fragment containing the β-gal gene region of the pSVβ / I vector was removed and a 160 bp Hin dIII / Bam HI fragment of pMSG (KCCM 10202) was inserted. The pPGM-1 vector was deposited with the Korea Microorganism Conservation Center and assigned accession number KCCM 10232.

Example 8: Preparation of pPGM-2 Vector

pPGM-2 vectors were prepared by complementing multiple cloning sites of the pPGM-1 vector.

The Hin dIII- Bam HI fragment of the pSVβ vector was replaced with the corresponding fragment from pMSG (KCCM 10202) and ring-opened with the Eco RI enzyme to link the interferon MAR factor. In addition, Nhe I and Xho I enzymes were processed, and fragments including the T7 promoter and multiple cloning sites were inserted therein. The fragment was constructed using PCR primers (SEQ ID NO: 20 and SEQ ID NO: 21) containing the recognition sequences of Avr II and Sal I restriction enzymes.

8 shows the structure of the pPGM-2 vector and the nucleotide sequence of the multiple cloning site. The vector was deposited at the Korea Microorganism Conservation Center and received accession number KCCM 10338.

Example 9: Preparation of pPGM-3 Vector

Hin dIII- Bam HI fragment of the pSVβ vector was replaced with the corresponding fragment from pMSG (KCCM 10202). After treatment with EcoRI and NheI, the SV40 promoter was removed, the site was replaced with a CMV promoter, and then the ring was opened with Eco RI enzyme to insert the interferon MAR factor. The structure of the pPGM-3 expression vector prepared as above is shown in Figure 9, the accession number is KCCM 10339.

Example 10 Recombinant Protein Production Using pPGM-1 Vector

pPGM-1 vectors were treated with NheI and XhoI enzymes to link the insertion genes. The gene is a cDNA encoding human growth hormone (Gene Bank #: E01424) or human interferon beta (Gene Bank #: V00534).

The expression vector prepared as described above was transduced into CHO DG44 cells and high expressing transformed cell lines were selected while increasing the MTX concentration added to the medium. Expression titers were determined by Western blot as shown in FIGS. 10 and 11.

1.08 X 10 ⁵ cells were placed in a 12-well plate, incubated for 48 hours, and the medium was recovered to perform Western blot. Lane 1 is 25 ng positive control protein and lanes 2-5 are cultures of different clones selected. The expression rate of human growth hormone was about 20 μg / ml / day, and the expression rate of interferon beta was about 15 μg / ml / day.

As mentioned above, the pPGM-1 vector, pPGM-2 vector, and pPGM-3 vector of the present invention improve the expression suppression of foreign genes by peripheral bases of host cells and increase expression. In addition, the expression vector of the present invention can be effectively applied to the production of a large amount of industrially useful protein, it is possible to produce a recombinant protein having the same structure and function as the original protein of higher animals.

1 illustrates the structure of a vector prepared by modifying a pSV-β-gal vector to prepare an expression vector of the present invention.

Figure 2 is a positive cell line increase by the interferon beta MAR factor observed by β-gal staining method,

3 shows the frequency of the positive cell lines expressing β-gal upon amplification of the transgene by MTX addition,

Figure 4 is a measure of the frequency and expression of β-gal expression in G418 selection medium,

5 is a measurement of β-gal expression frequency and expression amount in DHFR selection medium,

Figure 6 is a measure of the expression frequency and β-gal expression in the presence of the CMV promoter,

Figure 7 shows the structure of the pPGM-1 expression vector of the present invention,

Figure 8 shows the structure of the pPGM-2 expression vector of the present invention and the nucleotide sequence of the multiple cloning site,

Figure 9 shows the structure of the pPGM-3 expression vector of the present invention,

10 is a Western blot analysis of the expression titers of human growth hormone-expressing cell lines prepared using pPGM-1,

11 is a Western blot analysis of the expression titers of interferon beta-expressing cell lines prepared using pPGM-1.

<110> PAN-GEN BIOTECH LABORATORIES INC. <120> EXPRESSION VECTOR FOR ANIMAL CELL CONTAINING NUCLEAR MATRIX ATTACHMENT REGION OF INTERFERON BETA <150> KR 10-2000-77279 <151> 2001-12-15 <160> 21 <170> KopatentIn 1.71 <210> 1 <211> 5409 <212> DNA <213> Artificial Sequence <220> <223> pPGM-1 vector <220> <221> promoter (222) (1) .. (419) <223> SV40 virus <220> <221> gene 222 (449) .. (584) <223> small T antigen of SV40 virus <220> <221> gene (222) (1194) .. (2054) <223> beta-lactamase <220> <221> gene (222) (3225) .. (5397) <223> interferon beta MAR <400> 1 gcgcagcacc atggcctgaa ataacctctg aaagaggaac ttggttaggt accttctgag 60 gcggaaagaa ccagctgtgg aatgtgtgtc agttagggtg tggaaagtcc ccaggctccc 120 cagcaggcag aagtatgcaa agcatgcatc tcaattagtc agcaaccagg tgtggaaagt 180 ccccaggctc cccagcaggc agaagtatgc aaagcatgca tctcaattag tcagcaacca 240 tagtcccgcc cctaactccg cccatcccgc ccctaactcc gcccagttcc gcccattctc 300 cgccccatgg ctgactaatt ttttttattt atgcagaggc cgaggccgcc tcggcctctg 360 agctattcca gaagtagtga ggaggctttt ttggaggcct aggcttttgc aaaaagcttg 420 ctagcggccg cagatctgtt aactcgagaa cttgtttatt gcagcttata atggttacaa 480 ataaagcaat agcatcacaa atttcacaaa taaagcattt ttttcactgc attctagttg 540 tggtttgtcc aaactcatca atgtatctta tcatgtctgg atcggcatgc aagctggcac 600 tggccgtcgt tttacaacgt cgtgactggg aaaaccctgg cgttacccaa cttaatcgcc 660 ttgcagcaca tccccctttc gccagctggc gtaatagcga agaggcccgc accgatcgcc 720 cttcccaaca gttgcgcagc ctgaatggcg aatggcgcct gatgcggtat tttctcctta 780 cgcatctgtg cggtatttca caccgcatat ggtgcactct cagtacaatc tgctctgatg 840 ccgcatagtt aagccagccc cgacacccgc caacacccgc tgacgcgccc tgacgggctt 900 gtctgctccc ggcatccgct tacagacaag ctgtgaccgt ctccgggagc tgcatgtgtc 960 agaggttttc accgtcatca ccgaaacgcg cgagacgaaa gggcctcgtg atacgcctat 1020 ttttataggt taatgtcatg ataataatgg tttcttagac gtcaggtggc acttttcggg 1080 gaaatgtgcg cggaacccct atttgtttat ttttctaaat acattcaaat atgtatccgc 1140 tcatgagaca ataaccctga taaatgcttc aataatattg aaaaaggaag agtatgagta 1200 ttcaacattt ccgtgtcgcc cttattccct tttttgcggc attttgcctt cctgtttttg 1260 ctcacccaga aacgctggtg aaagtaaaag atgctgaaga tcagttgggt gcacgagtgg 1320 gttacatcga actggatctc aacagcggta agatccttga gagttttcgc cccgaagaac 1380 gttttccaat gatgagcact tttaaagttc tgctatgtgg cgcggtatta tcccgtattg 1440 acgccgggca agagcaactc ggtcgccgca tacactattc tcagaatgac ttggttgagt 1500 actcaccagt cacagaaaag catcttacgg atggcatgac agtaagagaa ttatgcagtg 1560 ctgccataac catgagtgat aacactgcgg ccaacttact tctgacaacg atcggaggac 1620 cgaaggagct aaccgctttt ttgcacaaca tgggggatca tgtaactcgc cttgatcgtt 1680 gggaaccgga gctgaatgaa gccataccaa acgacgagcg tgacaccacg atgcctgtag 1740 caatggcaac aacgttgcgc aaactattaa ctggcgaact acttactcta gcttcccggc 1800 aacaattaat agactggatg gaggcggata aagttgcagg accacttctg cgctcggccc 1860 ttccggctgg ctggtttatt gctgataaat ctggagccgg tgagcgtggg tctcgcggta 1920 tcattgcagc actggggcca gatggtaagc cctcccgtat cgtagttatc tacacgacgg 1980 ggagtcaggc aactatggat gaacgaaata gacagatcgc tgagataggt gcctcactga 2040 ttaagcattg gtaactgtca gaccaagttt actcatatat actttagatt gatttaaaac 2100 ttcattttta atttaaaagg atctaggtga agatcctttt tgataatctc atgaccaaaa 2160 tcccttaacg tgagttttcg ttccactgag cgtcagaccc cgtagaaaag atcaaaggat 2220 cttcttgaga tccttttttt ctgcgcgtaa tctgctgctt gcaaacaaaa aaaccaccgc 2280 taccagcggt ggtttgtttg ccggatcaag agctaccaac tctttttccg aaggtaactg 2340 gcttcagcag agcgcagata ccaaatactg ttcttctagt gtagccgtag ttaggccacc 2400 acttcaagaa ctctgtagca ccgcctacat acctcgctct gctaatcctg ttaccagtgg 2460 ctgctgccag tggcgataag tcgtgtctta ccgggttgga ctcaagacga tagttaccgg 2520 ataaggcgca gcggtcgggc tgaacggggg gttcgtgcac acagcccagc ttggagcgaa 2580 cgacctacac cgaactgaga tacctacagc gtgagctatg agaaagcgcc acgcttcccg 2640 aagggagaaa ggcggacagg tatccggtaa gcggcagggt cggaacagga gagcgcacga 2700 gggagcttcc agggggaaac gcctggtatc tttatagtcc tgtcgggttt cgccacctct 2760 gacttgagcg tcgatttttg tgatgctcgt caggggggcg gagcctatgg aaaaacgcca 2820 gcaacgcggc ctttttacgg ttcctggcct tttgctggcc ttttgctcac atgttctttc 2880 ctgcgttatc ccctgattct gtggataacc gtattaccgc ctttgagtga gctgataccg 2940 ctcgccgcag ccgaacgacc gagcgcagcg agtcagtgag cgaggaagcg gaagagcgcc 3000 caatacgcaa accgcctctc cccgcgcgtt ggccgattca ttaatgcagc tggcacgaca 3060 ggtttcccga ctggaaagcg ggcagtgagc gcaacgcaat taatgtgagt tagctcactc 3120 attaggcacc ccaggcttta cactttatgc ttccggctcg tatgttgtgt ggaattgtga 3180 gcggataaca atttcacaca ggaaacagct atgacatgat tacgaattca gcaaggtcgc 3240 cacgcacaag atcaatatta acaatcagtc atctctcttt agcaataaaa aggtgaaaaa 3300 ttacatttta aaaatgacac catagacgat gtatgaaaat aatctacttg gaaataaatc 3360 taggcaaaga agtgcaagac tgttacccag aaaacttaca aattgtaaat gagaggttag 3420 tgaagattta aatgaatgaa gatctaaata aacttataaa ttgtgagaga aattaatgaa 3480 tgtctaagtt aatgcagaaa cggagagaca tactatattc atgaactaaa agacttaata 3540 ttgtgaaggt atactttctt ttcacataaa tttgtagtca atatgttcac cccaaaaaag 3600 ctgtttgtta acttgtcaac ctcatttcaa aatgtatata gaaagcccaa agacaataac 3660 aaaaatattc ttgtagaaca aaatgggaaa gaatgttcca ctaaatatca agatttagag 3720 caaagcatga gatgtgtggg gatagacagt gaggctgata aaatagagta gagctcagaa 3780 acagacccat tgatatatgt aagtgaccta tgaaaaaaat atggcatttt acaatgggaa 3840 aatgatgatc tttttctttt ttagaaaaac agggaaatat atttatatgt aaaaaataaa 3900 agggaaccca tatgtcatac catacacaca aaaaaattcc agtgaattat aagtctaaat 3960 ggagaaggca aaactttaaa tcttttagaa aataatatag aagcatgcca tcatgacttc 4020 agtgtagaga aaaatttctt atgactcaaa gtcctaacca caaagaaaag attgttaatt 4080 agattgcatg aatattaaga cttattttta aaattaaaaa accattaaga aaagtcaggc 4140 catagaatga cagaaaatat ttgcaacacc ccagtaaaga gaattgtaat atgcagatta 4200 taaaaagaag tcttacaaat cagtaaaaaa taaaactaga caaaaatttg aacagatgaa 4260 agagaaactc taaataatca ttacacatga gaaactcaat ctcagaaatc agagaactat 4320 cattgcatat acactaaatt agagaaatat taaaaggcta agtaacatct gtggcaatat 4380 tgatggtata taaccttgat atgatgtgat gagaacagta ctttacccca tgggcttcct 4440 ccccaaaccc ttaccccagt ataaatcatg acaaatatac tttaaaaacc attaccctat 4500 atctaaccag tactcctcaa aactgtcaag gtcatcaaaa ataagaaaag tctgaggaac 4560 tgtcaaaact aagaggaacc caaggagaca tgagaattat atgtaatgtg gcattctgaa 4620 tgagatccca gaacagaaaa agaacagtag ctaaaaaact aatgaaatat aaataaagtt 4680 tgaactttag ttttttttaa aaaagagtag cattaacacg gcaaagtcat tttcatattt 4740 ttcttgaaca ttaagtacaa gtctataatt aaaaattttt taaatgtagt ctggaacatt 4800 gccagaaaca gaagtacagc agctatctgt gctgtcgcct aactatccat agctgattgg 4860 tctaaaatga gatacatcaa cgctcctcca tgttttttgt tttcttttta aatgaaaaac 4920 tttatttttt aagaggagtt tcaggttcat agcaaaattg agaggaaggt acattcaagc 4980 tgaggaagtt ttcctctatt cctagtttac tgagagattg catcatgaat gggtgttaaa 5040 ttttgtcaaa tgctttttct gtgtctatca atatgaccgt gtgattttct tctttaacct 5100 gttgatggga caaattacgt taattgattt tcaaacgttg aaccaccctt acatatctgg 5160 aataaattct acttggttgt ggtgtatatt ttttgataca ttcttggatt ctttttgcta 5220 atattttgtt gaaaatgttt gtatctttgt tcatgagaga tattggtctg ttgttttctt 5280 ttcttgtaat gtcattttct agttccggta ttaaggtaat gctggcctag ttgaatgatt 5340 taggaagtat tccctctgct tctgtcttct gaaagagatt gtagaaagtt gatacaaaag 5400 ccgaattcg 5409 <210> 2 <211> 5546 <212> DNA <213> Artificial Sequence <220> <223> pPGM-2 vector <220> <221> promoter (222) (1) .. (419) <223> SV40 virus <220> <221> primer_bind <222> (426) .. (445) <223> T7 promoter <220> <221> gene (222) (1331) .. (2191) <223> beta-lactamase gene <220> <221> gene <222> (3361) .. (5534) <223> Interferon beta MAR elemen <400> 2 gcgcagcacc atggcctgaa ataacctctg aaagaggaac ttggttaggt accttctgag 60 gcggaaagaa ccagctgtgg aatgtgtgtc agttagggtg tggaaagtcc ccaggctccc 120 cagcaggcag aagtatgcaa agcatgcatc tcaattagtc agcaaccagg tgtggaaagt 180 ccccaggctc cccagcaggc agaagtatgc aaagcatgca tctcaattag tcagcaacca 240 tagtcccgcc cctaactccg cccatcccgc ccctaactcc gcccagttcc gcccattctc 300 cgccccatgg ctgactaatt ttttttattt atgcagaggc cgaggccgcc tcggcctctg 360 agctattcca gaagtagtga ggaggctttt ttggaggcct aggcttttgc aaaaagcttg 420 ctaggtaata cgactcacta tagggagacc caagctggct agcgtttaaa cttaagcttg 480 gtaccgagct cggatccact agtccagtgt ggtggaattc tgcagatatc cagcacagtg 540 gcggccgctc gagtctagag ggcccgttta aacacgcgtg tcgagaactt gtttattgca 600 gcttataatg gttacaaata aagcaatagc atcacaaatt tcacaaataa agcatttttt 660 tcactgcatt ctagttgtgg tttgtccaaa ctcatcaatg tatcttatca tgtctggatc 720 ggcatgcaag ctggcactgg ccgtcgtttt acaacgtcgt gactgggaaa accctggcgt 780 tacccaactt aatcgccttg cagcacatcc ccctttcgcc agctggcgta atagcgaaga 840 ggcccgcacc gatcgccctt cccaacagtt gcgcagcctg aatggcgaat ggcgcctgat 900 gcggtatttt ctccttacgc atctgtgcgg tatttcacac cgcatatggt gcactctcag 960 tacaatctgc tctgatgccg catagttaag ccagccccga cacccgccaa cacccgctga 1020 cgcgccctga cgggcttgtc tgctcccggc atccgcttac agacaagctg tgaccgtctc 1080 cgggagctgc atgtgtcaga ggttttcacc gtcatcaccg aaacgcgcga gacgaaaggg 1140 cctcgtgata cgcctatttt tataggttaa tgtcatgata ataatggttt cttagacgtc 1200 aggtggcact tttcggggaa atgtgcgcgg aacccctatt tgtttatttt tctaaataca 1260 ttcaaatatg tatccgctca tgagacaata accctgataa atgcttcaat aatattgaaa 1320 aaggaagagt atgagtattc aacatttccg tgtcgccctt attccctttt ttgcggcatt 1380 ttgccttcct gtttttgctc acccagaaac gctggtgaaa gtaaaagatg ctgaagatca 1440 gttgggtgca cgagtgggtt acatcgaact ggatctcaac agcggtaaga tccttgagag 1500 ttttcgcccc gaagaacgtt ttccaatgat gagcactttt aaagttctgc tatgtggcgc 1560 ggtattatcc cgtattgacg ccgggcaaga gcaactcggt cgccgcatac actattctca 1620 gaatgacttg gttgagtact caccagtcac agaaaagcat cttacggatg gcatgacagt 1680 aagagaatta tgcagtgctg ccataaccat gagtgataac actgcggcca acttacttct 1740 gacaacgatc ggaggaccga aggagctaac cgcttttttg cacaacatgg gggatcatgt 1800 aactcgcctt gatcgttggg aaccggagct gaatgaagcc ataccaaacg acgagcgtga 1860 caccacgatg cctgtagcaa tggcaacaac gttgcgcaaa ctattaactg gcgaactact 1920 tactctagct tcccggcaac aattaataga ctggatggag gcggataaag ttgcaggacc 1980 acttctgcgc tcggcccttc cggctggctg gtttattgct gataaatctg gagccggtga 2040 gcgtgggtct cgcggtatca ttgcagcact ggggccagat ggtaagccct cccgtatcgt 2100 agttatctac acgacgggga gtcaggcaac tatggatgaa cgaaatagac agatcgctga 2160 gataggtgcc tcactgatta agcattggta actgtcagac caagtttact catatatact 2220 ttagattgat ttaaaacttc atttttaatt taaaaggatc taggtgaaga tcctttttga 2280 taatctcatg accaaaatcc cttaacgtga gttttcgttc cactgagcgt cagaccccgt 2340 agaaaagatc aaaggatctt cttgagatcc tttttttctg cgcgtaatct gctgcttgca 2400 aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg gatcaagagc taccaactct 2460 ttttccgaag gtaactggct tcagcagagc gcagatacca aatactgttc ttctagtgta 2520 gccgtagtta ggccaccact tcaagaactc tgtagcaccg cctacatacc tcgctctgct 2580 aatcctgtta ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg ggttggactc 2640 aagacgatag ttaccggata aggcgcagcg gtcgggctga acggggggtt cgtgcacaca 2700 gcccagcttg gagcgaacga cctacaccga actgagatac ctacagcgtg agctatgaga 2760 aagcgccacg cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg gcagggtcgg 2820 aacaggagag cgcacgaggg agcttccagg gggaaacgcc tggtatcttt atagtcctgt 2880 cgggtttcgc cacctctgac ttgagcgtcg atttttgtga tgctcgtcag gggggcggag 2940 cctatggaaa aacgccagca acgcggcctt tttacggttc ctggcctttt gctggccttt 3000 tgctcacatg ttctttcctg cgttatcccc tgattctgtg gataaccgta ttaccgcctt 3060 tgagtgagct gataccgctc gccgcagccg aacgaccgag cgcagcgagt cagtgagcga 3120 ggaagcggaa gagcgcccaa tacgcaaacc gcctctcccc gcgcgttggc cgattcatta 3180 atgcagctgg cacgacaggt ttcccgactg gaaagcgggc agtgagcgca acgcaattaa 3240 tgtgagttag ctcactcatt aggcacccca ggctttacac tttatgcttc cggctcgtat 3300 gttgtgtgga attgtgagcg gataacaatt tcacacagga aacagctatg acatgattac 3360 gaattcagca aggtcgccac gcacaagatc aatattaaca atcagtcatc tctctttagc 3420 aataaaaagg tgaaaaatta cattttaaaa atgacaccat agacgatgta tgaaaataat 3480 ctacttggaa ataaatctag gcaaagaagt gcaagactgt tacccagaaa acttacaaat 3540 tgtaaatgag aggttagtga agatttaaat gaatgaagat ctaaataaac ttataaattg 3600 tgagagaaat taatgaatgt ctaagttaat gcagaaacgg agagacatac tatattcatg 3660 aactaaaaga cttaatattg tgaaggtata ctttcttttc acataaattt gtagtcaata 3720 tgttcacccc aaaaaagctg tttgttaact tgtcaacctc atttcaaaat gtatatagaa 3780 agcccaaaga caataacaaa aatattcttg tagaacaaaa tgggaaagaa tgttccacta 3840 aatatcaaga tttagagcaa agcatgagat gtgtggggat agacagtgag gctgataaaa 3900 tagagtagag ctcagaaaca gacccattga tatatgtaag tgacctatga aaaaaatatg 3960 gcattttaca atgggaaaat gatgatcttt ttctttttta gaaaaacagg gaaatatatt 4020 tatatgtaaa aaataaaagg gaacccatat gtcataccat acacacaaaa aaattccagt 4080 gaattataag tctaaatgga gaaggcaaaa ctttaaatct tttagaaaat aatatagaag 4140 catgccatca tgacttcagt gtagagaaaa atttcttatg actcaaagtc ctaaccacaa 4200 agaaaagatt gttaattaga ttgcatgaat attaagactt atttttaaaa ttaaaaaacc 4260 attaagaaaa gtcaggccat agaatgacag aaaatatttg caacacccca gtaaagagaa 4320 ttgtaatatg cagattataa aaagaagtct tacaaatcag taaaaaataa aactagacaa 4380 aaatttgaac agatgaaaga gaaactctaa ataatcatta cacatgagaa actcaatctc 4440 agaaatcaga gaactatcat tgcatataca ctaaattaga gaaatattaa aaggctaagt 4500 aacatctgtg gcaatattga tggtatataa ccttgatatg atgtgatgag aacagtactt 4560 taccccatgg gcttcctccc caaaccctta ccccagtata aatcatgaca aatatacttt 4620 aaaaaccatt accctatatc taaccagtac tcctcaaaac tgtcaaggtc atcaaaaata 4680 agaaaagtct gaggaactgt caaaactaag aggaacccaa ggagacatga gaattatatg 4740 taatgtggca ttctgaatga gatcccagaa cagaaaaaga acagtagcta aaaaactaat 4800 gaaatataaa taaagtttga actttagttt tttttaaaaa agagtagcat taacacggca 4860 aagtcatttt catatttttc ttgaacatta agtacaagtc tataattaaa aattttttaa 4920 atgtagtctg gaacattgcc agaaacagaa gtacagcagc tatctgtgct gtcgcctaac 4980 tatccatagc tgattggtct aaaatgagat acatcaacgc tcctccatgt tttttgtttt 5040 ctttttaaat gaaaaacttt attttttaag aggagtttca ggttcatagc aaaattgaga 5100 ggaaggtaca ttcaagctga ggaagttttc ctctattcct agtttactga gagattgcat 5160 catgaatggg tgttaaattt tgtcaaatgc tttttctgtg tctatcaata tgaccgtgtg 5220 attttcttct ttaacctgtt gatgggacaa attacgttaa ttgattttca aacgttgaac 5280 cacccttaca tatctggaat aaattctact tggttgtggt gtatattttt tgatacattc 5340 ttggattctt tttgctaata ttttgttgaa aatgtttgta tctttgttca tgagagatat 5400 tggtctgttg ttttcttttc ttgtaatgtc attttctagt tccggtatta aggtaatgct 5460 ggcctagttg aatgatttag gaagtattcc ctctgcttct gtcttctgaa agagattgta 5520 gaaagttgat acaaaagccg aattcg 5546 <210> 3 <211> 5601 <212> DNA <213> Artificial Sequence <220> <223> pPGM-3 vector <220> <221> promoter (222) (1) .. (611) <223> CMV promoter <220> <221> gene (222) (1386). (2246) <223> beta-lactamase gene <220> <221> gene <222> (3417) .. (5589) <223> Interferon beta MAR element <400> 3 gaattcacta gtgattaggg cccgttgaca ttgattattg actagttatt aatagtaatc 60 aattacgggg tcattagttc atagcccata tatggagttc cgcgttacat aacttacggt 120 aaatggcccg cctggctgac cgcccaacga cccccgccca ttgacgtcaa taatgacgta 180 tgttcccata gtaacgccaa tagggacttt ccattgacgt caatgggtgg agtatttacg 240 gtaaactgcc cacttggcag tacatcaagt gtatcatatg ccaagtacgc cccctattga 300 cgtcaatgac ggtaaatggc ccgcctggca ttatgcccag tacatgacct tatgggactt 360 tcctacttgg cagtacatct acgtattagt catcgctatt accatggtga tgcggttttg 420 gcagtacatc aatgggcgtg gatagcggtt tgactcacgg ggatttccaa gtctccaccc 480 cattgacgtc aatgggagtt tgttttggca ccaaaatcaa cgggactttc caaaatgtcg 540 taacaactcc gccccattga cgcaaatggg cggtaggcgt gtacggtggg aggtctatat 600 aagcagagct cgctagcggc cgcagatctg ttaactcgag aacttgttta ttgcagctta 660 taatggttac aaataaagca atagcatcac aaatttcaca aataaagcat ttttttcact 720 gcattctagt tgtggtttgt ccaaactcat caatgtatct tatcatgtct ggatcggcat 780 gcaagctggc actggccgtc gttttacaac gtcgtgactg ggaaaaccct ggcgttaccc 840 aacttaatcg ccttgcagca catccccctt tcgccagctg gcgtaatagc gaagaggccc 900 gcaccgatcg cccttcccaa cagttgcgca gcctgaatgg cgaatggcgc ctgatgcggt 960 attttctcct tacgcatctg tgcggtattt cacaccgcat atggtgcact ctcagtacaa 1020 tctgctctga tgccgcatag ttaagccagc cccgacaccc gccaacaccc gctgacgcgc 1080 cctgacgggc ttgtctgctc ccggcatccg cttacagaca agctgtgacc gtctccggga 1140 gctgcatgtg tcagaggttt tcaccgtcat caccgaaacg cgcgagacga aagggcctcg 1200 tgatacgcct atttttatag gttaatgtca tgataataat ggtttcttag acgtcaggtg 1260 gcacttttcg gggaaatgtg cgcggaaccc ctatttgttt atttttctaa atacattcaa 1320 atatgtatcc gctcatgaga caataaccct gataaatgct tcaataatat tgaaaaagga 1380 agagtatgag tattcaacat ttccgtgtcg cccttattcc cttttttgcg gcattttgcc 1440 ttcctgtttt tgctcaccca gaaacgctgg tgaaagtaaa agatgctgaa gatcagttgg 1500 gtgcacgagt gggttacatc gaactggatc tcaacagcgg taagatcctt gagagttttc 1560 gccccgaaga acgttttcca atgatgagca cttttaaagt tctgctatgt ggcgcggtat 1620 tatcccgtat tgacgccggg caagagcaac tcggtcgccg catacactat tctcagaatg 1680 acttggttga gtactcacca gtcacagaaa agcatcttac ggatggcatg acagtaagag 1740 aattatgcag tgctgccata accatgagtg ataacactgc ggccaactta cttctgacaa 1800 cgatcggagg accgaaggag ctaaccgctt ttttgcacaa catgggggat catgtaactc 1860 gccttgatcg ttgggaaccg gagctgaatg aagccatacc aaacgacgag cgtgacacca 1920 cgatgcctgt agcaatggca acaacgttgc gcaaactatt aactggcgaa ctacttactc 1980 tagcttcccg gcaacaatta atagactgga tggaggcgga taaagttgca ggaccacttc 2040 tgcgctcggc ccttccggct ggctggttta ttgctgataa atctggagcc ggtgagcgtg 2100 ggtctcgcgg tatcattgca gcactggggc cagatggtaa gccctcccgt atcgtagtta 2160 tctacacgac ggggagtcag gcaactatgg atgaacgaaa tagacagatc gctgagatag 2220 gtgcctcact gattaagcat tggtaactgt cagaccaagt ttactcatat atactttaga 2280 ttgatttaaa acttcatttt taatttaaaa ggatctaggt gaagatcctt tttgataatc 2340 tcatgaccaa aatcccttaa cgtgagtttt cgttccactg agcgtcagac cccgtagaaa 2400 agatcaaagg atcttcttga gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa 2460 aaaaaccacc gctaccagcg gtggtttgtt tgccggatca agagctacca actctttttc 2520 cgaaggtaac tggcttcagc agagcgcaga taccaaatac tgttcttcta gtgtagccgt 2580 agttaggcca ccacttcaag aactctgtag caccgcctac atacctcgct ctgctaatcc 2640 tgttaccagt ggctgctgcc agtggcgata agtcgtgtct taccgggttg gactcaagac 2700 gatagttacc ggataaggcg cagcggtcgg gctgaacggg gggttcgtgc acacagccca 2760 gcttggagcg aacgacctac accgaactga gatacctaca gcgtgagcta tgagaaagcg 2820 ccacgcttcc cgaagggaga aaggcggaca ggtatccggt aagcggcagg gtcggaacag 2880 gagagcgcac gagggagctt ccagggggaa acgcctggta tctttatagt cctgtcgggt 2940 ttcgccacct ctgacttgag cgtcgatttt tgtgatgctc gtcagggggg cggagcctat 3000 ggaaaaacgc cagcaacgcg gcctttttac ggttcctggc cttttgctgg ccttttgctc 3060 acatgttctt tcctgcgtta tcccctgatt ctgtggataa ccgtattacc gcctttgagt 3120 gagctgatac cgctcgccgc agccgaacga ccgagcgcag cgagtcagtg agcgaggaag 3180 cggaagagcg cccaatacgc aaaccgcctc tccccgcgcg ttggccgatt cattaatgca 3240 gctggcacga caggtttccc gactggaaag cgggcagtga gcgcaacgca attaatgtga 3300 gttagctcac tcattaggca ccccaggctt tacactttat gcttccggct cgtatgttgt 3360 gtggaattgt gagcggataa caatttcaca caggaaacag ctatgacatg attacgaatt 3420 cagcaaggtc gccacgcaca agatcaatat taacaatcag tcatctctct ttagcaataa 3480 aaaggtgaaa aattacattt taaaaatgac accatagacg atgtatgaaa ataatctact 3540 tggaaataaa tctaggcaaa gaagtgcaag actgttaccc agaaaactta caaattgtaa 3600 atgagaggtt agtgaagatt taaatgaatg aagatctaaa taaacttata aattgtgaga 3660 gaaattaatg aatgtctaag ttaatgcaga aacggagaga catactatat tcatgaacta 3720 aaagacttaa tattgtgaag gtatactttc ttttcacata aatttgtagt caatatgttc 3780 accccaaaaa agctgtttgt taacttgtca acctcatttc aaaatgtata tagaaagccc 3840 aaagacaata acaaaaatat tcttgtagaa caaaatggga aagaatgttc cactaaatat 3900 caagatttag agcaaagcat gagatgtgtg gggatagaca gtgaggctga taaaatagag 3960 tagagctcag aaacagaccc attgatatat gtaagtgacc tatgaaaaaa atatggcatt 4020 ttacaatggg aaaatgatga tctttttctt ttttagaaaa acagggaaat atatttatat 4080 gtaaaaaata aaagggaacc catatgtcat accatacaca caaaaaaatt ccagtgaatt 4140 ataagtctaa atggagaagg caaaacttta aatcttttag aaaataatat agaagcatgc 4200 catcatgact tcagtgtaga gaaaaatttc ttatgactca aagtcctaac cacaaagaaa 4260 agattgttaa ttagattgca tgaatattaa gacttatttt taaaattaaa aaaccattaa 4320 gaaaagtcag gccatagaat gacagaaaat atttgcaaca ccccagtaaa gagaattgta 4380 atatgcagat tataaaaaga agtcttacaa atcagtaaaa aataaaacta gacaaaaatt 4440 tgaacagatg aaagagaaac tctaaataat cattacacat gagaaactca atctcagaaa 4500 tcagagaact atcattgcat atacactaaa ttagagaaat attaaaaggc taagtaacat 4560 ctgtggcaat attgatggta tataaccttg atatgatgtg atgagaacag tactttaccc 4620 catgggcttc ctccccaaac ccttacccca gtataaatca tgacaaatat actttaaaaa 4680 ccattaccct atatctaacc agtactcctc aaaactgtca aggtcatcaa aaataagaaa 4740 agtctgagga actgtcaaaa ctaagaggaa cccaaggaga catgagaatt atatgtaatg 4800 tggcattctg aatgagatcc cagaacagaa aaagaacagt agctaaaaaa ctaatgaaat 4860 ataaataaag tttgaacttt agtttttttt aaaaaagagt agcattaaca cggcaaagtc 4920 attttcatat ttttcttgaa cattaagtac aagtctataa ttaaaaattt tttaaatgta 4980 gtctggaaca ttgccagaaa cagaagtaca gcagctatct gtgctgtcgc ctaactatcc 5040 atagctgatt ggtctaaaat gagatacatc aacgctcctc catgtttttt gttttctttt 5100 taaatgaaaa actttatttt ttaagaggag tttcaggttc atagcaaaat tgagaggaag 5160 gtacattcaa gctgaggaag ttttcctcta ttcctagttt actgagagat tgcatcatga 5220 atgggtgtta aattttgtca aatgcttttt ctgtgtctat caatatgacc gtgtgatttt 5280 cttctttaac ctgttgatgg gacaaattac gttaattgat tttcaaacgt tgaaccaccc 5340 ttacatatct ggaataaatt ctacttggtt gtggtgtata ttttttgata cattcttgga 5400 ttctttttgc taatattttg ttgaaaatgt ttgtatcttt gttcatgaga gatattggtc 5460 tgttgttttc ttttcttgta atgtcatttt ctagttccgg tattaaggta atgctggcct 5520 agttgaatga tttaggaagt attccctctg cttctgtctt ctgaaagaga ttgtagaaag 5580 ttgatacaaa agccgaattc g 5601 <210> 4 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer for MAR of chicken lysozyme A <400> 4 ggatccataa tataactgta 20 <210> 5 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer for MAR of chicken lysozyme A <400> 5 aagcttaaaa gattgaagca 20 <210> 6 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer for chicken phi alpha-globin 5 'MAR <400> 6 aagcttttaa ccaacaaaaa 20 <210> 7 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer for chicken phi alpha-globin 5 'MAR <400> 7 ctgcagacct aacctgtcac 20 <210> 8 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer for CHO DHFR intron MAR <400> 8 tatacgtgaa tagtttttct 20 <210> 9 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer for CHO DHFR intron MAR <400> 9 gagttggaac tgagaagttc 20 <210> 10 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer for human HPRT intron MAR <400> 10 aagcttggtc aagaatgctg 20 <210> 11 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer for human HPRT intron MAR <400> 11 gctgggcgtg gtggtgcctg 20 <210> 12 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer for human CSP-B gene flanking SAR element <400> 12 ggatcccatt ctccttgatg 20 <210> 13 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer for human CSP-B gene flanking SAR element <400> 13 gaattcaaac aactcaatag 20 <210> 14 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer for human interferon-beta gene flanking SAR element <400> 14 gaattcagca aggtcgccac 20 <210> 15 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer for human interferon-beta gene flanking SAR element <400> 15 ttgtatcaac tttctacaat 20 <210> 16 <211> 31 <212> DNA <213> Artificial Sequence <220> <223> sense primer for constructing pSV-beta-gal / ver1 vector <400> 16 gcactagtcc cgggcccatg attacgaatt c 31 <210> 17 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> sense primer for constructing pSV-beta-gal / ver1 vector <400> 17 gtgccagctt gcatgcctgc aggtc 25 <210> 18 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 18 agggcccgtt gacattgatt attg 24 <210> 19 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 19 aagcttgcta gcgagctctg cttatataga cctccc 36 <210> 20 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 20 cctaggtaat acgactcact ataggg 26 <210> 21 <211> 31 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 21 gtcgacacgc gtgtttaaac gggccctcta g 31

Claims (13)

delete delete delete delete delete delete delete delete delete (a) a promoter selected from the group consisting of an SV40 promoter, a cytomegalovirus (CMV) promoter and a Mouse Mammary Tumor Virus (MMTV) promoter, and a MAR matrix (Nuclear matrix attachment region) of the interferon beta gene present 5 'above the promoter Inserting a target gene into an animal cell expression vector to control expression by the promoter; (b) transforming the animal cell expression vector and the dihydrofolate reductase (DHFR) gene into which the target gene is inserted into CHO (chinese hamster ovary) DG44 cells deficient in DHFR; And (c) selecting and culturing the transformant in a medium containing MTX (methotrexate) to produce a recombinant protein Recombinant protein production method using an animal cell expression vector comprising a. The method of claim 10, wherein the animal cell expression vector is selected from the group consisting of pPGM-1 (KCCM 10232), pPGM-2 (KCCM 10338) and pPGM-1 (KCCM 10339). delete The method of claim 10, wherein the MTX is contained in the medium at 10 nM to 50 nM.