KR100540200B1 - Method for suppression of sialidase expression in cell lines using sialidase antisense ??? - Google Patents

Abstract

The present invention relates to a method of inhibiting sialidase expression in a transformed host cell line using genetic recombination technology, ie antisense RNA technology. In the present invention, by inhibiting the expression of some or all of the sialidase gene, the glycoprotein has an intact glycemic structure by preventing the removal of sialic acid located at the end of the glycoprotein of the glycoprotein produced in a host cell line such as a hamster cell line. And ultimately to increase the yield of glycoproteins.

Description

Method for suppression of sialidase expression in cell lines using sialidase antisense NRNA}

1 is a schematic diagram showing the types of primers for obtaining sialidase genes and their positions in sialidase cDNA.

2 is agarose gel electrophoresis of the results of PCR of all or part of the sialidase cDNA using the synthesized primer.

3 is a schematic diagram of the pZeoSV2 (−) vector used to transform the cell line.

4 shows a replica assay of transformed cell lines. Here, (A) shows fluorescence by UV irradiation, and (B) shows the location of cells by protein staining.

5 shows the results of 4-MU assay of each transformed cell line.

The present invention relates to a method of inhibiting sialidase expression in a transformed host cell line using genetic recombination technology, ie antisense RNA technology. In the present invention, by inhibiting the expression of the sialidase gene in part or all to prevent the removal of sialic acid located at the sugar chain terminal of the glycoprotein produced in a host cell line, such as hamster cell line to prevent the glycoprotein intact glycostructure And ultimately increase the yield of glycoproteins.

In general, most proteins produced by genetic engineering have been known to have the same bioactivity because their primary structure, that is, their amino acid sequence, matches their natural form. However, proteins produced using microorganisms such as Escherichia coli are known to be deficient in sugar chain structure. Therefore, when the natural type of the protein is derived from eukaryotic cells, the genetically engineered protein produced in E. coli is natural in sugar chain structure. There is no choice but to show the difference.

In vitro activity of a genetically engineered protein expressed in E. coli is sufficiently identical to that of the natural type, but the in vivo activity may be significantly reduced compared to the natural type, and erythropoietin is currently known as a representative example. Therefore, in many cases, specific proteins are prepared by genetic engineering methods using yeast or higher animal cell lines, which may be identical or similar in sugar chain structure, as transformation hosts.

On the other hand, the most important role for the in vivo activity of the protein is the terminal sialic acid of the sugar chain structure, their presence is related to the stability in vivo rather than the activity of the glycoprotein itself. That is, the presence of sialic acid increases the in vivo half-life of glycoproteins and is known to play a very important clinical role.

However, even when the basic type of the sugar chain structure of the genetically engineered protein is expressed in the same manner, the host cell expresses even a small amount of sialidase in the cell, and the sialidase is located at the end of the sugar chain of the useful protein already expressed. Eliminating sialic acid results in undesirable consequences of reducing the final yield.

Accordingly, the present inventors can partially or completely inhibit the expression of sialidase in higher animal cells, which are well used as host cells of the useful protein, thereby reducing the removal of sialic acid after expression of the useful protein, thereby dramatically increasing the final yield. We have studied this method and have achieved this goal by using anti-sense RNA method which is one of genetic engineering techniques. Such antisense RNA methods include cDNA cloning of sialidase, insertion of part or all of the cloned cDNA into a specific higher animal expression vector, transduction of the constructed vector into host cells, transcription identification, and expression of sialidase. This entails a very arduous set of expertise in the identification of reduction and stability.

Hamster sialidase consists of a total of 379 amino acids. In order to use the PCR method most commonly used to obtain sialidase cDNA, oligonucleotide fragments of the N-terminal and C-terminal portions of sialidase cDNA were synthesized to a size of about 30 bases. At this time, EcoRI and BamHI recognition sites were introduced as linkers for cloning into expression vectors, and used as primers for gene amplification using PCR.

Synthesized oligonucleotides were purified by HPLC and used as primers, and PCR (Expand High Fidelity PCR system; BM) was performed using lambda cDNA library (Stratagene) of Chinese hamster egg as a template (FIG. 2).

PZeoSV2 (−) vector (Invitrogen, FIG. 3) is used to express antisense RNA in host cells. The expression vector pZeoSV2 (-) contains several restriction cloning sites, and the orientation of restriction enzyme recognition sites is opposite to pZeoSV2 (+). PCR amplified sialidase cDNA gene was eluted from agarose gel electrophoresis using Quagen kit, conjugated to pGEM-T vector (Promega), and transformed into E. coli NM522. Escherichia coli is grown overnight in LB-ampicillin solid medium plated with X-gal / IPTG, and then plasmid DNA is purified from white colonies and reacted with restriction enzymes (BamHI, EcoRI) to obtain sialidase cDNA gene. This was again agarose gel electrophoresis eluted by the above method and then conjugated with the pZeoSV2 (-) vector obtained by reacting with a restriction enzyme (BamHI, EcoRI) in advance and transformed into E. coli NM522. Only positive strains are obtained from a zeocin-containing medium and used for transformation of Chinese hamster host cells. On the other hand, the sequence of the obtained sialidase cDNA gene is analyzed and compared with the sequence described in the known literature to confirm the conformity of the nucleotide sequence.

Whether the antisense RNA performs its function in the host cell may depend on the antisense RNA used. Therefore, the present inventors have made a modified vector using a gene containing only a part of the sequence in addition to the entire sequence of sialidase cDNA, but the scope is not limited thereto (FIG. 1). That is, the inventors prepared and purified primers N4, sial7, sial8 and N8 as in Example 3.

PCR was carried out as in Example 2 using each of the obtained primers, and then cloned into pZeoSV (-) (Fig. 2, Fig. 3).

As a host cell for transformation, CHO tk-cell line -cfc33 (Accession No .: KCLRF-BP- 00007) was used and the expression vector was constructed using an expression cell line protocol using a kit (SuperFect Transfection Kit; Quagen). Perform the switch. Briefly described as follows. 5 μg of each DNA is used (minimum DNA concentration: 0.1 μg / μl) and the cell line is prepared to be 1-4 × 10 ⁵ cells / 60 mm dish (5 ml) one day before transformation. Mix the prepared plasmid DNA and 150µl of serum-free culture medium (DMEM / F-12) well. Add reagent (20 μl of SuperFect Transfectin Reagent), mix well, and react for 5 to 10 minutes at room temperature. Then, mix well with 1 ml of culture medium (5% serum, DMEM / F-12). This was added to the cells carefully washed with PBS and 37 ℃ / 5% CO ₂ React in the incubator. After 2-3 hours, the cell lines are washed 4 times with PBS and replaced with 5 ml of fresh culture medium (5% serum DMEM / F-12).

Culture medium (5% serum DMEM / F-12, zeocin 50㎍ / ml) was changed and grown for 2 to 3 days after transformation, followed by 25% cell division and 50㎍ / ml of zeocine Change the badge every day and grow for about 7 to 10 days. After confirming that all the negative cell lines are dead, the surviving colonies are transferred to a new plate and cultured, so that the cells become 400 cell / 100 mm plates again. After culturing for 24 hours, cover the surface of the cell line with the prepared Whatman paper (press with a cylinder to prevent the paper from moving). After incubating for about 11-14 days, Whatman paper is transferred to another plate, washed twice with PBS, and then the paper is dried with the contact surface of the cell line upward. The dried paper was treated with 0.1 mM 4-methylumberiferryl-NeuAc (4-methylumberiferryl-NeuAc; diluted with 0.1 M sodium acetate buffer, pH 5.0) for 1 to 2 hours, followed by fluorescence under UV light. . At this time, the fluorescence shows the activity of sialidase (FIG. 4). Even if no fluorescence is visible or weak, the cell line is less active due to less expression of sialidase, so only these are selected for a more quantitative experiment for the 4-methylumberriferyl-NeuAc (4MU) assay. The cultured supernatant and harvested cell lines are separated from the newly separated and selected cell lines, and the cell lines are crushed by a method such as Sonyation and centrifuged again to obtain only the supernatant. 100 μl of each of these samples was reacted with 400 μl of 0.1 mM 4MU (0.1 M sodium acetate buffer, pH5.0) for 2 to 5 hours, and 2 ml of glycine buffer was added to stop the reaction, followed by fluorometer. ) At 365 mm and 450 mm (FIG. 5).

The present inventors have succeeded in producing a desired sialidase-negative cell line through the above-described experiments, and as a result of confirming by a method such as IEF electrophoresis, these cell lines are prepared with most of the erythropoietin attached to the sialic acid of the sugar chain terminal. And it was found. Therefore, when a glycoprotein such as erythropoietin is prepared by genetic engineering method using the cell line obtained according to the present invention, the improvement of the stability of the desired protein and the yield increase are predicted.

Example 1 Cloning of Sialidase Gene

Oligonucleotide fragments of the N-terminal and C-terminal portions of the sialidase cDNA for use in the PCR method were synthesized as follows.

1) Synthesis of Oligonucleotide Fragments by DNA Synthesizer

Using a DNA synthesizer (model: Applied Biosystems 392 DNA Synthesizer), oligonucleotides were synthesized with a size of about 30 bases and used as primers for gene amplification using PCR. That is, in this example, N1 and N2 primers were synthesized, and EcoRI and BamHI recognition sites were introduced as linkers to be cloned into expression vectors.

2) Purification of Synthetic Oligonucleotides

The oligonucleotide solution synthesized by the DNA synthesizer was precipitated with butanol, and then dissolved in 150 μl of TE solution, and the synthesized oligonucleotide was purified using HPLC. The column used was Nucleogen-DEAE 60-7 (10 x 125 mm, Macherey-Nagel Cat.No. 736597), 20% acetonitrile and 20 mM sodium acetate solution from 20% acetonitrile and 20 mM sodium acetate solution (pH 6.0) as mobile phase. (pH 6.0) and a linear concentration gradient up to 1 M potassium chloride solution were used. The flow rate was 1 ml per minute and eluted for 120 minutes. The largest peak portion eluting near 90 minutes was taken and ethanol precipitated.

3) PCR amplification

In order to amplify the sialidase cDNA gene and to clone it into the expression vector, the synthesized N1 and N2 were used as primers, and as a template, the lambda cDNA library (Stratagene) of a Chinese hamster egg was used as an expand high fidelity PCR system (BM). PCR was performed under the conditions shown in Table 1 below using ().

  1st 1 cycle   2nd 29 cycles   3rd 1 cycle   94 ° C 1 minute 56 ° C 1 minute 72 ° C 1 minute 30 seconds   94 ° C 20 sec 56 ° C 1min 72 ° C 1min   94 ° C 30 sec 58 ° C 1min 72 ° C 5min

Example 2: Preparation of transforming expression vector pZEON1N2

PCR amplified sialidase cDNA gene according to Example 1 was eluted from agarose gel electrophoresed using Quagen kit and conjugated to pGEM-T vector (Promega) and then transformed into E. coli NM522. Growing overnight in LB-Ampicillin solid medium plated with X-gal / IPTG, purified plasmid DNA from white colonies and reacted with restriction enzymes (BamHI, EcoRI) to obtain sialidase cDNA gene. This was again eluted by agarose gel electrophoresis, and then conjugated with a pZeoSV2 (-) vector obtained by reaction with a restriction enzyme (BamHI, EcoRI) in advance. The vector construct was transformed into E. coli NM522 and only positive strains were obtained from zeocin containing medium and used for transformation of Chinese hamster host cells.

On the other hand, by analyzing the nucleotide sequence of the obtained sialidase cDNA gene, the agreement with the sialidase gene sequence disclosed in the known literature was confirmed.

Example 3: Preparation of transforming expression vector pZEO modified vector

In order to prepare various expression vectors of various functions, primers N4, sial7, sial8 and N8 were prepared and purified in the same manner as in Example 1, and the specific nucleotide sequences of each primer are shown in the sequence listing.

PCR was performed as in Example 2 using the synthesized primers, and then cloned into pZeoSV (−) to construct the vectors of Table 2 below (FIGS. 2 and 3).

primer Template Gene size Constructed Vector N1, N4 pZEON1N2 170 bp pZEON1N4 N1, sial8 900 bp pZEON18 sial7, N8 300 bp pZEO7N8 sial7, sial8 560 bp pZEO78

Example 4 Transformation of Host Cells

CHO tk-cell line -cfc33 (Accession Number: KCLRF-BP- 00007) was used as a host cell for transformation, and a SuperFect Transfection Kit (Quagen) was used as a transformation kit.

5 μg of each DNA was used (minimum DNA concentration: 0.1 μg / μl), and cell lines were prepared to be 1 to 4 × 10 ⁵ cells / 60 mm dish (5 mL) one day before transformation. 150 μl of the prepared plasmid DNA and serum-free culture medium (DMEM / F-12) were mixed well. After adding the reagent (SuperFect Transfectin Reagent, 20µl), the mixture was mixed well and reacted at room temperature for 5-10 minutes, and then mixed well with 1 ml of culture medium (5% serum, DMEM / F-12). This was added to the cells carefully washed with PBS and 37 ℃ / 5% CO ₂ The reaction was carried out in the incubator. After 2-3 hours, the cell lines were washed 4 times with PBS and replaced with 5 ml of fresh culture medium (5% serum DMEM / F-12).

Example 5: Selection of Transgenic Cell Lines

1) Antibiotic Treatment

Culture medium (5% serum DMEM / F-12, zeocin 50㎍ / ml) was changed and grown for 2 to 3 days after transformation, and then divided into 25% of cells and 50㎍ / ml of zeocine. The medium was changed every day and grown for about 7 to 10 days. After confirming that all negative cell lines died, the following experiment was performed.

2) Replica fluorescence observation

The colonies surviving after zeocin treatment were transferred to a new plate and cultured to form a 400 cell / 100 mm plate. After incubation for 24 hours, the surface of the cell line was covered with the prepared Whatman paper (press with a cylinder to prevent the paper from moving). After incubating for about 11-14 days, Whatman paper was transferred to another plate, washed twice with PBS, and the paper was dried with the contact surface of the cell line upward. The dried paper was treated with 0.1 mM 4-methylumberlyryl-NeuAc (0.1 M sodium acetate buffer, pH 5.0 diluted) for 1-2 hours followed by fluorescence under UV light. At this time, the fluorescence shows the activity of sialidase (FIG. 4). Even if the fluorescence is invisible or visible, the weak cell line has less expression of sialidase and thus is less active. Therefore, only these cells are selected for further quantitative experiments, and the 4-methylumbergeryl-NeuAc (4MU) assay is performed as follows. It was.

3) 4-Methylumberriferyl-NeuAc (4MU) Assay

No fluorescence or weak cells selected in step 2) were grown to perform a 4-MU assay as follows.

Each of the culture supernatants and harvested cell lines were separated from the newly isolated cultured selected cell lines, and the cell lines were disrupted by the sonication method and centrifuged again to obtain only the supernatants. 100 μl of each of these samples was reacted with 400 μl of 0.1 mM 4MU (0.1 M sodium acetate buffer, pH5.0) for 2 to 5 hours, and 2 ml of glycine buffer was added to stop the reaction, followed by fluorometer. ) At 365 mm and 450 mm (FIG. 5).

4) The amount of sialic acid at the end of the sugar chain was confirmed by IEF electrophoresis of erythropoietin produced in the obtained transformed cells, and the yield improvement in the production process was predicted.

<110> CHEILJEDANG Corporation <120> Method for suppression of sialidase expression in cell lines using sialidase antisense RNA <130> PC99-0249-JIJD <160> 6 <170> KOPATIN 1.5 <210> 1 <211> 30 <212> DNA <213> Artificial Sequence <220> Primer N1 <400> 1 ggatccatgg cgacttgccc tgtcctgcag 30 <210> 2 <211> 30 <212> DNA <213> Artificial Sequence <220> Primer N2 <400> 2 gaattctcac tgggcaccaa acactgctgg 30 <210> 3 <211> 21 <212> DNA <213> Artificial Sequence <220> Primer N4 <400> 3 gaattcgcaa aggccagcag g 21 <210> 4 <211> 24 <212> DNA <213> Artificial Sequence <220> Primer N8 <400> 4 gaattcatct aaggcatctg cctt 24 <210> 5 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> Primer sial 7 <400> 5 ggatccccct gcttatgcct atcggaaca 29 <210> 6 <211> 27 <212> DNA <213> Artificial Sequence <220> Primer sial 8 <400> 6 gaattcccaa attgcgggga gccatca 27

Claims (7)

A method for inhibiting expression of sialidase in a cell line using antisense RNA of a sialidase gene prepared using two primers selected and combined from among primers N1, N4, N8, sial 7 and sial 8. delete delete The method of claim 1, wherein the sialidase gene is inserted into the transformation vector pZeoSV2 (−). The method of claim 1, wherein the cell line is an animal cell line. The method of claim 5, wherein the animal cell line is CHO, COS or BHK. The method of claim 1, wherein the cell line is yeast.

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