CN107828738A - A kind of dnmt rna deficiency Chinese hamster ovary celI system and preparation method and application - Google Patents

Abstract

The invention relates to a DNA methyltransferase-deficient CHO cell line and its preparation method and application, belonging to the technical field of genetic engineering. The present invention uses CRISPR/Cas9 gene editing technology to knock out the DNA methyltransferase Dnmt3a gene of CHO cells, screen and identify DNA methyltransferase Dnmt3a-deficient CHO cells, and transfect eukaryotic expression vectors and screen stably expressing recombinant CHO cells A new CHO cell expression system based on DNA methyltransferase deficiency was established. The present invention establishes a recombinant gene CHO cell expression system through the genetic transformation of host CHO cells, which can significantly increase the expression level of recombinant proteins, overcome the problem of unstable expression of recombinant proteins, and improve the expression stability of recombinant proteins.

Description

A kind of DNA methyltransferase-deficient CHO cell line and its preparation method and application

technical field

The invention relates to a preparation method and application of a DNA methyltransferase-deficient Chinese hamster ovary (CHO) cell line, belonging to the technical field of genetic engineering.

Background technique

In the 1980s, tissue plasminogen activator (tissue type plasminogen activator, t-PA) was successfully expressed in recombinant Chinese hamster ovary (CHO) cells for the first time and was approved by the US FDA for clinical use. The era of biopharmaceuticals based on CHO cells is coming. At present, the recombinant drug protein produced by CHO cells has accounted for nearly 70% of the production of mammalian cells. However, the current CHO cell expression system has problems such as low expression, especially unstable expression, and cannot meet the growing demand for vaccine development, clinical treatment, and gene therapy. Therefore, using gene and cell engineering methods to study the recombinant expression of CHO cells is of great significance for improving the expression level and stability of transgenes and establishing a high-yielding and stable-yielding recombinant protein CHO cell expression system. Studies have demonstrated that insensitivity of transgenes to methylation by reducing CpG islands in the promoter is beneficial for efficient and sustained expression of recombinant proteins. For example, the mutation of C-179 to G upstream of the transcription start site of hCMV-MIE promoter can significantly improve the sustained expression of recombinant proteins in stably transfected CHO cells. Therefore, an in-depth study of the effect of DNA methylation on the expression of transgenes and its mechanism will help to screen recombinant CHO cell lines with high and stable yields.

DNA methylation (DNA methylation) is a common epigenetic modification in prokaryotic and eukaryotic genomes. It is also an important regulation method of gene expression in mammals and plays a key role in gene transcription repression: gene promoter region (Including transcription factor binding sites) The high methylation state of CpG islands can directly prevent the binding of transcription factors and lead to changes in chromatin structure, thereby limiting the binding of transcription factors to gene promoters, and further affecting gene transcription, splicing, translation and expression etc. have an impact. The research on the effect of DNA methylation on the expression of recombinant proteins in CHO cells mainly focuses on the modification of promoters, including point mutations of promoter methylation sites, CpG island-free promoters, the application of synthetic promoters, and core CpG islands. Component insertion, etc. DNA methylation requires the catalysis of DNA methyltransferases (DNA methyltransferase, Dnmt), including de novo methyltransferases Dnmt3a and Dnmt3b, and DNA methylation maintenance enzyme Dnmt1, especially Dnmt3a-mediated promoter methylation and transgene Unstable expression is closely related.

At present, the use of cell engineering technology to modify cells has been widely used, especially through gene knockout or gene silencing to affect the expression level of genes related to specific biological functions, so as to obtain cells with changed specific biological functions. Due to its technical advantages such as easy operation, stability, flexibility and high efficiency, it will be the main technical strategy for the current and future transformation of CHO cells to continuously improve their recombinant protein production capacity.

Contents of the invention

In order to solve the above technical problems, the present invention uses CRISPR/Cas9 gene editing technology to knock out the DNA methyltransferase Dnmt3a gene of CHO cells, screen and identify the DNA methyltransferase Dnmt3a-deficient CHO cells, and transfect the eukaryotic expression vector and A new methylation-free CHO cell expression system based on DNA methyltransferase Dnmt3a deficiency was established by screening stable recombinant CHO cell lines.

In order to achieve the above object, the technical scheme adopted in the present invention is as follows:

A CHO cell line deficient in the DNA methyltransferase Dnmt3a gene.

The genomic DNA sequence of the DNA methyltransferase Dnmt3a is GenBank: NW_003613640.1, and the genomic DNA sequence of Dnmt3a-deficient CHO cells is knocked out and 199 bases are deleted, that is, bases 41138-41336.

Further, the CHO cell line is selected from any one of CHO-K1, CHO-S, and CHO-DG44.

The present invention also provides a method for preparing the above-mentioned DNA methyltransferase Dnmt3a gene-deficient CHO cell line, which can be obtained by knocking out the CHO cell DNA methyltransferase Dnmt3a gene by using CRISPR/Cas9 gene editing technology; including using CRISPR/Cas9 gene The editing technology knocks out the DNA methyltransferase Dnmt3a gene, screens and identifies Dnmt3a-deficient CHO cells, and verifies whether the defective cell lines can undergo normal proliferation and subculture through detection of cell biological characteristics such as cell proliferation and apoptosis.

Specifically, the above method includes the following steps:

1) Determine the target site for the candidate gene: According to the genome sequence (No.NW_003613640.1) and mRNA sequence (No.XM_016964036.1) of the Chinese hamster DNA methyltransferase gene Dnmt3a in NCBI's GenBank, primers were designed for the corresponding genomic DNA gene Fragment PCR amplification, the PCR amplified gene fragment was cloned and sequenced to verify the accuracy of the sequence; the online tool (http://crispr.mit.edu/) was used to assist in the design of chimeric single-stranded guide RNA (chimeric single-stranded guide RNA) for the Dnmt3a gene. guide RNA, chimeric sgRNA) targeting site, respectively design two pairs of primers according to the target sequence to construct the targeting sgRNA vector expressing CRISPR/Cas9;

2) Construction of sgRNA vector: use Bbs I restriction endonuclease to linearize the px330 vector plasmid and recover the linear fragment; the sgRNA oligonucleotide single strand anneals to form a double strand; the linearized px330 vector and the double-stranded sgRNA are ligated overnight ; The ligation product was transformed into DH5α competent cells, screened with ampicillin resistance, picked a single colony, and shaken overnight at 37°C to extract the plasmid and perform sequencing verification; select the correctly sequenced positive clone plasmid for cell transfection;

3) CHO cell transfection, monoclonal selection and cell knockout identification

Before transfection, 2.0×105 CHO cells ^were seeded in a 24-well culture plate, and when the cell confluence reached 80%-90%, the above-mentioned recombinant plasmid containing sgRNA was added to co-transfect CHO cells; the transfected cells were serially diluted and limiting dilution to achieve the selection of monoclonal mutant cells with independent knockout; the genomic DNA of the obtained defective cell lines was extracted, and the Dnmt3a gene fragment was amplified by PCR and sequenced to confirm the success of gene knockout.

The present invention also provides a eukaryotic cell expression system comprising the above-mentioned DNA methyltransferase Dnmt3a gene-deficient CHO cell line.

The present invention also provides a method for preparing the eukaryotic cell expression system, comprising: inserting the target gene into the expression vector to construct a recombinant expression vector; transfecting the recombinant expression vector into the defective CHO cell line, and obtaining expression system.

Further, the constructed expression vector can be transfected into Dnmt3a-deficient and normal control CHO cells, polyclonal CHO recombinant cell lines can be screened and subcultured, and the transient expression and long-term stable expression of the target gene in the recombinant CHO cells can be detected and analyzed.

The present invention also provides the application of the above-mentioned DNA methyltransferase Dnmt3a gene-deficient CHO cell line and the above-mentioned eukaryotic cell expression system in preparing the target protein and the like.

The inability to continuously and efficiently express the target gene in the recombinant cell line or even the expression decline is a common and prominent problem in the use of CHO cells for recombinant protein production. At present, it is believed that DNA methylation is the main cause of this expression instability, especially the promoter methylation mediated by DNA methyltransferase DNMT3a is closely related to the unstable expression of transgenes. In recent years, the cell engineering transformation strategy is to use cell engineering technology, especially gene knockout, gene overexpression or gene silencing, to change the expression level of biological function genes related to the expression or activity of recombinant proteins, and to improve the production of recombinant proteins. and/or great potential for activity. Therefore, the present invention combines host cell genetic modification and expression vector optimization, and uses CRISPR/Cas9 gene editing technology to construct a new CHO cell expression system based on DNA methyltransferase deficiency, which can significantly improve the expression level of recombinant proteins and overcome the The current expression system has the problems of low expression level and unstable expression.

Description of drawings

Figure 1: Sequencing identification results of Dnmt3a gene knockout monoclonal Dnmt3a-30 in CHO cells; among them, the deleted 199 base sequence is the gene knockout deletion, the arrow points to the deletion mutation site, and the underline is the Dnmt3a gene fragment Amplification primers, the wavy line is the sgRNA targeting site.

Figure 2: CCK-8 method to detect the proliferation of Dnmt3a-deficient CHO cell monoclonal cells Dnmt3a-30 and Dnmt3a-40 and normal CHO-K1 cells.

Fig. 3: Apoptotic results of Dnmt3a-deficient CHO cells and normal CHO cells detected by flow cytometry.

Figure 4: Schematic diagram of eukaryotic expression vector pWTY-02 driven by CMV promoter.

Figure 5: The positive cell rate of eGFP gene driven by CMV promoter in Dnmt3a-deficient CHO cells and normal CHO cells.

Figure 6: CMV promoter-driven eGFP expression stability experiment results in Dnmt3a-deficient CHO cells and normal CHO cells.

Figure 7: Detection and analysis results of recombinant EPO protein expression in Dnmt3a-deficient CHO cells and normal CHO cells.

Detailed ways

The following examples are used to illustrate the present invention, but are not intended to limit the scope of the present invention. Those who do not indicate specific techniques or conditions in the embodiments, according to the techniques or conditions described in documents in the art, such as Sambrook and other molecular cloning experiment manuals (Sambrook J&Russell DW, Molecular Cloning: a Laboratory Manual, 2001), or according to the product Instructions are carried out. The reagents or instruments used were not indicated by the manufacturer, and they were all conventional products that can be purchased through formal channels.

Example 1 Knockout of CHO cell Dnmt3a gene

1.1 Determine the target site for the candidate gene

Primers (D3a-Ex1seq-L:5′-GACCACAAGAATTCCGGCTC-3′, D3a-Ex1seq-R: 5'-CGTGTGTGAATCTGTGTGGG-3') PCR amplification of the corresponding gene fragment was carried out, and the accuracy of the sequence was verified by cloning and sequencing of the PCR amplified fragment. The chimeric single-stranded guide RNA targeting site of the Dnmt3a gene assisted by the online tool (http://crispr.mit.edu/) is as follows:

D3a-Ex1-98rev: 5'-ATCATCCTCCCGCTCCAAAGTGG-3';

D3a-Ex1-308fw:5′-TTTGAGGGGTCATCCTTGCAGGG3′

Two pairs of primers were designed according to the target sequence to construct the targeting sgRNA vector expressing CRISPR/Cas9.

1.2 Construction of sgRNA vector

Use Bbs I restriction endonuclease to linearize the px330 vector plasmid and recover the linear fragment. The sgRNA oligonucleotide single strand anneals to form a double strand. The linearized px330 vector was ligated with double-stranded sgRNA overnight. The ligation product was transformed into DH5α competent cells, screened by ampicillin resistance, picked a single colony, and shaken overnight at 37°C to extract the plasmid and verify it by sequencing. The positive clone plasmids with correct sequencing were selected for cell transfection.

1.3 CHO cell transfection, monoclonal selection and cell knockout identification

Before transfection, 2.0×10 ⁵ CHO-K1 cells were seeded in a 24-well culture plate, and when the cell confluence reached 80%-90%, the above-mentioned recombinant plasmid containing sgRNA was added to co-transfect CHO-K1 cells. Transfected cells were subjected to serial dilution and limiting dilution to achieve selection of monoclonal mutant cells with independent knockouts. Using the CRISPR/Cas9 gene knockout method and PCR amplification verification results, we finally screened and obtained six Dnmt3a-deficient CHO cell clones (clones 30, 31, 32, 33, 40, 41). The sequencing results of the PCR amplification product of the Dnmt3a-deficient cell line clone 30 showed that the gene knockout resulted in a deletion mutation of 199 bases in the Dnmt3a gene, and the gene knockout was successful (see Figure 1 for the results).

Example 2 Verification of biological characteristics of Dnmt3a-deficient CHO cells

Using wild-type CHO-K1 cells as a control, the cell growth characteristics of the obtained Dnmt3a-deficient CHO cell clones were verified, including observation of cell morphology and growth status, detection of cell proliferation by CCK-8 method, flow cytometry (FCM) Cell apoptosis was detected to verify whether the Dnmt3a-deficient CHO cell line could undergo normal growth and passage. CCK-8 kit (Cell Counting Kit-8 kit, Beyotime Biological Company) detection of cell proliferation (results shown in Figure 2) and flow cytometry results of detection of cell apoptosis (results in Figure 3) suggest that the obtained Dnmt3a The defective CHO cell line can grow and pass on normally, and its biological characteristics such as cell growth state, shape, cell proliferation, and cell apoptosis are not significantly different from normal CHO cells.

Example 3 Construction of recombinant expression vectors driven by different promoters

In the present invention, the eukaryotic expression vector pIRESneo2 (Clontech Company) is used as the parent vector to construct the eukaryotic expression vector pWTY-02 driven by the CMV promoter to express green fluorescent protein (eGFP) (see FIG. 4 ).

Example 4 Expression analysis of target gene eGFP in recombinant CHO cells

4.1 CHO cell transfection

CHO-K1 cells were cultured, and one day before cell transfection, they were subcultured to fresh serum-free medium at a cell density of 1×10 ⁵ , and when the cell confluence reached 80%-90%, Liposome 3000 (Invitrogen, USA ) for transfection. Two groups of CHO cells were transfected: Dnmt3a-deficient and normal control CHO-K1 cells. Three replicate wells were transfected in parallel with each plasmid.

4.2 Transient expression observation of transfected cell lines

After 24 hours of transfection, the expression of eGFP in transiently transfected cells was observed with an inverted fluorescence microscope. The results showed that the transfection efficiency of the eukaryotic expression vector in Dnmt 3a-deficient and normal CHO-K1 cells was equivalent, and there was no significant difference in the positive cell rate of eGFP expression between the two (see Figure 5). This shows that the knockout of Dnmt3a gene has no effect on cell transfection efficiency.

4.3 Screening of stable expression polyclonal CHO cell lines and analysis of long-term stable expression of eGFP

Add G418 for selection, obtain stable transformed polyclonal cell lines after two weeks of selection and culture, and subculture for 50 generations with selection pressure (with G418) and without selection pressure (without G418), and each experimental group was divided into CHO cells for every 10 generations. The cells were analyzed by flow cytometry according to 10 ⁶ cells in each sample, and the mean fluorescence intensity (MFI) of eGFP was measured. The results of flow cytometry showed that regardless of the G418 screening pressure, eGFP expressed by the CMV promoter could be stably expressed in Dnmt3a-deficient CHO cells for a long time (see Figure 6).

Example 5 Expression Analysis of Erythropoietin (EPO) Target Protein in Recombinant CHO Cells

In order to further test the effect of Dnmt3a-deficient CHO cells on the stability of recombinant protein expression, we constructed a eukaryotic expression vector driven by CMV promoter to express erythropoietin (EPO) gene based on plasmid pWTY-02. The constructed plasmid containing EPO gene expression vector was transfected into Dnmt3a-deficient and normal CHO-K1 cells respectively. The transfected cells were cultured in a medium containing G418 (800 μg/mL) for 15 days to select a stably transfected recombinant cell pool, and the recombinant CHO cells were subcultured every 3 days to passage 50. The recombinant CHO cells were then cultured in 125 mL culture shake flasks with 25 mL of protein-free, serum-free, chemically defined CD CHO medium (Life Technologies, containing 8 mM L-glutamine) for 5 days until the cell count When the concentration reached 8×10 ⁶ , the supernatant was collected by centrifugation for detection and analysis of recombinant EPO protein expression.

Western blot was used to analyze the expression of recombinant EPO protein in Dnmt3a-deficient and normal CHO-K1 cell pools. Specifically, the supernatant containing recombinant EPO was added to 5×SDS sample buffer and boiled in a water bath for 10 minutes to fully denature. 10 μL of protein samples were separated by 10% SDS-polyacrylamide gel electrophoresis, and transferred to nitrocellulose membrane by wet transfer method. Block the membrane with 5% BSA for 2 hours, incubate overnight at 4°C with 1:1000 diluted anti-EPO primary antibody (Santa Cruz, CA), and add 1:5000 diluted HRP-labeled anti-mouse IgG secondary antibody (Santa Cruz, CA) after washing the membrane with TBST , CA) incubate at 37°C for 1 hour, wash the membrane with TBST and add chemiluminescence color development solution to develop, observe and collect image results on a gel imager, and analyze the gray value of the target protein to indicate the expression level of the protein to be tested. The results of Western blot detection showed that the average expression level (860.5 ± 42.9 mg/L) of recombinant EPO protein in the Dnmt3a-deficient CHO cell pool was significantly higher than the average expression level (373.7 ± 29.6 mg/L) in the non-deficient CHO cell pool. L). The expression levels of recombinant EPO protein in the recombinant Dnmt3a-deficient CHO cell pool and non-defective CHO cell pool stably cultured for 50 passages were 672.7±35.1mg/L and 157.9±16.2mg/mL, respectively. The differences in expression levels were statistically significant (see Figure 7, P<0.05). These results indicated that Dnmt3a-deficient CHO cells could significantly increase the expression level and long-term expression stability of recombinant EPO protein.

Although the present invention has been described in detail with general descriptions and specific embodiments above, it is obvious to those skilled in the art that some modifications or improvements can be made on the basis of the present invention. Therefore, the modifications or improvements made on the basis of not departing from the spirit of the present invention all belong to the protection scope of the present invention.

sequence listing

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Claims (10)

1. A kind of 1. Chinese hamster ovary celI system of dnmt rna Dnmt3a gene defection types.
2. 2. Chinese hamster ovary celI system according to claim 1, it is characterised in that the dnmt rna Dnmt3a genomes DNA sequence dna is GenBank：NW_003613640.1, Dnmt3a deficiency Chinese hamster ovary celI genomic dna sequence knock out missing 199 Base is 41138-41336 bit bases；And/or

   Preferably, any one of the Chinese hamster ovary celI system in CHO-K1, CHO-S, CHO-DG44.
3. 3. the preparation method of the Chinese hamster ovary celI system of claim 1 or 2, it is characterised in that utilize CRISPR/Cas9 gene editings Technology knocks out Chinese hamster ovary celI dnmt rna Dnmt3a genes and obtained.
4. 4. preparation method according to claim 3, it is characterised in that comprise the following steps：

   1) target practice site is determined for candidate gene：According to Chinese hamster dnmt rna gene in NCBI GenBank Dnmt3a genome sequences No.NW_003613640.1 and mRNA sequence No.XM_016964036.1 design primers carry out corresponding Genomic DNA genetic fragment PCR is expanded, and PCR amplification genes fragment is carried out to the accuracy of cloning and sequencing checking sequence；Apply The chimeric single-chain guide RNA target practices site of the Line tool Computer Aided Design Dnmt3a genes, separately design two pairs according to targeting sequence and draw Thing is with construction expression CRISPR/Cas9 target practice sgRNA carriers；

   2) structure of sgRNA carriers：Use Bbs I restriction enzymes linearisation px330 vector plasmids and the linear piece of glue reclaim Section；The single-stranded annealing of sgRNA oligonucleotides forms double-strand；Linearisation px330 carriers and double-strand sgRNA are attached overnight；Even Thing of practicing midwifery converts DH5 α competent cells, the screening of ammonia benzyl resistance coated plate, and picking single bacterium colony, 37 DEG C of shaking tables shake bacterium and extract plasmid overnight And carry out sequence verification；Choose and correct positive colony plasmid progress cell transfecting is sequenced；

   3) Chinese hamster ovary celI is transfected, monoclonal is selected and cell knocks out identification；

   Preferably, by 2.0 × 10 before transfection⁵Individual Chinese hamster ovary celI is inoculated in 24 well culture plates, when cell fusion degree reaches 80%- The above-mentioned recombinant plasmid corotation transfected cho cell containing sgRNA is added when 90%；Transfectional cell is subjected to gradient dilution and the limit is dilute Release, the monoclonal mutant cell with independently knocking out is picked out to reach；To obtain the defects of type cell line extract its gene Group DNA, expand Dnmt3a genetic fragments using PCR and be sequenced to determine gene knockout success.
5. 5. preparation method according to claim 4, it is characterised in that for Dnmt3a genomic DNA fragments PCR amplifications Primer is as follows：

   D3a-Ex1seq-L:5′-GACCACAAGAATTCCGGCTC-3′；

   D3a-Ex1seq-R:5′-CGTGTGTGAATCTGTGTGGG-3′；And/or

   The chimeric single-chain guide RNA target practices site is as follows：

   D3a-Ex1-98rev:5′-ATCATCCTCCCGCTCCAAAGTGG-3′；

   D3a-Ex1-308fw:5′-TTTGAGGGGTCATCCTTGCAGGG3′。
6. 6. the Chinese hamster ovary celI system prepared comprising any one of the Chinese hamster ovary celI system of claim 1 or 2 or claim 3-5 methods described Eukaryotic cell expression system.
7. 7. the preparation method of eukaryotic cell expression system described in claim 6, it is characterised in that including：Target gene is inserted In expression vector, structure obtains recombinant expression carrier；The recombinant expression carrier is transfected to the Chinese hamster ovary celI for entering the deficiency System, expression system is obtained through screening.
8. 8. eukaryotic cell expression system described in the Chinese hamster ovary celI system or claim 6 described in claim 1 or 2 is preparing purpose egg The application of white aspect.
9. 9. the primer for Chinese hamster ovary celI dnmt rna gene Dnmt3a genomic DNA fragments PCR amplifications：

   D3a-Ex1seq-L:5′-GACCACAAGAATTCCGGCTC-3′；

   D3a-Ex1seq-R:5′-CGTGTGTGAATCTGTGTGGG-3′。
10. 10. for selectively targeted Chinese hamster ovary celI dnmt rna gene Dnmt3a chimeric single-chain guide RNA, its target practice position Point is as follows：

    D3a-Ex1-98rev:5′-ATCATCCTCCCGCTCCAAAGTGG-3′；

    D3a-Ex1-308fw:5′-TTTGAGGGGTCATCCTTGCAGGG3′。