DNMT3B GENE-DEFICIENT CHO CELL LINE, PREPARATION AND APPLICATIONS THEREOF AND RECOMBINANT PROTEIN EXPRESSION SYSTEM USING THE SAME

Abstract

The invention relates to genetic engineering, and more particularly to a Dnmt3b gene-deficient CHO cell line, a preparation method and an application thereof and a recombinant protein expression system using the same. The invention adopts a CRISPR/Cas9 gene editing technique to knock out the Dnmt3b gene from the CHO cells to produce the Dnmt3b gene-deficient CHO cell line, which can significantly improve the expression level and stability of the target gene in CHO cells, overcoming the defects existing in the current CHO cell expression system, such as low expression level and stability. It has been demonstrated that using the CHO line provided herein to express a recombinant adalimumab can significantly increase the expression level of the recombinant adalimumab, indicating that the CHO cell line can be widely used to enhance the expression of target proteins.

Claims

1. A Dnmt3b gene-deficient CHO cell line, wherein the Dnmt3b gene in the Dnmt3b gene-deficient CHO cell line is lost in function, and the Dnmt3b gene has a sequence as shown in SEQ ID NO: 1.

2. The Dnmt3b gene-deficient CHO cell line of claim 1, wherein the Dnmt3b gene-deficient CHO cell line is derived from a CHO cell line selected from the group consisting of CHO-K1, CHO-S and CHO-DG44.

3. A method of preparing the Dnmt3b gene-deficient CHO cell line of claim 1, comprising: knocking out the Dnmt3b gene from CHO cells by using a CRISPR/Cas9 gene editing technique to produce the Dnmt3b gene-deficient CHO cell line.

4. The method of claim 3, wherein the step of knocking out the Dnmt3b gene from CHO cells comprises: (1) designing sgRNA sequences I and II as target sites according to the sequence of the Dnmt3b gene; (2) adding a first sticky end and a second sticky end respectively to the sgRNA sequences I and II; synthesizing two pairs of primers from the sgRNA sequences I and II; subjecting the two pairs of primers to annealing to correspondingly produce double-stranded DNA fragments; and respectively ligating the double-stranded DNA fragments into two CRISPR/Cas9 expression vectors respectively carrying fluorescent reporter genes I and II to construct two CRISPR/Cas9-sgRNA vectors; and (3) co-transfecting the two CRISPR/Cas9-sgRNA vectors into the CHO cells; selecting monoclonal cells containing the two fluorescent reporter genes I and II by flow cytometry for culture; and subjecting the monoclonal cells to knockout verification to obtain the Dnmt3b gene-deficient CHO cell line.

5. The method of claim 4, wherein in step (1), the sgRNA sequences I and II are respectively shown as follows: TABLE-US-00007 D3b-Ex1-31fw: (SEQIDNO:5) 5-GAGGAATGTCTCATCGTCAATGG-3; and D3b-Ex1-105fw: (SEQIDNO:6) 5-CTTGGAGGCAATGTGCACAGAGG-3.

6. The method of claim 5, wherein in step (2), the two pairs of primers are respectively shown as follows: TABLE-US-00008 D3b-Ex1-31fw-1: (SEQIDNO:7) 5-CACCGAGGAATGTCTCATCGTCAATGG-3; D3b-Ex1-31fw-2: (SEQIDNO:8) 5-AAACCCATTGACGATGAGACATTCCTC-3; D3b-Ex1-105fw-3: (SEQIDNO:9) 5--CACCGCTTGGAGGCAATGTGCACAGAGG-3; and D3b-Ex1-105fw-4: (SEQIDNO:10) 5-AAACCCTCTGTGCACATTGCCTCCAAGC-3.

7. The method of claim 5, wherein in step (3), primers used in the knockout verification are shown as follows: TABLE-US-00009 Dnmt3b-Ex1PCR-L: (SEQIDNO:3) 5-GTGCCCCCATTTCTCCTACT-3; and Dnmt3b-Ex1PCR-R: (SEQIDNO:4) 5-AGACCCAATGTGCTGGTCTC-3.

8. A use of the Dnmt3b gene-deficient CHO cell line of claim 1 in the preparation of a target protein.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0038] FIG. 1 shows the screening of a Dnmt3b gene-knockout CHO monoclonal cell line of the invention by PCR amplification.

[0039] FIG. 2 shows the detection of the proliferation of Dnmt3b gene-deficient CHO monoclonal cell lines 3b-2 and 3b-7 and normal CHO-K1 cells.

[0040] FIG. 3 shows the detection of apoptosis of Dnmt3b-deficient CHO cells and normal CHO cells by flow cytometry.

[0041] FIG. 4a is a fluorescence image showing the transient expression of eGFP gene driven by the CMV promoter in Dnmt3b-deficient CHO cells;

[0042] FIG. 4b is a white light image showing the transient expression of eGFP gene driven by the CMV promoter in Dnmt3b-deficient CHO cells;

[0043] FIG. 4c is a fluorescence image showing the transient expression of eGFP gene driven by the CMV promoter in normal CHO-K1 cells; and

[0044] FIG. 4d is a white light image showing the transient expression of eGFP gene driven by the CMV promoter in normal CHO-K1 cells.

[0045] FIG. 5 shows the expression stability of eGFP gene driven by the CMV promoter in Dnmt3b-deficient CHO cells (3b-7) and normal CHO-K1 cells.

[0046] FIG. 6 shows Western Blot results of recombinant adalimumab in Dnmt3b-deficient CHO cells (3b-7) and normal CHO cells.

DETAILED DESCRIPTION OF EMBODIMENTS

[0047] The invention will be described in detail below with reference to the embodiments, and these embodiments are not intended to limit the invention. Unless otherwise specified, instruments and reagents used in the following examples and experimental examples are all commercially available.

Example 1 Preparation of Methyltransferase Dnmt3b Gene-Deficient CHO Cell Line

[0048] Provided herein was a method of preparing a DNA methyltransferase Dnmt3b gene-deficient CHO cell line, which was specifically described as follows.

[0049] 1. Determination of Target Sites for a Candidate Gene

[0050] (1) Partial Amplification of Sequence of DNA Methyltransferase Dnmt3b Gene

[0051] Primers for amplification were designed according to the sequence of the Chinese hamster DNA methyltransferase Dnmt3bgene (No. NW_006879210) recorded in GenBank of NCBI, and were shown as follows:

TABLE-US-00004 Dnmt3b-Ex1PCR-L: (SEQIDNO:3) 5-GTGCCCCCATTTCTCCTACT-3; and Dnmt3b-Ex1PCR-R: (SEQIDNO:4) 5-AGACCCAATGTGCTGGTCTC-3.

[0052] The Dnmt3b gene fragment was amplified by PCR, and the amplified product was cloned and sequenced for verification. The desired amplified sequence was shown in SEQ ID NO: 2.

[0053] (2) Determination of sgRNA Sequences of Target Sites

[0054] The sgRNA sequences of target sites for the Dnmt3b gene were designed with the help of an online tool (http://crispr.mit.edu/), and were shown as follows:

TABLE-US-00005 D3b-Ex1-31fw: (SEQIDNO:5) 5-GAGGAATGTCTCATCGTCAATGG-3; and D3b-Ex1-105fw: (SEQIDNO:6) 5-CTTGGAGGCAATGTGCACAGAGG-3.

[0055] 2. Construction of a sgRNA Expression Vector

[0056] (1) Designing and Synthesis of Primers

[0057] Two pairs of primers were designed and synthesized according to the above sgRNA sequences of target sites, and respectively added with a sticky end at the 5 end. The primers were shown as follows:

TABLE-US-00006 D3b-Ex1-31fw-1: (SEQIDNO:7) 5-CACCGAGGAATGTCTCATCGTCAATGG-3; D3b-Ex1-31fw-2: (SEQIDNO:8) 5-AAACCCATTGACGATGAGACATTCCTC-3; D3b-Ex1-105fw-3: (SEQIDNO:9) 5-CACCGCTTGGAGGCAATGTGCACAGAGG-3; and D3b-Ex1-105fw-4: (SEQIDNO:10) 5-AAACCCTCTGTGCACATTGCCTCCAAGC-3.

[0058] Since a U6 promoter was used in the sgRNA expression vector, the gene expression will be significantly up-regulated in the presence of a guanine (G) in the starting site of the gene transcription. Therefore, during the designing process of the primers, if the starting base at 5 end of the forward primer was G, it was required to additionally add a guanine to ensure a high expression level. In this case, a cytosine (C) was required to be added at the 3 end of the corresponding reverse primer.

[0059] (2) Preparation of Double-Stranded DNA Fragments by Annealing

[0060] The two pairs of primers (D3b-Ex1-31fw-1+D3b-Ex1-31fw-2; D3b-Ex1-105fw-3+D3b-Ex1-105fw-4) obtained in step (1) were respectively subjected to annealing to produce double-stranded DNA fragments both with a sticky end. Specifically, the two pairs of primers were respectively phosphorylated and then transferred to a PCR instrument for denaturation and annealing, where the phosphorylation was performed through the steps of: mixing 1.0 L of respective primers (100 M), 1.0 L of 10T4 Ligation Buffer (NEB), 0.5 L of T4Polynucleotide Kinase (NEB M0201S) and 6.5 L of ddH.sub.2O uniformly to produce a phosphorylation system (10 L); and incubating the phosphorylation system at 37 C. for 30 min to complete the phosphorylation; the denaturation was performed at 95 C. for 5 min; and the annealing was performed by reducing the temperature from 95 C. to 25 C. at 5 C./min.

[0061] (3) Linearization of CRISPR/Cas9 Expression Vectors

[0062] Two CRISPR/Cas9 expression vectors pX458-ECFP carrying the gene of fluorescent protein ECFP and pX458-DsRed2 carrying the gene of fluorescent protein DsRed2 were linearized in the presence of endonucleaseBbs I, purified and recovered to obtain DNA fragments of the vectors, where the digestion was performed through the steps of: mixing 1.0 g of the pX458-DsRed2 or pX458-ECFP vector, 3.0 L of 10NEB Buffer 2.1 and 1.0 L of Bbs I (NEB) followed by addition of ddH.sub.2O to a volume of 30.0 L; and incubating the digestion system at 37 C. for 2 h to complete the digestion; and the digested product was purified using a QIAquick PCR Purification Kit and dissolved with 30.0 L of ddH.sub.2O for recovery.

[0063] (4) Construction of sgRNA Expression Vectors

[0064] CRISPR/Cas9 expression vectors containing sgRNA were obtained by ligation, transformation and screening, where the ligation was performed through the steps of mixing 0.5 L of the double-stranded DNA fragment with the sticky end obtained in step (2), 2.0 L of the vector DNA with the same sticky end obtained in step (3), 0.5 L of T4DNA ligase (NEB M0202S) and 1.0 L of 10T4 Ligation Buffer (NEB) followed by addition of ddH.sub.2O to a volume of 10.0 L to produce a ligation system; and reacting the ligation system for 1 h to complete the ligation; the transformation and screening were performed through the steps of transforming the ligated product into E. coli DH5a cells; spreading the cells on a ampicillin-resistant plate; incubating the plate at 37 C. overnight; and picking up a single colony for sequencing verification to obtain the expression vectors pX458-3b-1 and pX458-3b-2 respectively capable of expressing ECFP and DsRed2.

[0065] 3. Transfection of CHO Cells, and Screening and Identification of Gene-Knockout Monoclonal Cell Line

[0066] The pX458-3b-1 and pX458-3b-2 expression vectors were mixed in equal weight and transfected into CHO cells in a liposome-mediated manner. Then the CHO cells were screened to obtain Dnmt3b gene-knockout monoclonal cell line.

[0067] The above process was specifically described as follows. CHO-K1 cells were cultured in a DMEM medium containing 10% inactivated fetal bovine serum at 37 C. and 5% CO.sub.2. Before the transfection, 2.010.sup.5 CHO-K1 cells were seeded in a 24-well culture plate and cultured for 24 h. When the confluency reached about 90%, the cells were used for the transfection. The pX458-3b-1 and pX458-3b-2 vectors each for 1.5 g were diluted with 150.0 L of a reduced serum media (Opti-MEM). 0.75 L of a liposome Lipofectamine 3000 was diluted with 150.0 L of the reduced serum media (Opti-MEM) and added to the diluted expression vector medium. Then the reaction mixture was fully mixed and incubated at room temperature for 20 min. The medium in the 24-well plate was discarded, and then the CHO-K1 cells in respective wells were added with 300 L of the mixture of the vector and the liposome. Another three wells were treated in the same manner and used as parallel controls, and the wells in which the cells were not added with the transfection mixture were used as negative control. All cells were cultured at 37 C. and 5% CO.sub.2 for 1.5 h, and then the medium was replaced for continuous culture.

[0068] In order to obtain stable Dnmt3b gene-knockout monoclonal cells, single cells with double positive of DsRed2 and ECFP were sorted by flow cytometry into a 96-well plate containing 150 L of fresh medium after 72 h of the transfection, and cultured for 14 d. After that, the resulting monoclonal cells were transferred to a 48-well plate for enlarged culture and subsequent PCR verification.

[0069] The extraction of genomic DNA from monoclonal cells was performed as follows. A small number of cells (about 110.sup.6-10.sup.7) were collected and centrifuged at 350g for 5 min. The supernatant was discarded, and the cells were added with 20.0 L of a cell lysis solution containing 100 mM KCl, 20 mM Tris-HCl (pH 9.0), 0.3% Triton X-100 and 1.0 mg/mL proteinase K, gently mixed using a pipette and incubated at 55 C. for 15 min for complete lysis. Then the system was incubated at 95 C. for 10 min to denature the proteinase K, and the resulting lysate containing the genomic DNA of the cells can be used as a PCR template and stored at 20 C. for use.

[0070] Primers Dnmt3b-Ex1PCR-L and Dnmt3b-Ex1PCR-R were used to amplify the target fragment containing the target site by PCR, and sequencing analysis was performed to determine whether the base deletion or insertion occurred in the monoclonal cell lines. Results of the agarose gel electrophoresis detection for PCR products were shown in FIG. 1, where PC indicated positive plasmid control; NC indicated blank negative control; and M indicated DNA molecular weight marker. It can be seen from FIG. 1 that single-cell clones 1, 2, 4, and 7 were homozygotes with deletion of a large fragment. The single-cell clones 1, 2, 4, and 7 were further identified by sequencing analysis to be Dnmt3b gene-knockout cell lines. Then these Dnmt3b gene-knockout monoclonal cell lines were subjected to enlarged culture and cryopreserved in liquid nitrogen.

[0071] 4. Identification of Biological Characteristics of Dnmt3b-Deficient CHO Cells

[0072] Whether the Dnmt3b gene-deficient cell line can perform normal growth and passage was demonstrated by examining biological characteristics of the cells such as cell proliferation and apoptosis.

[0073] Wild CHO-K1 cells were used as control to verify the growth characteristics of the obtained Dnmt3b-deficient CHO monoclonal cells, determining whether the Dnmt gene-deficient CHO cell line can perform normal growth and passage. Specifically, the verification included observation of cell morphology and growth status, detection of cell proliferation by CCK-8 assay and the examination of cell apoptosis by flow cytometry (FCM), where Cell Counting Kit-8 (CCK-8) was employed; the results of the detection of cell proliferation were shown in FIG. 2; and the results of the examination of cell apoptosis were shown in FIG. 3.

[0074] It can be seen from the results that there was no significant difference between the Dnmt3b-deficient CHO cell line and the normal CHO cells in the biological characteristics such as growth status, morphology, proliferation and apoptosis, indicating that the Dnmt3b-deficient CHO cell line can perform normal growth, proliferation and passage.

Example 2 Application of the Dnmt3b Gene-Deficient CHO Cell Line

[0075] Expression of Target Gene eGFP in Recombinant CHO Cells

[0076] 1. Transfection of CHO Cells

[0077] Dnmt3b gene-deficient CHO cells (3b-7) and normal CHO cells (CHO-K1) were used herein. These two types of CHO cells were cultured, and inoculated into a fresh DMEM medium containing 10% inactivated fetal bovine serum at a density of 210.sup.5 cells one day before the transfection. When the conflency reached 90%, Lipofectamine 3000 was used to perform the transfection, where the plasmid used herein was an eukaryotic expression vector pWTY-02 constructed by our laboratory, in which the expression of enhanced green fluorescent protein (eGFP) was driven by the CMV promoter. The transfection was performed in three parallel replicates.

[0078] 2. Transient Expression of Transfected Cell Lines

[0079] 48 h after the transfection, the transient expression of eGFP in the two groups of cells was observed by an inverted fluorescence microscope, and the results were shown in FIGS. 4A-4D, where FIG. 4A was a fluorescence image showing the transient expression of eGFP gene in Dnmt3b-deficient CHO cells; FIG. 4B was a white light image showing the transient expression of eGFP gene in Dnmt3b-deficient CHO cells; FIG. 4C was a fluorescence image showing the transient expression of eGFP gene in normal CHO-K1 cells; and FIG. 4D was a white light image showing the transient expression of eGFP gene in normal CHO-K1 cells.

[0080] It can be concluded from the results that the expression vector had comparable transfection efficiency in Dnmt 3b-deficient and normal CHO-K1 cells, and there was no significant difference in the ratio of the eGFP-positive cells between the two groups, which indicated that the knockout of Dnmt 3b gene showed no significant effect on the transfection efficiency of cells.

[0081] 3. Screening of a Polyclonal CHO Cell Line with Stable Expression and Analysis of Long-Term Stable Expression of eGFP

[0082] The cells were screened in the presence of G418, and stably-transformed polyclonal cell pools were obtained after two weeks of screening. Then the cell pools were cultured to passage 30 respectively in the presence (G418+) and absence (G418-) of G418, and 10.sup.6 CHO cells from respective groups were analyzed by flow cytometry to measure the mean fluorescence intensity (MFI) of eGFP. It can be seen from the results that according to the criterion for evaluating the expression stability (whether the expression level of eGFP in CHO cells at passage 30 was greater than 70% of that in CHO cells at passage 1), the expression stability of eGFP whose expression was driven by the CMV promoter in recombinant Dnmt3b-deficient CHO cells was significantly higher that in the normal CHO cells (see FIG. 5).

Example 3 Another Application of the Dnmt3b Gene-Deficient CHO Cell Line

[0083] Expression of Recombinant Antibody (Adalimumab) in Recombinant CHO Cells

[0084] An eukaryotic expression vector, in which the expression of adalimumab was driven by the CMV promoter, was constructed based on the plasmid pWTY-02 to further test the effect of Dnmt3b-deficient CHO cells on the expression stability of recombinant protein (antibody). The expression vector plasmid was respectively transfected into Dnmt3b-deficient cells (3b-7) and normal CHO cells (CHO-K1), and then the transfected cells were cultured in a medium containing 800 g/mL of G418 for 15 days to screen stably-transfected recombinant cell pools. The recombinant CHO cells were passaged every other 3 days and cultured to passage 30. Subsequently, the recombinant CHO cells were cultured in a 125 mL shaking flask containing 30 mL of a protein-free, serum-free, chemically-defined (CD) CHO medium with 8 mM L-glutamine (Life Technologies Co., Ltd.) for 6 days to the cell number of 1.510.sup.7, where the cells were collected every day and detected by a Countstar BioTech cell counter (Shanghai Ruiyu Biotechnology Co., Ltd.) for the density and viability, and the supernatant was collected by centrifugation on the 6th day and then analyzed for the expression of the recombinant adalimumab.

[0085] The expression of adalimumab in the recombinant Dnmt3b-deficient cells (3b-7) and normal CHO cells (CHO-K1) was analyzed by Western Blot. Specifically, the supernatant containing adalimumab was added to a 5SDS sample buffer and boiled in a water bath for 10 min for complete denaturation. 25 L of the denatured protein sample was separated by 10% SDS-polyacrylamide gel electrophoresis and then transferred to a nitrocellulose membrane by a wet transfer method. The membrane was blocked with 5% BSA for 2 h, incubated with goat anti-human secondary antibody diluted by 1:6000 at room temperature for 1.5 h, washed three times with TBST and developed with a chemiluminescent detection reagent. The image was observed and collected using a gel imager to analyze the gray value of the target protein, obtaining the expression level of the protein to be tested.

[0086] It can be seen from the results that the expression level of the recombinant adalimumab in Dnmt3b-deficient CHO cell pool (1217.149.9 mg/L) was significantly higher than that in the normal CHO cell pool (392.637.3 mg/L). Moreover, after cultured to passage 30, the expression levels of the recombinant adalimumab in Dnmt3b-deficient CHO cell pool and normal CHO cell pool were respectively 985.858.5 mg/L and 160.926.6 mg/mL, indicating that the difference in expression level was of statistical significance (P<0.05, see FIG. 6). These results demonstrated that the expression and stability of recombinant adalimumab in Dnmt3b-deficient CHO cells were significantly improved.

Example 4 Preparation of Recombinant Protein Expression System

[0087] The recombinant protein expression system provided herein was prepared as follows. Specifically, the target gene was inserted into an expression vector to construct a recombinant protein expression vector. Then the recombinant protein expression vector was transfected into the above Dnmt3b-deficient CHO cell line and screened to obtain the recombinant protein expression system capable of expressing the target protein.