Application of ANXA1 gene as recombinant protein expression regulation target, nucleic acid fragment and plasmid for regulating gene and prepared CHO cell line

Abstract

The invention relates to the technical field of recombinant protein expression and cell culture. The invention provides an application of an ANXA1 gene as a recombinant protein expression regulation target, a nucleic acid fragment and a plasmid for regulating the gene, and a prepared CHO cell line. By silencing / inhibiting ANXA1 gene expression of CHO cells, the cell proliferation capacity is regulated and controlled, and the yield of recombinant protein is increased; the method specifically comprises the following steps: inserting and integrating an shRNA fragment of a targeted ANXA1 gene into a piggyBac transposon vector, and constructing an shRNA transposon vector; the shRNA transposon carrier is continuously and efficiently catalyzed by PBase to be integrated into the genome of the CHO cell, so that the continuous interference effect of the gene in the CHO cell is realized. The CHO cell line constructed by the invention can significantly reduce the mRNA and protein expression level of ANXA1, and as a host cell for recombinant protein expression, the CHO cell line multiplies the expression quantity of recombinant protein.

Description

Technical Field

[0001] The present invention relates to the technical field of recombinant protein expression and cell culture, and in particular to the application of ANXA1 gene as a target for regulating recombinant protein expression, and regulating the nucleic acid fragment and plasmid of the gene and the prepared CHO cell line. Background Art

[0002] Chinese hamster ovary (CHO) cells are currently the most widely used mammalian cell expression system. Its advantages are: it can guide the correct folding of recombinant proteins, provide complex N-glycosylation and accurate O-glycosylation and other post-translational processing functions, and the expression products are closest to humanized protein molecules in terms of molecular structure, physicochemical properties and biological functions. However, this expression system still has the problem of low recombinant protein expression, which is mainly due to the clonal heterogeneity of transgenic expression, productivity loss in long-term culture, and low secretion efficiency of recombinant proteins. Therefore, in order to meet the growing demand for therapeutic recombinant proteins (antibodies), it is of great significance and application value to optimize and improve the CHO recombinant protein expression system using genetic and cell engineering methods to improve its transgenic expression level and expression stability, which is of great significance and application value for establishing a high-yield and stable recombinant protein CHO cell expression system.

[0003] In recent years, genes that regulate cell proliferation and apoptosis have been confirmed to regulate the yield and quality of recombinant proteins. Studies have shown that growth properties such as cell proliferation and apoptosis are regulated by a variety of protein factors, and cell proliferation, apoptosis and cell cycle regulation can be achieved by regulating the expression of related protein factors. Annexin A1 (ANXA1) belongs to the calcium-dependent phospholipid binding protein family and is involved in a variety of biological functions, such as cell differentiation and proliferation, cell death signal regulation, and phagocytosis and clearance of apoptotic cells. The function and role of ANXA1 in mammalian cell expression systems have not yet been elucidated. Therefore, whether the expression of the ANXA1 gene in the CHO cell line can be regulated to increase the expression level of recombinant proteins and provide a new cell engineering solution for establishing an efficient CHO cell expression system is the core technical problem that the present invention needs to solve. Summary of the invention

[0004] The purpose of the present invention is to provide the application of ANXA1 gene as a target for regulating the expression of recombinant protein, and to regulate the nucleic acid fragment, plasmid and prepared CHO cell line of the gene, and to provide the application of cell lines with low expression of ANXA1 gene as host cells for recombinant protein expression, so as to increase the expression amount of recombinant protein and the yield of recombinant protein.

[0005] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0006] The present invention provides application of ANXA1 gene as a target for regulating recombinant protein expression.

[0007] The present invention also provides a shRNA nucleic acid fragment targeting the ANXA1 gene, and the nucleotide sequence of the shRNA nucleic acid fragment is shown in SEQ ID NO.1.

[0008] The present invention also provides the use of the shRNA nucleic acid fragment in silencing / inhibiting ANXA1 gene expression.

[0009] The present invention also provides a shRNA expression vector targeting the ANXA1 gene, wherein the shRNA expression vector is constructed by inserting the shRNA nucleic acid fragment between the ITR fragments of the starting vector.

[0010] Preferably, the starting vector is a piggyBac vector; the shRNA nucleic acid fragment is inserted downstream of the U6 promoter of the starting vector.

[0011] The present invention also provides the use of the shRNA expression vector in constructing a cell line for silencing / inhibiting the ANXA1 gene.

[0012] The present invention also provides the use of the shRNA nucleic acid fragment or the shRNA expression vector in regulating cell proliferation ability and improving the yield of recombinant protein.

[0013] The present invention also provides a cell line for silencing / inhibiting ANXA1 gene, wherein the cell line is constructed by transferring the shRNA expression vector into a recombinant protein expression cell.

[0014] The present invention also provides a method for constructing the cell line, comprising the following steps: mixing the recombinant protein expression cells, the shRNA expression vector, and the PBase transposase plasmid and then transfecting the mixture to obtain a cell line for silencing / inhibiting the ANXA1 gene.

[0015] The present invention also provides the use of the cell line in producing recombinant proteins, wherein the recombinant proteins include adalimumab and human serum albumin.

[0016] The present invention provides the application of ANXA1 gene as a target for regulating the expression of recombinant proteins, as well as nucleic acid fragments, plasmids and prepared CHO cell lines for regulating the gene. The present invention regulates the cell proliferation ability and improves the recombinant protein yield by silencing / inhibiting the expression of ANXA1 gene in CHO cells; specifically, the shRNA fragment targeting the ANXA1 gene is inserted and integrated into the piggyBac transposon vector to construct the shRNA transposon vector; the shRNA transposon vector is continuously and efficiently catalyzed by PBase to be integrated into the genome of CHO cells, thereby achieving the continuous interference effect of the gene in CHO cells. The CHO cell line constructed by the present invention can significantly reduce the mRNA and protein expression levels of ANXA1, and as a host cell for recombinant protein expression, it can double the expression of recombinant proteins. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 Schematic diagram of the ANXA1 gene knockdown transposon vector constructed in Example 1 of the present invention;

[0018] Figure 2 This is a graph showing the results of knocking down the mRNA expression of the ANXA1 gene in CHO cells by qPCR in Example 3 of the present invention;

[0019] Figure 3 This is a graph showing the results of Western blot detection of ANXA1 gene protein expression knockdown in CHO cells in Example 3 of the present invention;

[0020] Figure 4 The figure is a graph of CHO cell proliferation (upper figure) and a graph of viable cell density (lower figure) in Example 4 of the present invention in which the ANXA1 gene was knocked down using the CCK-8 method;

[0021] Figure 5 This is a schematic diagram of the comparison of the protein expression yield of recombinant adalimumab in normal CHO cells and ANXA1 knockdown CHO cell lines detected by Western blot in Example 5 of the present invention. DETAILED DESCRIPTION

[0022] The present invention provides application of ANXA1 gene as a target for regulating recombinant protein expression.

[0023] The present invention also provides a shRNA nucleic acid fragment targeting the ANXA1 gene, the nucleotide sequence of the shRNA nucleic acid fragment is shown in SEQ ID NO.1;

[0024] SEQ ID NO. 1: 5'-TAGCAGATAACAAGCTAATTA-3'.

[0025] The present invention also provides the use of the shRNA nucleic acid fragment in silencing / inhibiting ANXA1 gene expression.

[0026] The present invention also provides a shRNA expression vector targeting the ANXA1 gene, wherein the shRNA expression vector is constructed by inserting the shRNA nucleic acid fragment between the ITR fragments of the starting vector.

[0027] Preferably, the starting vector is a piggyBac vector; the shRNA nucleic acid fragment is inserted downstream of the U6 promoter of the starting vector.

[0028] The present invention also provides the use of the shRNA expression vector in constructing a cell line for silencing / inhibiting the ANXA1 gene.

[0029] The present invention also provides the use of the shRNA nucleic acid fragment or the shRNA expression vector in regulating cell proliferation ability and improving the yield of recombinant protein.

[0030] The present invention also provides a cell line for silencing / inhibiting ANXA1 gene, wherein the cell line is constructed by transferring the shRNA expression vector into a recombinant protein expression cell.

[0031] The present invention also provides a method for constructing the cell line, comprising the following steps: mixing the recombinant protein expression cells, the shRNA expression vector, and the PBase transposase plasmid and then transfecting the mixture to obtain a cell line for silencing / inhibiting the ANXA1 gene.

[0032] The present invention also provides the use of the cell line in producing a recombinant protein, wherein the recombinant protein is adalimumab.

[0033] The technical solutions provided by the present invention are described in detail below in conjunction with the embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0034] Example 1 Construction of a transposon vector for knocking down the ANXA1 gene

[0035] This embodiment provides a transposon vector for knocking down the ANXA1 gene, and the construction method thereof is as follows:

[0036] 1) According to the sequence of Chinese hamster ANXA1 in NCBI GenBank (No.: NW003614889.1) and the design principles of shRNA vectors, the sequence of the nucleic acid fragment encoding shRNA was designed to be: 5'-TAGCAGATAACAAGCTAATTA-3' (SEQ ID NO. 1);

[0037] 2) Using the PiggyBac vector (purchased from Yunzhou Biotechnology Co., Ltd.) as the starting vector, the nucleic acid fragment synthesized in step 1) was inserted into the downstream of the U6 promoter of the starting vector by enzyme digestion and ligation, thus obtaining a transposon vector for knocking down the ANXA1 gene. The schematic diagram of its structure is shown in FIG. Figure 1 shown.

[0038] Example 2 Construction of a CHO cell line with low expression of the ANXA1 gene

[0039] This example provides a CHO cell line with low expression of the ANXA1 gene, and the construction process thereof is as follows:

[0040] 1) Cell culture preparation: CHO-S cells were grown in DMEM / F12 medium containing 10% inactivated fetal bovine serum at 37°C and 5% CO2. Before transfection, 2.0×10 5 CHO-S cells were seeded in 24-well culture plates and used for cell transfection when the cell confluence reached 85% after 24 hours of culture.

[0041] 2) Cell transfection: Add 50 μL of DMEM / F12 medium to each centrifuge tube, and add 2 μL of lip2000 to 50 μL of DMEM / F12 medium and mix gently to make a lip2000 dilution, and let stand at room temperature for 5 minutes. 50 μL of DMEM / F12 medium, 0.6 μg of the transposon vector constructed in Example 1, and 0.2 μg of PBase transposase plasmid (Beijing Weijin Biotechnology Co., Ltd.) were added to each well of the test group. The mass ratio of the transposon vector to the PBase transposase was 3:1, and wild CHO-S cells were used as the control group; the culture medium of the inoculated cells in the above 24-well plate was discarded, and the incubated plasmid DNA and liposome mixture were mixed and placed at room temperature for 20 minutes. The DNA-lip2000 complex was added to the inoculated cells. All cells were incubated in a 37°C and 5% CO2 incubator for 4 h, the culture medium was discarded, and fresh culture medium was replaced for continued culture; 48 h after transfection, Geneticin and geneticin (G418) were used for screening, and when the cell confluence reached 80%, the cells were passaged at a ratio of 1:3 and the drug screening culture was continued for 14 days.

[0042] The following experimental groups were as follows: the shRNA group was the CHO cell line with low expression of the ANXA1 gene constructed in Example 2; and the Control group was the wild-type CHO-S cell line.

[0043] Example 3 Detection of the efficiency of ANXA1 gene silencing / inhibition expression

[0044] Experimental method: The cells of the shRNA group and the control group were collected respectively, and the expression level of the ANXA1 gene in each group was detected at the mRNA level by qPCR experiment. The specific experimental method is as follows.

[0045] 1) qPCR experiments

[0046] Primers for qPCR detection of target gene ANXA1 and internal reference gene β-actin were designed and synthesized respectively. The cultured wild-type (Control) and shRNA group CHO cell lines (shANXA1) were collected, and the total RNA of each group of cells was extracted with Trizol. Then, the total RNA was reverse transcribed into cDNA using the All-in-one 1st Strand cDNA Synthesis SuperMix Kit (purchased from Nearshore Protein Technology Co., Ltd.). cDNA was diluted at a ratio of 1:4 and used as a template; a 20μL reaction system was used: 10μL SYBRGreenMix, 2μL diluted template, 1μL each of primers (F+R), and 6μL enzyme-free water were added. The reaction conditions were: pre-denaturation at 95℃ for 30s, amplification stage (95℃ for 10s, 60℃ for 30s, 45 cycles). The melting curve stage was 95℃ for 15s, 60-95℃ for 1min. Finally, according to 2 -△△CT The algorithm was used to calculate the relative expression of ANXA1 gene mRNA. Figure 2 As shown, the results showed that the mRNA expression level of ANXA1 gene in the CHO cell line of the shANXA1 group was significantly reduced to 6±0.017 times that of the wild-type CHO cells (P<0.001).

[0047] 2) Western blot experiment

[0048] Western blot was used to analyze the expression of ANXA1 protein in the shANXA1 group and wild-type CHO cell pool. Specifically, the cells were washed twice with ice-cold PBS and lysed with RIPA buffer. The supernatants of the two groups of CHO cells were added to 5×SDS sample buffer and boiled in a water bath for 10 min to fully denature. 10 μL of protein samples were taken respectively and separated by 10% SDS-PAGE gel electrophoresis and then transferred to nitrocellulose membrane by wet transfer. The membrane was blocked in 5% skim milk TBST buffer for 2 h, and incubated with Anti-ANXA1 (1:1000) and Anti-β-actin (1:1000) overnight, and then incubated with Anti-Rabbit IgG (1:10000) for 2 h. After washing the membrane three times with TBST, the chemiluminescent colorimetric solution was added for development, and the image results were observed and collected on a gel imager to analyze the expression level of the protein to be tested.

[0049] like Figure 3 As shown, the expression level of ANXA1 protein in ANXA1 knockdown CHO cells was significantly decreased compared with that in wild-type CHO cells.

[0050] Example 4 Verification of biological characteristics of ANXA1 knockdown CHO cells

[0051] The cell biological characteristics such as cell proliferation were tested to verify whether the ANXA1 knockdown CHO cell line could grow and subculture normally. Using wild-type CHO-S cells as a control, the cell growth characteristics of the ANXA1 knockdown CHO cells were verified, including observation of cell morphology and growth status, detection of cell proliferation by CCK-8 method, and detection of viable cell density (VCD) by suspension counting. The CCK-8 kit (Cell Counting Kit-8, Beyotime Biotechnology Co., Ltd.) was used to detect cell proliferation; wild-type and low ANXA1 gene CHO cells were cultured at 5×10 5 / mL suspension for 7 days, and cells were collected every day. VCD was detected using a BioTech cell counter (Shanghai Ruiyu Biotechnology Co., Ltd.).

[0052] like Figure 4 As shown, the results showed that the biological characteristics of the CHO cell line with ANXA1 gene knockdown, such as cell growth state, morphology, and live cell density, were not significantly different from those of wild-type CHO cells, but the cell proliferation ability was higher than that of wild-type CHO cells, indicating that the CHO cell line with ANXA1 knockdown can improve the cell proliferation ability while growing and subculturing normally.

[0053] Example 5 Expression of recombinant protein in CHO cells

[0054] In this example, the ANXA1 gene knockdown CHO cell line screened and verified in Example 2 was applied to recombinant protein expression, including construction of a recombinant protein expression system. The specific construction process is as follows:

[0055] 1) Construction of recombinant adalimumab eukaryotic expression vector

[0056] The element sequences required for constructing the eukaryotic expression vector include u6 promoter, SV40 polyA, IRES, hPGK promoter, HC chain (GenBank No: LQ506328.1) and LC chain (GenBank No: LQ506329.1) of adalimumab, blasticidin (BSD) resistance gene, and puromycin (Puro) resistance gene sequence, which were synthesized by General Biotechnology (Anhui) Co., Ltd.

[0057] First, the LC and HC sequences were cloned into The control eukaryotic expression vector pCHO-HL was obtained by inserting the promoters EF2 / CMV and CMV / EF1 of the pCHO1.0 expression vector (Thermo Fisher Scientific). Subsequently, the pEF2 / CMV-LC-polyA-pCMV / EF1 and Puro sequences in the pCHO-HL expression vector were replaced with the CMV promoter and the BSD resistance gene sequence, respectively, to construct the vector pHC-BSD. Then, the HC and BSD resistance gene sequences in the pHC-BSD vector were replaced with the LC and Puro resistance gene sequences, respectively, to construct the pLC-Puro vector. Finally, the LC-polyA-EF-1α expression cassette was cloned into the pHC-BSD vector to construct the pLC-pHC dual promoter eukaryotic expression vector expressing adalimumab.

[0058] 2) Transfection and recombinant protein expression detection

[0059] The eukaryotic expression vector constructed in step 1) was transfected into the shRNA group cells and the control group cells respectively, and the transfected cells were cultured in a medium containing oryzacidin (1 mg / mL) for 15 days to screen the stably transfected recombinant cell pool. The recombinant CHO cells were then cultured in a six-well plate with 3 mL of protein-free, serum-free, chemically defined CD CHO medium (Henan Punoyi Biological Products Research Institute Co., Ltd.) for 6 days until the cell number reached 1×10 7 Cells / mL were collected every day and the cell density and viability were detected by BioTech cell counter (Shanghai Ruiyu Biotechnology Co., Ltd.). The supernatant was collected by centrifugation on the 7th day and quantified with the same cell number for the detection and analysis of the expression of recombinant adalimumab.

[0060] Western blot was used to analyze the expression of adalimumab in recombinant wild-type CHO cells and ANXA1 knockdown cells. Figure 5 As shown, the results showed that CHO cells with knockdown of ANXA1 gene expression could significantly increase the expression level of recombinant adalimumab, with the expression level increasing by 4.0±1.4 times (P<0.05).

[0061] As can be seen from the above examples, the present invention provides the application of ANXA1 gene as a target for regulating the expression of recombinant protein, as well as the nucleic acid fragment, plasmid and prepared CHO cell line for regulating the gene. The present invention regulates the cell proliferation ability and improves the recombinant protein yield by silencing / inhibiting the expression of ANXA1 gene in CHO cells; specifically, the shRNA fragment targeting the ANXA1 gene is inserted and integrated into the piggyBac transposon vector to construct the shRNA transposon vector; PBase continuously and efficiently catalyzes the integration of the shRNA transposon vector into the genome of CHO cells to achieve the continuous interference effect of the gene in CHO cells. The CHO cell line constructed by the present invention can significantly reduce the mRNA and protein expression levels of ANXA1, and as a host cell for recombinant protein expression, it can double the expression of recombinant protein.

[0062] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. Application of ANXA1 gene as a target for regulating recombinant protein expression.

2. A shRNA nucleic acid fragment targeting the ANXA1 gene, characterized in that: The nucleotide sequence of the shRNA nucleic acid fragment is shown in SEQ ID NO.

1.

3. Use of the shRNA nucleic acid fragment according to claim 2 in silencing / inhibiting ANXA1 gene expression.

4. A shRNA expression vector targeting ANXA1 gene, characterized in that: The shRNA expression vector is constructed by inserting the shRNA nucleic acid fragment described in claim 2 between the ITR fragments of the starting vector.

5. The shRNA expression vector according to claim 4, characterized in that The starting vector is a piggyBac vector; the shRNA nucleic acid fragment is inserted downstream of the U6 promoter of the starting vector.

6. Use of the shRNA expression vector according to claim 4 or 5 in constructing a cell line for silencing / inhibiting the ANXA1 gene.

7. Use of the shRNA nucleic acid fragment according to claim 2 or the shRNA expression vector according to any one of claims 4 to 5 in regulating cell proliferation ability and increasing the yield of recombinant proteins.

8. A cell line for silencing / inhibiting the ANXA1 gene, characterized in that: The cell line is constructed by transferring the shRNA expression vector according to claim 4 or 5 into a recombinant protein expression cell.

9. The method for constructing a cell line according to claim 8, characterized in that: The method comprises the following steps: mixing recombinant protein expression cells, the shRNA expression vector according to claim 4 or 5, and the PBase transposase plasmid, and then transfecting the mixture to obtain a cell line for silencing / inhibiting the ANXA1 gene.

10. Use of the cell line according to claim 8 in producing recombinant proteins, characterized in that: The recombinant protein includes adalimumab.

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