Application of Aars1 gene in regulation and control of expression of exogenous recombinant protein

Abstract

The invention provides application of an Aars1 gene in regulation and control of expression of an exogenous recombinant protein, and belongs to the technical fields of recombinant protein expression, gene engineering and cell culture. According to the CHO cell line, shRNA fragments of targeted Aars1 genes are inserted and integrated into piggyBac transposon vectors, a recombinant vector for inhibiting / silencing the Aars1 genes and an Aars1 low-expression CHO cell line are constructed, the CHO cell line can remarkably reduce the mRNA expression level of the Aars1, meanwhile, the CHO cell line serves as a host cell for recombinant protein expression, and compared with a wild type CHO cell line, the CHO cell line has the advantages that the expression level of the CHO cell line is greatly improved; the recombinant adalimumab can improve the expression quantity of recombinant protein (the expression quantity of the recombinant adalimumab is increased by 3.1 + / -0.18 times), effectively overcomes the problem of low expression level of the existing CHO cell expression system, and can be used for further constructing a recombinant protein expression system or kit.

Description

Technical Field

[0001] The invention belongs to the technical fields of recombinant protein expression, genetic engineering and cell culture, and particularly relates to application of Aars1 gene in regulating the expression of exogenous recombinant protein. Background Art

[0002] Medicinal recombinant proteins are currently the most widely used type of biological drugs in clinical applications, with good therapeutic effects and few side effects. However, in the process of obtaining medicinal recombinant proteins, there are still prominent problems such as low recombinant protein expression and heterogeneity, which cannot meet the growing demand for the development, preparation of clinical therapeutic drugs and gene therapy of medicinal recombinant proteins such as vaccines, monoclonal antibodies, bispecific antibodies, cytokines, and growth factors. Therefore, the use of genetic and cell engineering methods to study the expression of host cell recombinant proteins is of great significance and application value for improving the transgenic expression level and expression stability and establishing a high-yield and stable recombinant protein host cell expression system.

[0003] The inventors have used a cell line with low expression of the RASSF5 gene as a host cell to construct a recombinant protein expression system (application number 202410889174.1, patent name: cell line with low expression of the RASSF5 gene, recombinant protein expression system, kit, construction method, application), which can increase the expression of recombinant adalimumab by 2.1±0.28 times, effectively overcoming the problem of low expression level of the current CHO cell expression system. However, the inventors still hope to further improve the expression level of the recombinant protein.

[0004] Alanyl-tRNA synthetase / lactyltransferase (Aars1) is an important cytoplasmic enzyme that is widely involved in various cellular biological processes, including RNA modification, protein modification, cell proliferation, apoptosis, autophagy, and remodeling of the extracellular matrix. It plays a role in cell proliferation and survival by regulating multiple cell signaling pathways. However, there are no reports on the effects of Aars1 on recombinant protein expression in cells. Summary of the invention

[0005] The purpose of the present invention is to provide a method that can significantly improve the expression level of recombinant proteins, and to provide the use of Aars1 gene and related biological materials in regulating the expression of exogenous recombinant proteins.

[0006] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0007] The invention provides application of Aars1 gene in regulating the expression of exogenous recombinant protein.

[0008] Preferably, the expression of exogenous recombinant proteins in host cells is promoted by silencing / inhibiting Aars1 gene expression or knocking out Aars1 gene.

[0009] Preferably, the method for silencing / inhibiting the expression of the Aars1 gene is: taking the Aars1 gene as the target gene, designing a shRNA fragment, and inserting the shRNA fragment into the downstream position of the U6 promoter of the starting vector to obtain a recombinant vector for inhibiting / silencing the Aars1 gene, and then transfecting the recombinant vector for inhibiting / silencing the Aars1 gene into a host cell to obtain a cell line with low expression of the Aars1 gene.

[0010] Preferably, the sequence of the shRNA fragment is shown as SEQ ID NO.1;

[0011] SEQ ID NO.1: 5'-GAAATCCGAGAGCGGTTTATA-3';

[0012] The starting vector is a piggyBac vector.

[0013] Preferably, the host cell comprises a mammalian cell, a yeast cell, an insect cell or a plant cell.

[0014] Preferably, the host cell is a CHO cell.

[0015] Preferably, the exogenous recombinant protein includes a vaccine, a monoclonal antibody, a bispecific antibody, a cytokine or a growth factor.

[0016] The invention provides a recombinant vector for silencing / inhibiting the expression of Aars1 gene.

[0017] The present invention provides a cell line with low expression of Aars1 gene or a cell line with knockout of Aars1 gene.

[0018] The present invention also provides an exogenous recombinant protein expression system, wherein the exogenous recombinant protein expression system uses the above-mentioned Aars1 gene low-expression cell line or Aars1 gene knockout cell line as a host cell containing an expression vector containing an exogenous recombinant protein encoding gene.

[0019] Beneficial Effects

[0020] The present invention finds that the gene Aars1 encoding lactate transferase has the effect of increasing the expression level of exogenous recombinant proteins in mammalian cells. Therefore, the present invention constructs a recombinant vector for inhibiting / silencing the Aars1 gene and a CHO cell line with low expression of Aars1. After using the recombinant protein expression system or kit to construct the recombinant protein expression system or kit, the expression amount of the recombinant protein can be increased exponentially.

[0021] The present invention inserts and integrates a shRNA fragment targeting the Aars1 gene into a piggyBac transposon vector to construct a recombinant vector for inhibiting / silencing the Aars1 gene; the recombinant vector for inhibiting / silencing the Aars1 gene is integrated into the genome of a CHO cell through continuous and efficient catalysis by PBase, thereby achieving a continuous interference effect of the gene in the CHO cell, and constructing a CHO cell line with low expression of Aars1. The CHO cell line can significantly reduce the mRNA expression level of Aars1, and the CHO cell line is used as a host cell for recombinant protein expression. Compared with a wild-type CHO cell line, the expression amount of the recombinant protein can be increased (the expression amount of the recombinant adalimumab is increased by 3.1±0.18 times), thereby effectively overcoming the problem of low expression level of the current CHO cell expression system. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 A schematic diagram of the structure of the Aars1 inhibition / silencing vector constructed in Example 1 of the present invention;

[0023] Figure 2 This is a graph showing the results of detecting the relative expression of the mRNA of the Aars1 gene inhibited / silenced in CHO cells by RT-qPCR in Example 3 of the present invention;

[0024] Figure 3 This is a graph showing the results of using a cell counter to detect the viable cell density, cell viability and cell diameter of Aars1 inhibition / silencing in CHO cells in Example 4 of the present invention;

[0025] Figure 4 This is a graph showing the expression results of recombinant adalimumab in normal CHO cells and CHO cells with Aars1 gene inhibited / silenced in Example 5 of the present invention. DETAILED DESCRIPTION

[0026] 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.

[0027] Example 1

[0028] The construction of the recombinant vector for silencing / inhibiting the expression of the gene Aars1 is as follows:

[0029] 1) According to the sequence of Chinese hamster Aars1 in NCBI GenBank (Gene ID: 100766178) and the design principles of shRNA vectors, the sequence of the nucleic acid fragment encoding shRNA was designed to be: 5'-GAAATCCGAGAGCGGTTTATA-3' (SEQ ID NO. 1);

[0030] 2) Using 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 to obtain a recombinant vector for silencing / inhibiting the expression of the gene Aars1. The schematic diagram of its structure is shown in Figure 1 shown.

[0031] Example 2

[0032] The construction process of host cells with low expression of Aars1 gene is as follows:

[0033] 1) Cell culture preparation: CHO-S cells were cultured in DMEM / F12 medium containing 10% inactivated fetal bovine serum at 37°C and 5% CO 2 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 about 85% after 24 hours of culture.

[0034] 2) Cell transfection: Add 0.5 ml serum-free medium, 0.6 μg vector and 0.2 μg PBase transposase to each well (the mass ratio of vector to transposase is 3:1), add 50 μL DMEM / F12 medium, and add 2 μL lipo2000 to 50 μL DMEM / F12 medium and mix gently. Discard the medium of the cultured cells in the above 24-well plate, mix the incubated plasmid DNA and liposome mixture, and place at room temperature for 5 minutes. All cells were incubated at 37°C and 5% CO 2 After incubation in the incubator for 5 hours, the culture medium was discarded and replaced with fresh complete culture medium for continued culture; 48 hours after transfection, Geneticin and geneticin (G418) were used for screening, and when all non-transfected cell lines were killed by G418, a CHO cell line with low expression of the Aars1 gene was obtained.

[0035] 3) Experimental groups: recombinant vector for silencing / inhibiting the expression of gene Aars1 (Example 1) + PBase transposase (CHO+KD group) and blank control group (CHO group); the blank control group is a blank transfection in which no vector is added but only PBase transposase is added.

[0036] Example 3 Detection of the efficiency of silencing / inhibiting expression of the Aars1 gene

[0037] The CHO cell lines with low expression of the Aars1 gene screened after transfection in Example 2 (CHO+KD group) and the blank transfection control group cell lines (CHO group) were collected respectively, and the expression level of the Aars1 gene in each group was detected at the mRNA level by RT-qPCR experiment. The specific experimental method is as follows.

[0038] RT-qPCR experiment; primers for RT-qPCR detection of target gene Aars1 and internal reference gene β-actin were designed and synthesized respectively, and the cultured CHO cell lines of the Control group and shRNA group were collected, and the total RNA of each group of cells was extracted with Trizol, and then the total RNA was reversely transcribed into cDNA using the All-in one 1st Strand cDNA Synthesis Super Mix Kit (purchased from Nearshore Protein Technology Co., Ltd.). cDNA was diluted at a ratio of 1:4 and used as a template; a 20 μL reaction system was used: 10 μL of SYBR Green Mix, 2 μL of diluted template, 1 μL of each primer (primer sequence as shown in Table 1), and 6 μL of enzyme-free water were added.

[0039] Table 1 Primer sequences

[0040]

[0041]

[0042] The reaction conditions were: 95°C pre-denaturation for 30 seconds, amplification phase (95°C for 10 seconds, 60°C for 30 seconds, 45 cycles). Melting curve phase was 95°C for 15 seconds, 60°C for 1 minute. Finally, according to 2 -△△CT The algorithm was used to calculate the relative expression of Aars1 gene mRNA.

[0043] The results are as follows Figure 2 As shown, the mRNA expression of Aars1 gene in the shRNA group CHO cell line was significantly reduced by 9 times compared with the Control group CHO cell line. The above test results show that the expression of Aars1 gene in the CHO cell line with low expression of Aars1 gene constructed in Example 2 of the present invention was significantly inhibited, and the CHO cell line with low expression of Aars1 gene was successfully constructed.

[0044] Example 4

[0045] Verification of biological characteristics of CHO cell lines with low expression of Aars1 gene Cell proliferation and other cell biological characteristics were tested to verify whether CHO cells with knockdown of Aars1 gene could grow and subculture normally; CHO cells in CHO group and CHO+KD group were cultured at 5×10 5 / mL initial inoculation concentration was inoculated into the suspension medium, suspended for 6 days, and cells were collected every day to detect the cell growth status using a BioTech cell counter (Shanghai Ruiyu Biotechnology Co., Ltd.). Specifically, the cell growth characteristics of the CHO cell lines in the Control group and the shRNA group were compared and verified, including total cell density (Cell density), viable cell ratio (Cell viability) and cell diameter (Cell diameter).

[0046] like Figure 3 As shown, the biological characteristics such as cell growth state, morphology, and viable cell density of the CHO cells with low expression of the Aars1 gene (CHO+KD group) obtained in Example 3 were not significantly different from those of the CHO group cells, indicating that the CHO cell line with knockdown of Aars1 can grow, proliferate, and be subcultured normally.

[0047] Example 5 Expression of exogenous recombinant protein in CHO cell line with low expression of Aars1 gene

[0048] In this example, the CHO cell line with low expression of the Aars1 gene 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:

[0049] 1) Construction of recombinant adalimumab eukaryotic expression vector

[0050] 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.

[0051] First, the HC sequence was cloned into the promoters EF2 / CMV and CMV / EF1 of the pCHO1.0 expression vector (Thermo Fisher Scientific) to obtain the control eukaryotic expression vector pCHO-HL. Subsequently, the pEF2 / CMV-LC-polyA-pCMV / EF1 and Puro sequences in the pCHO-HL expression vector were replaced with the CMV promoter and BSD resistance gene sequences, respectively, to construct the vector pHC-BSD.

[0052] 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.

[0053] 2) Transfection and recombinant protein expression detection

[0054] The eukaryotic expression vector constructed in step 1) was transfected into the CHO cell line with low expression of Aars1 gene (CHO+KD group) and CHO group, and the transfected cells were cultured in a medium containing oryzacidin (15 μg / mL) to screen the stably transfected recombinant cell pool. The recombinant CHO cells were then cultured in a suspension culture bottle with 30 mL of protein-free, serum-free, chemically defined CD CHO medium (Henan Punoyi Biological Products Research Institute Co., Ltd.) for 6 days, and the cells were collected every day. The cell density and cell viability were detected by BioTech cell counter (Shanghai Ruiyu Biotechnology Co., Ltd.). The supernatant was collected by centrifugation on the 6th day, and the same cell number was used for quantitative analysis of the expression of recombinant adalimumab. The expression of adalimumab in the shRNA group and the Control group was analyzed by Western blot.

[0055] like Figure 4 As shown in Figure 2, compared with the CHO group, the expression level of recombinant adalimumab in the CHO+KD group was significantly increased, with the expression level increasing by 3.1±0.18 times ( Figure 4 B). This indicates that the CHO cell line with knockdown of Aars1 gene expression can be used as a host cell for recombinant protein production to construct a recombinant protein expression system, which can increase the yield of recombinant protein.

[0056] 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 Aars1 gene in regulating the expression of exogenous recombinant proteins.

2. The use according to claim 1, characterized in that: The expression of exogenous recombinant proteins in host cells can be promoted by silencing / inhibiting Aars1 gene expression or knocking out Aars1 gene.

3. The use according to claim 2, characterized in that: The method for silencing / inhibiting the expression of the Aars1 gene is as follows: taking the Aars1 gene as the target gene, designing a shRNA fragment, and inserting the shRNA fragment into the downstream position of the U6 promoter of the starting vector to obtain a recombinant vector for inhibiting / silencing the Aars1 gene, and then transfecting the recombinant vector for inhibiting / silencing the Aars1 gene into a host cell to obtain a cell line with low expression of the Aars1 gene.

4. The use according to claim 3, characterized in that: The sequence of the shRNA fragment is shown in SEQ ID NO. 1: 5'-GAAATCCGAGAGCGGTTTATA-3'; The starting vector is a piggyBac vector.

5. The use according to claim 2, characterized in that: The host cells include mammalian cells, yeast cells, insect cells or plant cells.

6. The use according to claim 5, characterized in that The host cell is a CHO cell.

7. The use according to claim 6, characterized in that The exogenous recombinant protein includes a vaccine, a monoclonal antibody, a bispecific antibody, a cytokine or a growth factor.

8. The recombinant vector for silencing / inhibiting the expression of Aars1 gene as claimed in claim 3 or 4.

9. A cell line with low expression of Aars1 gene or a cell line with knockout of Aars1 gene according to any one of claims 3 to 7.

10. An exogenous recombinant protein expression system, characterized in that: The exogenous recombinant protein expression system uses the Aars1 gene low-expression cell line or Aars1 gene knockout cell line described in claim 9 as a host cell of an expression vector containing an exogenous recombinant protein encoding gene.

Images