Application of TP63 gene in improving expression quantity of exogenous recombinant protein
By constructing a TP63 overexpression vector in CHO cells, the problem of low recombinant protein expression level in CHO cells was solved, the expression level of recombinant protein in the CHO cell line was significantly increased, and the production efficiency of recombinant antibodies was improved.
Patent Information
- Application Number
- CN202511041400.1
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-07-28
- Publication Date
- 2025-10-14
AI Technical Summary
The expression level of exogenous recombinant proteins in CHO cells is low, which is difficult to meet industrial needs.
A TP63 gene overexpression vector was constructed and integrated into the CHO cell locus to establish a TP63 overexpression CHO cell line. The stable overexpression of TP63 in CHO cells was improved by liposome transfection technology.
The expression level of recombinant proteins in CHO cells was significantly increased, and the production efficiency of recombinant antibodies was improved.
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Figure CN120775918A_ABST
Abstract
Description
Technical Field
[0001] The present invention belongs to the field of genetic engineering technology, and specifically relates to an application of the TP63 gene in increasing the expression level of exogenous recombinant proteins. Background Art
[0002] Currently, nearly 89% of approved recombinant therapeutic antibodies are produced in CHO cell systems. CHO cells are not susceptible to human viral infection, are adaptable to industrial-scale suspension expression, easily and stably integrate target genes into the genome, and exhibit efficient gene replication and expression levels. Furthermore, recombinant antibodies produced in CHO cells exhibit glycosylation similar to that of human cells. However, some proteins are expressed at very low levels in CHO cells, or are even difficult to express. Therefore, there is an urgent need to provide new strategies to increase the expression of exogenous proteins. Summary of the Invention
[0003] The present invention provides a TP63 The application of genes in increasing the expression of exogenous recombinant proteins is achieved by constructing TP63 The overexpression vector TP63 Integration into the CHO cell locus resulted in an overexpression TP63 Gene-mediated CHO cell line, achieving TP63 The stable overexpression of CHO can improve the expression level of recombinant antibodies by applying it to the recombinant antibody expression system, which provides a direction for the construction of high-yield CHO cell lines and the improvement of recombinant antibody production.
[0004] The technical solution adopted in the present invention is: The present invention provides a TP63 Application of genes in increasing the expression of exogenous recombinant proteins, TP63 The nucleotide sequence of the gene is shown in SEQ ID NO.1.
[0005] Preferably, by constructing the TP63 Gene overexpression vector, using overexpression vector to prepare overexpression TP63 The recombinant CHO cell line of the gene is then transferred into the recombinant CHO cell line to increase the expression level of the exogenous recombinant protein.
[0006] Preferably, the preparation method of the overexpression vector is as follows: synthesis TP63 Gene; Will TP63 The gene is cloned into the expression vector after enzyme digestion to obtain the overexpression vector.
[0007] Preferably, the expression vector is pWTY-His-WPRE-NeoR.
[0008] Preferably, the restriction endonucleases used for enzymatic digestion of the expression vector are KpnI and NheI.
[0009] Preferably, the preparation method of the recombinant CHO cell line is as follows: CHO-S cells were pre-cultured; The overexpression vector is transfected into pre-cultured CHO-S cells, and the cells are screened using a resistance gene to obtain the recombinant CHO cell line.
[0010] Preferably, the pre-culture process is: CHO-S cells were cultured at 37°C, v / v 5% CO2 to the logarithmic growth phase; 1×10 per well 5 The cells were inoculated and cultured until the cell confluence reached 85%.
[0011] Preferably, the transfection process is: The overexpression vector was added to DMEM / F12 medium, mixed, and allowed to stand at room temperature for 5 minutes to obtain a mixed solution 1; the mass volume ratio of the overexpression vector to the DMEM / F12 medium was 2 μg:125 μL; Add the transfection reagent to the DMEM / F12 medium, mix well, and let stand at room temperature for 5 minutes to obtain mixed solution 2; the volume ratio of the transfection reagent to the DMEM / F12 medium is 1:25; the mass volume ratio of the overexpression vector to the transfection reagent is 2 μg:5 μL; Add mixed solution 2 to mixed solution 1, mix well, and let stand at room temperature for 20 minutes to obtain the transfection solution; The transfection solution was added to the pre-cultured CHO-S cells, and after incubation for 5 hours, the culture medium was replaced with a complete culture medium, which was a DMEM / F12 culture medium containing 10% v / v fetal bovine serum.
[0012] Preferably, the transfection reagent is Lipofectamine 3000.
[0013] Compared with the prior art, the present invention has the following beneficial effects: The present invention provides a TP63 Application of genes in increasing the expression of exogenous recombinant proteins, TP63 The nucleotide sequence of the gene is shown in SEQ ID NO.1. TP63 The coding gene nucleic acid sequence was cloned into the vector to construct TP63 The overexpression vector was integrated into the CHO cell gene after drug screening to achieve TP63 The present invention creatively overexpresses TP63 Overexpression vectors are used to construct recombinant protein expression systems. TP63 Overexpression vector was transfected into CHO cells to construct overexpression TP63 CHO cell line that can significantly increase TP63 Protein expression level. At the same time, this CHO cell line, as a host cell for recombinant protein expression, can increase the expression level of recombinant proteins compared to wild-type CHO cell lines, effectively overcoming the problem of low antibody expression levels in current CHO cell expression systems. BRIEF DESCRIPTION OF THE DRAWINGS
[0014] Figure 1 Schematic diagram of the structure of the overexpression vector.
[0015] Figure 2 for TP63 Overexpression efficiency detection results, A: Western blot results; B: statistical results of Figure A.
[0016] Figure 3 The results of the detection of recombinant antibody expression levels are shown in Figure 1. A: Western blot results; B: statistical results of Figure 1.
[0017] Figure 4 To detect the expression level of the reporter gene EGFP. DETAILED DESCRIPTION
[0018] The present invention will be further described below by way of specific examples, but the scope of the present invention is not limited thereto. The details and forms of the technical solution of the present invention may be modified or replaced without departing from the spirit and scope of the present invention, but such modifications or replacements fall within the scope of protection of the present invention.
[0019] The inventive concept of the present invention is as follows: The screening and use of transcription factors can simultaneously activate or inhibit a series of key genes, improving the gene expression related to growth, metabolism, cycle, proliferation, pluripotency and apoptosis in CHO cells, as well as the overall fitness of the cells. The use of transcription factors is an important strategy for improving CHO cell productivity. Therefore, screening transcription factors, constructing cell lines, and thus establishing an efficient CHO cell expression system are of great significance.
[0020] The present invention detects high monoclonal cell lines and medium monoclonal cell lines of the transposon system through RNA-Seq, and screens out the transcription factor TP63 that is upregulated in the high-expression monoclonal cell lines. TP63 belongs to the p53 family, and the transcription factors of this family are a group of important tumor suppressors that can regulate various cell functions, such as cell cycle arrest, differentiation, DNA repair, and cell apoptosis. TP63Whether overexpression can increase the expression level of recombinant antibodies in CHO cell lines has not been reported.
[0021] In order to make those skilled in the art better understand the technical solution of the present invention and be able to implement it, the present invention is further described below in conjunction with specific examples. In the description of the present invention, if not otherwise specified, the reagents used are all commercially available and the methods used are all conventional techniques in the art.
[0022] Example 1 TP63 The application of genes in increasing the expression of exogenous recombinant proteins is as follows: 1. Build TP63 Gene overexpression vector.
[0023] According to the transcriptome sequencing results, TP63 Gene sequence, synthesis TP63 The nucleic acid fragment sequence of the CDS region was synthesized by General Bio (Anhui) Co., Ltd. TP63 The nucleic acid fragment sequence of the CDS region is shown in SEQ ID NO.1. TP63 The CDS region of the WT was then cloned into the pWTY-His-WPRE-NeoR expression vector to obtain TP63 The overexpression vector, the structural diagram of the overexpression vector is shown in FIG. Figure 1 The construction process of the overexpression vector is as follows:
[0024] synthesis TP63 Gene, KpnI and NheI TP63 The gene fragment and the pWTY-His-WPRE-NeoR expression vector were digested with enzymes at 37°C for 3 h. The double enzyme digestion system is shown in Tables 1 and 2 below.
[0025] Table 1 TP63 Gene fragment double enzyme digestion system Table 2 Expression vector double enzyme digestion system After enzyme digestion, electrophoresis was performed and DNA was recovered using a DNA gel recovery kit. TP63 Gene fragment and expression vector; double enzyme digestion with T4 DNA ligase TP63 The gene fragment and the linearized vector pWTY-His-WPRE-NeoR were ligated at 16°C overnight. The ligation system is shown in Table 3.
[0026] Table 3 Connection system The ligation product was transformed into DH5α competent cells; clone colonies were picked from the plate, plasmids were extracted and identified to select positive clones; sequencing was performed to verify that this vector was named pWTY-His-TP63, which is an overexpression vector.
[0027] SEQ ID NO.1:
[0028] 2. Construct overexpression TP63 recombinant CHO cell lines.
[0029] CHO-S cell pre-culture: CHO-S cells were cultured in DMEM / F12 medium containing 10% v / v inactivated fetal bovine serum at 37°C and 5% v / v CO2 until the logarithmic growth phase. 5 The cells were seeded into 12-well cell culture plates for 36 h and used for cell transfection when the cell confluence reached about 85%.
[0030] The overexpression vector constructed above was transfected into the CHOS cells by lipofectamine transfection. DMEM / F12 antibiotic-free culture medium was added with transfection reagent and TP63 The overexpression vector was transfected into the above-mentioned CHOS cells as the overexpression group, denoted as the TP63 group. Meanwhile, blank CHOS cells were transfected with the blank control vector as the control group.
[0031] The specific liposome transfection method is as follows: (1) Taking a 24-well plate as an example, take a 1.5 mL centrifuge tube and mark the vector name. Add 50 μL of basal DMEM / F12 culture medium into the tube, then pipette 0.8 μg of overexpression vector into the tube, mix thoroughly, and let it stand at room temperature for 5 minutes.
[0032] (2) Take another centrifuge tube, mark it as the transfection reagent tube, add 50 μL of basal DMEM / F12 medium to the tube, then add 2 μL of transfection reagent Lipofectamine 3000 to the tube, mix thoroughly, and let it stand at room temperature for 5 minutes. Lipofectamine 3000 was purchased from Thermo Fisher Scientific.
[0033] (3) Add the mixture in step (2) dropwise to the tube in step (1), mix thoroughly, and let stand at room temperature for 20 min.
[0034] (4) Remove the cells plated for transfection the day before from the incubator, discard the old culture medium, wash each well twice with 1× PBS buffer, and then add 500 μL of new DMEM / F12 reduced serum medium without double antibody.
[0035] (5) Add the mixed solution in step (3) dropwise to the cells, gently shake the culture plate to evenly distribute the mixed solution, and then place it in the incubator for culture. After incubation for 5 hours, remove the culture plate and replace with fresh complete culture medium to continue culture; 48 hours after cell transfection, use 800µg / mL G418 for cell screening. After the drug treatment is continued until the cells no longer die, count the cell suspension and inoculate 1 cell per well into a 96-well plate, and wait for cell proliferation. When the cell fusion rate in the 96-well plate reaches about 90%, expand the culture and proceed to the subsequent passaging and screening steps.
[0036] 3. TP63 Detection of overexpression efficiency.
[0037] After taking the cells of the Control group and TP63 group out of the incubator, they were placed on an ice box, the original culture medium was discarded, and the cells were washed three times with pre-cooled PBS on ice to remove the residual cell culture medium. Protein lysis buffer RIPA was added to fully lyse the cells. Using an ultra-low temperature centrifuge, centrifuge at 1000rpm for 5 minutes, add 5×SDSLoading sample buffer to the collected cell culture supernatant, and incubate at 100°C for 10 minutes to fully denature it. Take 10 μL of protein samples respectively and separate them by SDSPAGE gel electrophoresis. AntiTP63 was diluted 1:3000 as the primary antibody, and Antiβactin was diluted 1:10000 as the secondary antibody for detection. The ECL chemiluminescence colorimetric solution was used for development, and the expression level of the protein to be tested was observed and analyzed on a gel imager. ECL chemiluminescence colorimetric solution was purchased from Biyuntian Biotechnology Co., Ltd.
[0038] The results are as follows Figure 2 As shown in the figure, compared with the CHO cells in the Control group, the expression level of TP63 protein in the CHO cells in the TP63 group was significantly increased.
[0039] 4. Expression of recombinant antibodies.
[0040] The eukaryotic expression vector of the monoclonal antibody in1-1 was transfected into the CHO cells of the TP63 group and the CHO cells of the control group. The construction of the eukaryotic expression vector of the monoclonal antibody in1-1 of the present invention was commissioned to a biotechnology company.
[0041] 48 h after transfection, CHO cells were cultured in 3 mL of chemically defined CD CHO medium in six-well plates for 6 days until the cell count reached 1 × 10 7Cells were collected daily and cell density and viability were measured using a Countstar BioTech cell counter. The supernatant was collected by centrifugation on the 7th day and quantified using the same cell number for analysis of recombinant in1-1 mAb expression. Western blot analysis was performed to analyze the expression of in1-1 in CHO cells in the TP63 group and the control group. Figure 3 As shown, the results showed that the CHO cells in the TP63 group could significantly increase the expression level of recombinant in1-1, with the expression level increasing by 2.05 times.
[0042] In addition, the expression plasmid carrying the reporter gene EGFP was transfected into the CHO cells of the TP63 group and the CHO cells of the control group. 48 hours after transfection, the EGFP fluorescence was observed under a fluorescence microscope and photographed. Figure 4 As shown, TP63-overexpressing CHO cells can significantly enhance the fluorescence intensity of EGFP.
[0043] The technical features of the above-mentioned embodiments can be combined arbitrarily. In order to make the description concise, not all possible combinations of the technical features in the above-mentioned embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.
[0044] The above-described embodiments merely illustrate several implementations of the present invention. While the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the present invention. It should be noted that a person skilled in the art would be able to make numerous variations and improvements without departing from the spirit of the present invention, and all such variations and improvements fall within the scope of protection of the present invention.
Claims
1. TP63 The application of a gene in increasing the expression of an exogenous recombinant protein is characterized in that: described TP63 The nucleotide sequence of the gene is shown in SEQ ID NO.
1.
2. The use according to claim 1, characterized in that By constructing the TP63 Gene overexpression vector, using overexpression vector to prepare overexpression TP63 The recombinant CHO cell line of the gene is then transferred into the recombinant CHO cell line to increase the expression level of the exogenous recombinant protein.
3. The use according to claim 2, characterized in that The preparation method of the overexpression vector is as follows: synthesis TP63 Gene; Will TP63 The gene is cloned into the expression vector after enzyme digestion to obtain the overexpression vector.
4. The use according to claim 3, characterized in that The expression vector is pWTY-His-WPRE-NeoR.
5. The use according to claim 4, characterized in that The restriction endonucleases used to digest the expression vector were KpnI and NheI.
6. The use according to claim 2, characterized in that The preparation method of the recombinant CHO cell line is as follows: CHO-S cells were pre-cultured; The overexpression vector is transfected into pre-cultured CHO-S cells, and the cells are screened using a resistance gene to obtain the recombinant CHO cell line.
7. The use according to claim 6, characterized in that The process of the pre-cultivation is: CHO-S cells were cultured to the logarithmic growth phase; Inoculate and culture until the cell confluence reaches more than 85%.
8. The use according to claim 6, characterized in that The transfection process is as follows: The overexpression vector was added to DMEM / F12 medium, mixed, and allowed to stand at room temperature to obtain a mixed solution 1; the mass volume ratio of the overexpression vector to the DMEM / F12 medium was 2 μg:125 μL; Add the transfection reagent to the DMEM / F12 medium, mix well, and let stand at room temperature to obtain mixed solution 2; the volume ratio of the transfection reagent to the DMEM / F12 medium is 1:25; the mass volume ratio of the overexpression vector to the transfection reagent is 2 μg:5 μL; Add mixed solution 2 to mixed solution 1, mix well, and let stand at room temperature to obtain the transfection solution; The transfection solution was added to the pre-cultured CHO-S cells, and after incubation, the culture medium was replaced with a complete culture medium, and then the culture was continued; the complete culture medium was a DMEM / F12 culture medium containing fetal bovine serum.
9. The use according to claim 8, characterized in that The transfection reagent is Lipofectamine 3000.