Tcf7l1 overexpression vector, cell line, system, kit, construction method and application

Abstract

The invention relates to the technical field of recombinant protein expression and cell culture, in particular to a Tcf7l1 overexpression vector, a cell line, a system, a kit, a construction method and application. The Tcf7l1 transcription factor is creatively selected as a genetic engineering modification target of recombinant protein expression, for example, a Tcf7l1 overexpression vector is transfected to a CHO cell, stable overexpression of CHO cell Tcf7l1 is achieved, the overexpressed Tcf7l1 transcription factor regulates the growth performance of the CHO cell, and when the Tcf7l1 transcription factor serves as a host cell for recombinant protein expression, the yield of recombinant protein is increased; compared with a wild type CHO cell line, the Tcf7l1 overexpression cell line constructed by the invention is used as a foreign protein recombinant expression system, so that the recombinant protein expression quantity is increased, and the problem of low expression level of the existing CHO cell expression system is effectively solved.

Description

Technical Field

[0001] The present invention relates to the field of recombinant protein expression and cell culture technology, and in particular to a Tcf7l1 overexpression vector, a cell line, a system, a kit, a construction method and an application. Background Art

[0002] Currently, nearly 70% of the approved recombinant therapeutic proteins (RTPs) are produced by the CHO cell system because CHO cells have the ability to produce glycosylation similar to that of human cells. In addition, CHO cells are not susceptible to human virus infection, can adapt to industrial large-scale suspension expression, are easy to stably integrate target genes into the genome, and show efficient gene replication ability and expression levels. However, the expression level of some proteins in CHO cells is still very low or even difficult to express. Further optimization strategies are needed to increase the expression level and effectively express "difficult to express" proteins in CHO cells.

[0003] Mammalian cells have a sophisticated regulatory mechanism for protein expression, including transcription, translation, and modification regulation. Among them, the regulation of transcription includes the regulation of the production of downstream proteins based on the upstream gene transcription level, which largely determines the regulation of downstream protein production, and also includes the regulation by reducing or overexpressing transcription factors that affect the growth performance of mammalian cells. There have been many reports on the transformation of the CHO recombinant protein expression system based on the regulatory mechanism of upstream gene transcription levels on downstream proteins. Using transcription factors, a series of key genes are activated or inhibited at the same time to improve CHO cell growth, secretion, and the overall fitness of cells. However, there are few reports on the transformation strategies to improve CHO cell productivity, and there is no industrial application. Therefore, it is of great significance to further screen transcription factors, optimize vectors based on transcription factors, and transform cell lines to construct an efficient CHO cell expression system. Summary of the invention

[0004] In order to overcome the defects of the prior art, one of the objectives of the present invention is to provide a Tcf7l1 overexpression vector, which can be used to construct a recombinant protein expression system and increase the yield of recombinant protein.

[0005] The second object of the present invention is to provide an application of a cell line constructed by a Tcf7l1 overexpression vector as a host cell for recombinant protein expression, thereby increasing the yield of recombinant protein.

[0006] The third object of the present invention is to provide an application of the Tcf7l1 overexpression vector in constructing a recombinant protein expression system to increase the yield of the recombinant protein.

[0007] A fourth object of the present invention is to provide a kit for constructing a recombinant protein expression system, comprising the Tcf7l1 overexpression vector provided by the present invention.

[0008] A fifth object of the present invention is to provide a method for constructing the recombinant protein expression system provided by the present invention.

[0009] In order to achieve the above object, the technical solution adopted by the present invention is as follows:

[0010] Application of Tcf7l1 overexpressing cell lines as host cells for recombinant protein expression.

[0011] The application of the Tcf7l1 overexpression vector in constructing a recombinant protein expression system includes first transfecting Tcf7l1 overexpression into a host cell to construct a Tcf7l1 overexpression cell line, and then transfecting the recombinant protein expression vector into the Tcf7l1 overexpression cell line;

[0012] Alternatively, the Tcf7l1 overexpression vector and the recombinant protein expression vector are co-transfected into the host cells.

[0013] The Tcf7l1 overexpression vector comprises a Tcf7l1 gene encoding nucleotide sequence, and the nucleotide sequence is shown in SEQ ID NO.1.

[0014] In a specific embodiment of the present invention, the Tcf7l1 overexpression vector is constructed by inserting the Tcf7l1 gene encoding nucleotide sequence between the PB513B-Myc-T2A-Puro plasmid promoter and the Myc element.

[0015] The Tcf7l1 overexpression cell line is constructed by transfecting the above-mentioned Tcf7l1 overexpression vector into host cells.

[0016] A recombinant protein expression system, using the above cell line as a host cell,

[0017] A kit for constructing a recombinant protein expression system comprises a host cell, the above-mentioned Tcf7l1 overexpression vector, and a recombinant protein expression vector.

[0018] In a specific embodiment of the present invention, the recombinant protein expression vector is an infliximab expression vector. It should be understood that the present invention creatively constructs a Tcf7l1 overexpressing cell line, regulates the growth performance of the cell line by the Tcf7l1 transcription factor, and then regulates the recombinant protein yield of the cell line as a host cell for recombinant protein expression. Therefore, from a theoretical logic analysis, in the case where the embodiment of the present invention has verified that the Tcf7l1 overexpressing cell line can increase the recombinant expression yield of infliximab, the Tcf7l1 overexpressing cell line should also be able to increase the recombinant expression yield of other similar protein products.

[0019] In a specific embodiment of the present invention, the host cell is a CHO cell.

[0020] A method for constructing a recombinant protein expression system comprises firstly transfecting a Tcf7l1 overexpression vector into a host cell to construct a Tcf7l1 overexpression cell line; then transfecting a recombinant protein expression vector into the Tcf7l1 overexpression cell line to construct the system; or co-transfecting a Tcf7l1 overexpression vector and a recombinant protein expression vector into a host cell to construct the system.

[0021] Beneficial effects of the present invention:

[0022] 1) The present invention creatively selects Tcf7l1 transcription factor as a genetic engineering target for recombinant protein expression, for example, by transfecting a Tcf7l1 overexpression vector into CHO cells to achieve stable overexpression of Tcf7l1 in CHO cells. The overexpressed Tcf7l1 transcription factor regulates the growth performance of CHO cells, and when used as a host cell for recombinant protein expression, the yield of recombinant protein is increased;

[0023] 2) The Tcf7l1 overexpression cell line constructed by the present invention is used as an exogenous protein recombinant expression system. Compared with the wild-type CHO cell line, it improves the expression amount of the recombinant protein, and effectively overcomes the problem of low expression level in the current CHO cell expression system. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 Schematic diagram of the vector structure for overexpressing Tcf7l1;

[0025] Figure 2 The expression level of Tcf7l1 protein in the CHO cell line overexpressing Tcf7l1 constructed in Example 2;

[0026] Figure 3 The relative expression level of infliximab recombinantly expressed in the CHO cell line overexpressing Tcf7l1 in Example 4;

[0027] Figure 4 This is the fluorescence intensity of EGFP recombinantly expressed in the CHO cell line overexpressing Tcf7l1 in Example 4. DETAILED DESCRIPTION

[0028] The present invention is further described below in conjunction with specific implementation modes, but the protection scope of the present invention is not limited thereto.

[0029] Unless otherwise specified, the vector construction and vector transfection into cells involved in the following embodiments are all achieved by conventional means by those skilled in the art.

[0030] Specifically, the liposome transfection method in the following embodiment is specifically as follows:

[0031] (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 basic DMEM / F12 medium into the tube, then pipette 0.8 μg of the vector to be transfected into the tube, mix thoroughly, and let stand at room temperature for five minutes.

[0032] (2) Take another centrifuge tube, mark it as the transfection reagent tube, add 50 μL of basic DMEM / F12 medium into the tube, then add 2 μL of transfection reagent Lipofectamine 3000 into the tube, mix thoroughly, and let stand at room temperature for five minutes;

[0033] (3) Add the mixed solution in step (2) dropwise to the vector plasmid tube in step (1), mix thoroughly, and let stand at room temperature for 20 min;

[0034] (4) The cells plated for transfection the day before were removed from the incubator, the old culture medium was discarded, each well was washed twice with 1× PBS buffer, and then 500 μL of new DMEM / F12 culture medium without dual antibodies was added;

[0035] (5) Add the mixed solution in step (3) dropwise into the wells, gently shake the culture plate to evenly distribute the mixed solution, and then place it in an incubator for culture. After incubation for 5 hours, remove the culture plate and replace with fresh complete culture medium to continue culture.

[0036] Example 1 Construction of Tcf7l1 overexpression vector:

[0037] The specific test method is: based on the sequence of Tcf7l1 gene, the hTcf7l1 CDS region nucleic acid fragment sequence was synthesized (commissioned to General Biotechnology (Anhui) Co., Ltd. for synthesis), as shown in SEQ ID NO.1.

[0038] The synthesized hTcf7l1 CDS region nucleic acid fragment was cloned and inserted into the PB513B-Myc-T2A-Puro plasmid (commissioned by General Biotechnology (Anhui) Co., Ltd.) to obtain the hTcf7l1 overexpression vector. The structural diagram of the vector is shown in Figure 1 shown.

[0039] Example 2 Construction of a CHO cell line overexpressing Tcf7l1:

[0040] CHO-S cells (purchased from Gibco) were cultured in DMEM / F12 medium containing 10% inactivated fetal bovine serum at 37°C and 5% CO2 until the logarithmic growth phase. 5The cells were inoculated into a 12-well cell culture plate for 24 hours and used for cell transfection when the cell confluence reached about 85%.

[0041] 50 μL of DMEM / F12 medium was added to 0.5 mL of serum-free medium (purchased from Henan Punuoyi Biological Products Research Institute Co., Ltd.), and the hTcf7l1 overexpressing vector constructed in Example 1 was transfected into the CHO-S cells obtained above at 1.5 μg / well and 1 μg / well, respectively, by liposome transfection. 48 hours after cell transfection, the optimal Purinomycin concentration was used for cell screening. After drug treatment, the cells no longer died, the cell suspension was counted, and 1 cell per well was inoculated into a 96-well plate, and the cells were allowed to proliferate. When the cell fusion rate in the 96-well plate reached about 90%, the culture was expanded, and subsequent passaging and screening steps were performed.

[0042] CHO cell lines overexpressing hTcf7l1 were constructed and used as overexpression groups (Tcf7l1 (1.5 μg) group and Tcf7l1 (1 μg) group). Meanwhile, blank CHO-S cells were used as control group (Ctrl group).

[0043] Example 3 Detection of hTcf7l1 overexpression efficiency

[0044] After taking out the two groups of cells (Ctrl group and Tcf7l1 (1.5 μg) group, Tcf7l1 (1 μg) group) from 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, and RIPA protein lysis buffer was added. The cells were shaken back and forth to fully fuse the lysis solution with the cells in the culture bottle, and the cells were fully lysed. The ultra-low temperature centrifuge was used to centrifuge at 1000 rpm for 5 minutes, and 5×SDS sample buffer was added to the collected cell culture supernatant, and incubated at 100°C for 10 minutes to fully denature it. 10 μL of protein samples were taken respectively for separation by 10% SDS-PAGE gel electrophoresis, and Anti-Myc (1:3000) and Anti-β-actin (1:3000) were used for detection, and the ECL chemiluminescent colorimetric solution (purchased from Biyuntian Biotechnology Co., Ltd.) was used for development, and the expression level of the tested protein was observed and analyzed on a gel imager.

[0045] The results are as follows Figure 2 As shown, compared with the CHO cells in the Ctrl group, the expression level of hTcf7l1 protein in the CHO cells in the Tcf7l1 (1.5 μg) group and the Tcf7l1 (1 μg) group was significantly increased, and as the transfection amount of the Tcf7l1 overexpression vector increased, the expression level of Tcf7l1 increased, indicating that Example 2 successfully constructed and obtained Tcf7l1 overexpressing CHO cells.

[0046] Example 4 Recombinant protein expression

[0047] The eukaryotic expression vector of infliximab constructed in the laboratory was transfected into CHO cells in the Tcf7l1 (1.5 μg) group, Tcf7l1 (1 μg) group, and Ctrl group. 48 h after transfection, the recombinant CHO cells were cultured in 3 mL of protein-free, serum-free, chemically defined CD CHO medium (Henan Punuoyi Biological Products Research Institute Co., Ltd.) in a six-well plate for 6 days until the cell number reached 1×10 7 Cells were collected every day and the cell density and viability were detected by Countstar 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 infliximab.

[0048] Western blot was used to analyze the expression of infliximab in CHO cells in the Tcf7l1 (1.5 μg) group, Tcf7l1 (1 μg) group, and Control group. Figure 3 As shown, the results showed that CHO cells overexpressing Tcf7l1 could significantly increase the expression level of recombinant infliximab, with the expression level increasing by 2.2 times.

[0049] In addition, the expression plasmid carrying the reporter gene EGFP was transfected into the CHO cells of the Tcf7l1 group (1 μg) and the Control group. The EGFP fluorescence was observed under a fluorescence microscope 48 hours after transfection and photographed. Figure 4 As shown, Tcf7l1-overexpressing CHO cells can significantly enhance the fluorescence intensity of EGFP.

[0050] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some of the technical features therein. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. Application of Tcf7l1 overexpressing cell lines as host cells for recombinant protein expression.

2. Application of Tcf7l1 overexpression vector in constructing a recombinant protein expression system, characterized in that: The method comprises first transfecting Tcf7l1 overexpression into a host cell to construct a Tcf7l1 overexpression cell line, and then transfecting a recombinant protein expression vector into the Tcf7l1 overexpression cell line; Alternatively, the Tcf7l1 overexpression vector and the recombinant protein expression vector are co-transfected into the host cells.

3. Tcf7l1 overexpression vector, characterized in that: It contains a Tcf7l1 gene encoding nucleotide sequence, and its nucleotide sequence is shown in SEQ ID NO.

1.

4. The Tcf7l1 overexpression vector according to claim 3, characterized in that It was constructed by inserting the Tcf7l1 gene encoding nucleotide sequence between the PB513B-Myc-T2A-Puro plasmid promoter and the Myc element.

5. A Tcf7l1 overexpressing cell line, characterized in that The method comprises transfecting the Tcf7l1 overexpression vector as described in claim 3 or 4 into a host cell.

6. A recombinant protein expression system, characterized in that: The cell line according to claim 5 is used as a host cell.

7. A kit for constructing a recombinant protein expression system, characterized in that: It includes host cells, Tcf7l1 overexpression vectors, and recombinant protein expression vectors.

8. The kit for constructing a recombinant protein expression system according to claim 7, wherein: The Tcf7l1 overexpression vector includes a Tcf7l1 gene encoding nucleotide sequence, and the nucleotide sequence is shown in SEQ ID NO.1; Optionally, the Tcf7l1 overexpression vector is constructed by inserting the Tcf7l1 gene encoding nucleotide sequence between the PB513B-Myc-T2A-Puro plasmid promoter and the Myc element; Optionally, the recombinant protein expression vector is an infliximab expression vector.

9. The use according to any one of claims 1 to 2, or the cell line according to claim 5, or the expression system according to claim 6, or the kit according to any one of claims 7 to 8, characterized in that: The host cell is a CHO cell.

10. A method for constructing a recombinant protein expression system, characterized in that: The method comprises firstly transfecting a Tcf7l1 overexpression vector into a host cell to construct a Tcf7l1 overexpression cell line; and then transfecting a recombinant protein expression vector into the Tcf7l1 overexpression cell line to construct the cell line; Alternatively, the Tcf7l1 overexpression vector and the recombinant protein expression vector are co-transfected into the host cells to construct; Optionally, the host cell is a CHO cell; Optionally, the recombinant protein expression vector is an infliximab expression vector.

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