Novel plasmid vector capable of increasing CHO foreign protein expression quantity and construction and application of novel plasmid vector

Abstract

The invention discloses a novel plasmid vector capable of increasing CHO foreign protein expression quantity and construction and application thereof, and belongs to the technical field of gene engineering. The plasmid vector pCHO6GS belongs to a brand-new plasmid system, and the expression quantity of a common monoclonal antibody and recombinant protein (such as H5HA trimer) difficult to express in CHO can be increased by at least more than one time, so that the expression quantity of foreign protein is increased beyond expectation through the novel plasmid vector; and the method also plays an extremely important role in reducing cost and improving efficiency of various protein products taking CHO as a cell factory.

Description

Technical Field

[0001] The present invention belongs to the field of genetic engineering technology, and specifically relates to a novel plasmid vector capable of increasing the expression amount of CHO exogenous proteins, and the construction and application thereof. Background Art

[0002] Chinese hamster ovary (CHO) cells are the predominant eukaryotic cell expression system for modern commercial production of recombinant proteins. This family of cells originated in the laboratory of Dr. Theodore T. Puck in 1957. Since CHO cells were first used in bioreactors to produce complex large-molecule biopharmaceuticals in 1971, a variety of CHO cell-derived biologics have been approved for marketing, including monoclonal antibodies, interferon, and erythropoietin. However, compared to bacterial or yeast-based expression systems, the production of biopharmaceuticals using mammalian cell expression systems remains subject to certain limitations, such as limited resistance to environmental stress, low productivity, and high culture reagent costs.

[0003] With the continuous emergence of blockbuster biopharmaceuticals, the demand for biopharmaceutical APIs has surged from grams to kilograms and even tons, requiring a simultaneous increase in target protein unit production. By leveraging methods such as large-scale cell (and culture medium) screening based on expression levels, vector design based on optimized target gene expression enhancers or promoters, and metabolic pathway-based cell engineering, researchers have increased the expression level of monoclonal antibody drug engineering cell lines from approximately one gram per liter in the 1990s to an average of three to five grams per liter in recent years, with some even reaching over ten grams per liter.

[0004] Furthermore, technologies such as safe harbor regulation based on site-specific integration, regulation of apoptosis, key factors in the cell cycle and metabolism, and factors involved in protein secretion pathways have also been used in cell engineering research and have achieved considerable progress. The application of these new technologies and methods will pave the way for rationalized CHO cell line development and advanced biomanufacturing of complex proteins.

[0005] With the development of the emerging biotechnology pharmaceutical industry, typical monoclonal antibodies are being gradually replaced by novel drug molecules. An increasing number of complex novel protein drugs, such as viral antigens, recombinant trimeric subunit vaccines, and bispecific antibodies, are entering the industrial production pipeline. In many cases, these biomolecules are difficult to express, requiring customized solutions to improve transient or stable expression yields. For example, patent CN 118638868 A discloses an adenosine transporter ENT1 overexpression plasmid, its construction method, and applications. The ENT1 overexpression plasmid obtained in this invention can express a fluorescent fusion protein, which can indirectly characterize the expression of the exogenous ENT1 protein by observing its expression. The plasmid design improves the expression efficiency of the exogenous protein, increasing the expression level of ENT1 by approximately threefold. This reliable expression of ENT1 protein provides tools for subsequent upstream and downstream research on ENT1. This invention specifically designs a solution specifically for ENT1, specifically targeting the intracellular expression of the ENT1 protein.

[0006] There are many types of recombinant protein drugs, and increasing the production of exogenous recombinant proteins is particularly important for reducing drug costs and increasing drug efficiency. Current approaches focus on developing highly integrated, high-throughput screening platforms to identify more suitable cell clones, better culture media and feeds, and optimal cell culture conditions, thereby increasing the expression level of the target exogenous recombinant protein. High yields of exogenous proteins have not yet been achieved by overexpressing endogenous factors in CHO host cells on plasmids.

[0007] The CHO genome was sequenced by Xu et al. in 2011. After more than a decade of development, CHO gene annotation and other work have become increasingly sophisticated. Recent advances in multi-omics technologies, such as transcriptomics, proteomics, and metabolomics, have enabled the systematic analysis and characterization of important intracellular pathways and processes in bioprocessing, thereby identifying new target molecules and revealing their potential mechanisms of action. These targets can be identified through tools such as gain-of-function and loss-of-function techniques, laying the foundation for the development of superior cell-based production plants for difficult-to-express complex proteins.

[0008] However, from a cell engineering perspective, systematically transforming the protein synthesis, assembly and folding, and secretion pathways of CHO cells to make them an efficient expression system for "difficult-to-express" proteins is a more fundamental solution. This solution is currently receiving increasing attention from the scientific community, but research is still in its early stages and no better technical means have been discovered. Summary of the Invention

[0009] The purpose of the present invention is to provide a novel plasmid vector capable of increasing the expression of exogenous proteins in CHO. The plasmid vector pCHO6GS can increase the expression of general monoclonal antibodies and difficult-to-express recombinant proteins (such as H5HA trimer) in CHO by at least 1-fold, thus achieving an unexpected increase in the expression of exogenous proteins through the novel plasmid vector. It also plays an extremely important role in reducing costs and increasing efficiency of various protein products using CHO as cell factories.

[0010] The present invention is achieved through the following technical solutions:

[0011] A novel plasmid vector capable of increasing the expression of exogenous proteins in CHO cells, comprising:

[0012] The novel recombinant protein expression plasmid vector pCHO6GS is constructed by inserting no more than two target exogenous protein gene fragments into the vector;

[0013] The novel recombinant protein expression plasmid vector pCHO6GS is composed of a replication origin site, an ampicillin resistance gene, an SV40 early promoter, a glutamine synthetase gene, an SV40 intron and a transcription tailing signal, an mCMV promoter, an SV40 tailing signal, an mCMV promoter, an SV40 tailing signal, an SV40 early promoter, different isoforms of Bag6 protein, a ribosome insertion site, a blasticide resistance gene and an SV40 tailing signal connected in series in sequence.

[0014] Preferably, the target exogenous protein gene fragment includes any one or more of an antibody, an antibody fragment and a difficult-to-express recombinant protein.

[0015] Preferably, the target exogenous protein gene fragment is a secretory protein.

[0016] Preferably, the order of inserting the target exogenous protein gene fragment 1 and / or the target exogenous protein gene fragment 2 in the novel recombinant protein expression plasmid vector pCHO6GS into the novel recombinant protein expression plasmid vector pCHO6GS is:

[0017] Replication origin, ampicillin resistance gene, SV40 early promoter, glutamine synthetase gene, SV40 intron and transcription tailing signal, mCMV promoter, target exogenous protein gene fragment 1, SV40 tailing signal, mCMV promoter, target exogenous protein gene fragment 2, SV40 tailing signal, SV40 early promoter, different isoforms of Bag6 protein, ribosome insertion site, blasticidin resistance gene and SV40 tailing signal.

[0018] Preferably, the different isoforms of Bag6 protein include Bag6 isoforms X1 to X13.

[0019] Preferably, the different isomers of Bag6 protein are any one of Bag6 isomers X2, X3, X4, X5, X8 and X9.

[0020] A method for constructing a novel plasmid vector capable of increasing the expression of exogenous proteins in CHO cells comprises the following steps:

[0021] The fragment containing the replication origin and the ampicillin resistance gene, the glutamine synthetase gene, and the blasticidin gene fragment were obtained by PCR from the vectors pRRL-EGFP and PLV-Bla, respectively. The mCMV promoter, introns, and SV40 tailing signal were then linked in using seamless cloning technology. Different isoforms of the Bag6 protein were then ligated between the XbaI / XmaI residues to form the pCHO6GS vector.

[0022] The target exogenous protein gene fragment 1 (GOI-1) is connected between ClaI / NdeI;

[0023] The target exogenous protein gene fragment 2 (GOI-2) is connected between MuI / SpeI.

[0024] The above-mentioned vectors pRRL-EGFP and pLV-Bla are vectors owned by the laboratory of the applicant of the technical solution of this application. For details, please refer to the literature doi:10.1158 / 1541-7786.MCR-18-0718 and doi:10.1186 / 1742-4690-7-79.

[0025] A novel plasmid vector capable of increasing the expression of CHO exogenous proteins is used to increase the expression of exogenous proteins.

[0026] Compared with the prior art, the present invention has at least the following technical effects:

[0027] (1) The present invention provides a novel plasmid vector capable of increasing the expression of exogenous proteins in CHO cells. The plasmid vector pCHO6GS can increase the expression of general monoclonal antibodies and difficult-to-express recombinant proteins (such as H5HA trimer) in CHO cells by at least 1-fold. This achieves an unexpected increase in the expression of exogenous proteins through the novel plasmid vector, and also plays an extremely important role in reducing costs and increasing efficiency of various protein products using CHO as cell factories.

[0028] (2) This novel plasmid vector, capable of increasing the expression of exogenous proteins in CHO cells, represents a novel plasmid system that significantly increases the production of exogenous proteins after transfection into CHO cells. This plasmid incorporates the CHO cell's own bag6 gene (CHO-bag6). By co-expressing the target exogenous protein gene with the CHO-bag6 gene, the production of the target exogenous protein can be increased by 1-3 times.

[0029] (3) This novel plasmid vector for increasing the expression of exogenous proteins in CHO has the following advantages:

[0030] ① The universality of increasing the expression of exogenous proteins; ② Experimental verification shows that different isoforms of Bag6 can increase the expression of exogenous proteins, which facilitates the selection and construction of plasmid vectors with higher precision; ③ The plasmid carries the glutamine synthetase (GS) gene, which is compatible with GS-deficient cells and can be cloned and screened using glutamine-free culture medium; ④ The blasticidin resistance gene is introduced after the Bag6 gene through the ribosomal insertion site, and cloning and screening can be performed by adding the basticidin antibiotic; ⑤ The plasmid can express up to two exogenous genes at the same time, such as the heavy and light chain genes of an antibody. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 This is the plasmid map of pCHO6GS constructed in Example 1;

[0032] Figure 2 This is the plasmid map of pRRL-GPI-EGFP constructed in Example 2;

[0033] Figure 3 The difference in the effect of different bag6 isomers on the expression level of green fluorescent protein in Example 2

[0034] Figure 4 This is the plasmid map of pLV-bla-H5HA constructed in Example 3;

[0035] Figure 5 Comparison of the relative yields of H5HA trimer protein by different plasmids in Example 3

[0036] Figure 6 Comparison of recombinant H5HA trimer expression levels in Example 4;

[0037] Figure 7 Comparison of the expression levels of recombinant IVD antibodies in Example 5. DETAILED DESCRIPTION

[0038] The embodiments of the present invention will be described in detail below with reference to the examples. However, those skilled in the art will understand that the following examples are only used to illustrate the present invention and should not be construed as limiting the scope of the present invention. Specific conditions not specified in the examples are carried out according to conventional conditions or conditions recommended by the manufacturer. Reagents or instruments used without indicating the manufacturer are all conventional products that can be purchased commercially.

[0039] The technical solution of a specific embodiment of the present invention is:

[0040] The technical solution of this application is to conduct genomic and proteomic analysis on a CHO cell clone expressing a monoclonal antibody at a level of 6 g / L, and preliminarily obtain the chromosomal insertion site of the target exogenous gene, as well as some key host factor genes related to protein synthesis, degradation, folding, and secretion.

[0041] The discovery that one of the CHO host factor proteins, Bag6, was highly expressed in this high-yielding clone led to a systematic study of Bag6, which concluded that overexpressing Bag6 in CHO cells can effectively increase the expression levels of various recombinant proteins.

[0042] Therefore, a new recombinant protein expression vector pCHO6GS was further constructed, and it was found that this plasmid can increase the expression level of general monoclonal antibodies and the difficult-to-express recombinant protein H5HA trimer in CHO by at least 1 times.

[0043] At the same time, Bag6 has 13 different isoforms, and overexpression of isoforms 2, 3, 4, 5, 8 and 9 has the highest level of improvement in the expression of exogenous proteins.

[0044] Example 1: Construction of plasmid vector pCHO6GS

[0045] like Figure 1 Shown is the constructed pCHO6GS plasmid map.

[0046] pCHO6GS is composed of a replication origin (ori), an ampicillin resistance gene (AmpR), an SV40 early promoter (SV40E), a glutamine synthetase gene (GS cDNA), an SV40 intron and transcriptional cloning signal (SV40 intron+poly-A), an mCMV promoter (mCMV), a target foreign protein gene fragment 1 (GOI1), an SV40 cloning signal (SV40 poly-A), an mCMV promoter (mCMV), a target foreign protein gene fragment 2 (GOI2), an SV40 cloning signal (SV40 poly-A), an SV40 early promoter (SV40E), genes for different isoforms of the Bag6 protein (Bag6X1-X13), an ribosome insertion site (IRES), a blasticidin resistance gene (Blasticidin), and an SV40 cloning signal (SV40 poly-A) in series.

[0047] The pCHO6GS plasmid has the following features: 1) It contains the glutamine synthetase (GS) gene, making it compatible with GS-deficient cells and enabling cloning and screening using glutamine-free culture medium; 2) A blasticidin resistance gene is introduced after the Bag6 gene via a ribosomal insertion site, enabling cloning and screening by adding the antibiotic basticidin; and 3) the plasmid can simultaneously express up to two exogenous genes, such as the heavy and light chain genes of an antibody.

[0048] The nucleotide sequence of each of the above elements is shown below:

[0049] The nucleotide sequences of the elements of the pCHO6GS plasmid, 5'-3': SV40E, GS cDNA, SV40 intron+poly-A, mCMV, SV40 Poly-A, IRES, and Blasticidin are shown in SEQ ID NOs. 1-9.

[0050] The nucleotide sequences of Bag6 protein different isoform genes: Bag6X1-X13, 5'-3': are SEQ ID NO.10-22, respectively.

[0051] The nucleotide sequence of the pRRL-GPI-EGFP plasmid is shown in SEQ ID NO. 23;

[0052] The nucleotide sequence of the pLV-bla-H5HA plasmid is shown in SEQ ID NO.24.

[0053] Example 2: Comparison of the enhancement of exogenous protein expression by different Bag6 isoforms

[0054] Thirteen plasmids expressing Bag6 isoforms X1 to X13 were constructed and named pCHO6GS-X1 to pCHO6GS-X13, respectively. The EGFP-GPI gene was then inserted between ClaI and NdeI, and the plasmids were finally named pCHO6GS-X1-GPI-EGFP to pCHO6GS-X13-GPI-EGFP.

[0055] EGFP-GPI anchors enhanced green fluorescent protein (EGFP) to the cell surface through glycosylphosphatidylinositol (GPI).

[0056] like Figure 2 As shown, pRRL-GPI-EGFP is a lentiviral transfer vector carrying the EGFP gene. The plasmid is 7545 bp in size and the nucleotide sequence is shown in SEQ ID NO. 23. It serves as a control plasmid for the above-mentioned pCHO6GS-X1~X13-GPI-EGFP plasmid and is used to transfect CHO cells and express the EGFP protein as a participating plasmid.

[0057] The above 14 plasmids (13 Bag6 isoform plasmids and 1 pRRL-GPI-EGFP control plasmid) were transiently transfected into CHO cells, and the fluorescence intensity of EGFP expression was analyzed by flow cytometry. The relative fluorescence intensity was calculated using the formula: relative fluorescence intensity = fluorescence (pCHO6GS-Xn-EGPF) / fluorescence (pRRL-GPI-EGFP).

[0058] like Figure 3 Shown are the differences in the effects of different Bag6 isoforms on the expression level of green fluorescent protein.

[0059] Figure 3 The results showed that different Bag6 isoforms had different effects on enhancing the expression level of exogenous proteins, and isoforms 2, 3, 4, 5, 8, and 9 were preferred.

[0060] Example 3: Transient transfection of CHO cells with pCHO6GS vector and lentiviral transfer vector pLV-bla to analyze the difference in expression of secretory recombinant H5HA trimer protein

[0061] In order to verify the universality of bag6 in increasing the expression of exogenous proteins, the hemagglutinin protein H5HA of influenza A (H5N1) virus was selected and its trimer form was constructed. The expression level of this protein is low and it is a "difficult to express" protein.

[0062] The optimal pCHO6GS-X2 plasmid framework was selected to construct the plasmid pCHO6GS-X2-H5HA, and the lentiviral transfer vector pLV-bla-H5HA was used as a control.

[0063] like Figure 4 The nucleotide sequence of the constructed pLV-bla-H5HA plasmid is shown in SEQ ID NO. 24.

[0064] like Figure 5 Shown is a comparison of the relative yields of H5HA trimer protein by different plasmids.

[0065] Figure 5 The results showed that after CHO cells were transfected with Lipo2000 and cultured for 3 days, the cell supernatant was taken and the target protein content was detected using FortebioOctet. It was found that the protein expression level was significantly increased (about 1.8 times) compared with the control.

[0066] Example 4: Construction of stable transfectants using pCHO6GS vector and pLV-bla lentiviral vector to analyze differences in expression of recombinant H5HA trimer protein

[0067] To eliminate the possible differences in transfection efficiency during transient transfection, stable cell lines were constructed to evaluate the effects of the new plasmid on the expression level of exogenous proteins. The pCHO6GS-X2-H5HA plasmid was transferred into CHO cells using an electroporator. H5HA was then transferred into CHO cells using a four-plasmid lentiviral vector system (pLV-bla-H5HA, pLP1, pLP2, and pVSVG). Stable cell lines were screened using blasticidin.

[0068] like Figure 6 Shown is a comparison of the expression levels of recombinant H5HA trimers.

[0069] Figure 6 The results showed that after the stably transfected cells were cultured in a fed-batch culture method for 14 days, the target protein content was detected using the Fortebio Octet assay, and it was found that the protein expression level was significantly increased (approximately 2.66 times) compared with the control.

[0070] Example 5: Construction of stable transfectants using pCHO6GS vector and traditional pLV-bla lentiviral vector to analyze the difference in expression levels of recombinant IVD antibody 1 and IVD antibody 2

[0071] To verify the universality of bag6 in enhancing the expression level of exogenous proteins, other exogenous proteins were further tested.

[0072] Two in vitro diagnostic antibodies were selected and the pCHO6GS-X2-Ab1 and pCHO6GS-X2-Ab2 vectors were constructed, respectively. The pLV-bla-Ab1 and pLV-bla-Ab2 lentiviral vectors were used as controls.

[0073] like Figure 7 Shown is a comparison of the expression levels of recombinant IVD antibodies.

[0074] The same method as in Example 4 was used to construct a stable cell line.

[0075] Figure 7 The results showed that after the stably transfected cells were cultured using a fed-batch method for 14 days, the target protein content on the cells was detected using Fortebio Octet, and it was found that the expression levels of the two antibody proteins were significantly increased compared with the control.

[0076] Finally, it should be noted that the above description is only a preferred embodiment of the present invention and is not intended to limit the scope of protection of the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention shall be included in the scope of protection of the present invention.

Claims

1. A novel plasmid vector capable of increasing the expression of exogenous proteins in CHO cells, characterized in that: include: The novel recombinant protein expression plasmid vector pCHO6GS is constructed by inserting no more than two target exogenous protein gene fragments into the vector; The novel recombinant protein expression plasmid vector pCHO6GS is composed of a replication origin site, an ampicillin resistance gene, an SV40 early promoter, a glutamine synthetase gene, an SV40 intron and a transcription tailing signal, an mCMV promoter, an SV40 tailing signal, an mCMV promoter, an SV40 tailing signal, an SV40 early promoter, different isoforms of Bag6 protein, a ribosome insertion site, a blasticide resistance gene and an SV40 tailing signal connected in series in sequence.

2. A novel plasmid vector capable of increasing the expression of exogenous proteins in CHO cells according to claim 1, characterized in that: The target exogenous protein gene fragment includes any one or more of antibodies, antibody fragments and difficult-to-express recombinant proteins.

3. A novel plasmid vector capable of increasing the expression of exogenous proteins in CHO cells according to claim 2, characterized in that: The target exogenous protein gene fragment is a secretory protein.

4. A novel plasmid vector capable of increasing the expression of exogenous proteins in CHO cells according to claim 1, characterized in that: The order of inserting the target exogenous protein gene fragment 1 and / or the target exogenous protein gene fragment 2 in the novel recombinant protein expression plasmid vector pCHO6GS into the novel recombinant protein expression plasmid vector pCHO6GS is: Replication origin, ampicillin resistance gene, SV40 early promoter, glutamine synthetase gene, SV40 intron and transcription tailing signal, mCMV promoter, target exogenous protein gene fragment 1, SV40 tailing signal, mCMV promoter, target exogenous protein gene fragment 2, SV40 tailing signal, SV40 early promoter, different isoforms of Bag6 protein, ribosome insertion site, blasticidin resistance gene and SV40 tailing signal.

5. The novel plasmid vector for increasing the expression of CHO exogenous proteins according to claim 1, characterized in that: The different isoforms of the Bag6 protein include Bag6 isoforms X1 to X13.

6. A novel plasmid vector capable of increasing the expression of exogenous proteins in CHO cells according to claim 5, characterized in that: The different isomers of Bag6 protein are any one of Bag6 isomers X2, X3, X4, X5, X8 and X9.

7. A method for constructing a novel plasmid vector capable of increasing the expression of exogenous proteins in CHO cells according to any one of claims 1 to 6, characterized in that: The steps include: The fragment containing the replication origin and ampicillin resistance gene, the glutamine synthetase gene, and the blasticidin gene fragment were obtained by PCR from the vectors pRRL-EGFP and PLV-Bla, respectively; and the gene fragments including the mCMV promoter, intron, and SV40 tailing signal were linked in by seamless cloning technology. Then different isoforms of Bag6 protein were connected between XbaI / XmaI to finally form the pCHO6GS vector; The target exogenous protein gene fragment 1 is connected between ClaI / NdeI; The target exogenous protein gene fragment 2 is connected between MuI / SpeI.

8. Use of the novel plasmid vector according to any one of claims 1 to 6 for increasing the expression of exogenous proteins in CHO cells.

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