A novel plasmid vector with improved expression of CHO exogenous proteins and construction and application thereof

Abstract

The application discloses a novel plasmid vector with improved CHO exogenous protein expression amount and construction and application thereof, and belongs to the technical field of genetic engineering. The plasmid vector pCHO6GS belongs to a novel plasmid system, and can at least improve the expression amount of general monoclonal antibodies and difficult-to-express recombinant proteins (such as H5HA trimer) in CHO by more than 1 times, that is, the unexpected improvement of the exogenous protein expression amount is realized by the novel plasmid vector, and the novel plasmid vector has an extremely important role in cost reduction and efficiency increase of various protein products with CHO as a cell factory.

Description

Technical Field

[0001] This invention belongs to the field of genetic engineering technology, specifically relating to a novel plasmid vector that enhances the expression of exogenous CHO proteins, its construction, and its application. Background Technology

[0002] Chinese hamster ovary (CHO) cells are the most important eukaryotic cell expression system for the modern commercial production of recombinant proteins. This family of cells was first developed in 1957 in the laboratory of Dr. Theodore T. Puck. Since 1971, when CHO cells were used in bioreactors to produce complex macromolecular biopharmaceuticals, various CHO cell-derived biopharmaceuticals have been approved for marketing, including monoclonal antibodies, interferons, and erythropoietin. However, compared to bacterial or yeast-based expression systems, the use of mammalian cell expression systems for biopharmaceutical production still faces certain limitations, such as limited resilience to environmental stress, low productivity, and high costs of culture reagents.

[0003] With the continuous emergence of blockbuster biopharmaceuticals, the demand for biopharmaceutical raw materials has been leaping from the gram level to the kilogram level and even the ton level, which requires a corresponding increase in the unit yield of target proteins. Researchers have used methods such as large-scale cell (and culture medium) screening based on expression levels, vector design based on the optimization of target gene expression enhancers or promoters, and cell engineering based on metabolic pathways to increase the expression level of monoclonal antibody drugs in engineered cell lines from about one gram per liter in the 1990s to an average of three to five grams per liter in recent years, with some even reaching more than ten grams per liter.

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

[0005] With the development of the emerging biopharmaceutical industry, typical monoclonal antibody drugs are gradually being 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 industrial production pipelines. In most cases, these biomolecules are difficult to express, requiring tailored solutions to improve their transient or stable expression yields. For example, patent CN 118638868 A discloses an ENT1 overexpression plasmid, its construction method, and its applications. The ENT1 overexpression plasmid obtained by this invention can express fluorescent fusion proteins, and the expression of exogenous ENT1 proteins can be indirectly characterized by observing the expression of fluorescent proteins. The plasmid design improves the expression efficiency of exogenous proteins, increasing the expression level of ENT1 protein by approximately three times. ENT1 protein expression is reliable, providing tool support for subsequent upstream and downstream research on ENT1. This invention specifically designs a solution for the ENT1 protein, and this customized solution is specifically for the intracellular expression of the ENT1 protein.

[0006] Recombinant protein drugs are diverse, and increasing the yield of exogenous recombinant proteins is crucial for cost reduction and efficacy improvement. Current methods primarily focus on developing highly integrated, high-throughput screening platforms to identify more suitable cell clones, better culture media and feed, and optimal cell culture conditions, thereby increasing the expression level of the target exogenous recombinant protein. There is currently no evidence of achieving high-yield exogenous proteins through overexpression of endogenous factors in CHO host cells on plasmids.

[0007] The CHO genome sequencing was completed by Xu et al. in 2011, and after more than a decade of development, CHO gene annotation and other related 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 biological processing, thereby identifying new target molecules and revealing their potential mechanisms of action. The identification of these target molecules can be achieved through gain-of-function or loss-of-function techniques, laying the foundation for establishing superior cellular production facilities for complex proteins that are difficult to express.

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

[0009] The purpose of this invention is to provide a novel plasmid vector that can enhance the expression of exogenous proteins in CHO. This plasmid vector pCHO6GS can increase the expression of common monoclonal antibodies and difficult-to-express recombinant proteins (such as H5HA trimer) in CHO by at least 1 time. This achieves an unexpected increase in the expression of exogenous proteins through this novel plasmid vector, and also plays an extremely important role in reducing costs and increasing efficiency for various protein products using CHO as a cell factory.

[0010] This invention is achieved through the following technical solution:

[0011] A novel plasmid vector capable of enhancing the expression of exogenous CHO proteins includes:

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

[0013] The novel recombinant protein expression plasmid vector pCHO6GS is composed of the replication origin site, ampicillin resistance gene, SV40 early promoter, glutamine synthase gene, SV40 intron and transcription tailing signal, mCMV promoter, SV40 tailing signal, mCMV promoter, SV40 tailing signal, SV40 early promoter, different isoforms of Bag6 protein, ribosome intervention site, blast fungicide resistance gene and SV40 tailing signal in sequence.

[0014] Preferably, the target exogenous protein gene fragment includes any one or more of antibodies, antibody fragments, and recombinant proteins that are difficult to express.

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

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

[0017] Replication origin site, ampicillin resistance gene, SV40 early promoter, glutamine synthase 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 intervention site, blast fungicide resistance gene and SV40 tailing signal.

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

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

[0020] A method for constructing a novel plasmid vector that enhances the expression level of exogenous CHO proteins includes the following steps:

[0021] Fragments containing the replication origin and ampicillin resistance gene, glutamine synthase gene, and blast fungicide gene fragment were obtained by PCR from vectors pRRL-EGFP and PLV-Bla, respectively. Then, gene fragments such as mCMV promoter, intron, and SV40 tailing signal were ligated into the vector using seamless cloning technology. Finally, different isoforms of Bag6 protein were ligated between XbaI and XmaI to form the pCHO6GS vector.

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

[0023] The target exogenous protein gene fragment 2 (GOI-2) is ligated between MuliI and SpeI.

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

[0025] Application of a novel plasmid vector that enhances the expression of exogenous CHO protein in enhancing the expression of exogenous proteins.

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

[0027] (I) This invention provides a novel plasmid vector that can enhance the expression of exogenous proteins in CHO. The plasmid vector pCHO6GS can increase the expression of common monoclonal antibodies and difficult-to-express recombinant proteins (such as H5HA trimer) in CHO by at least 1 time. This novel plasmid vector achieves an unexpected increase in the expression of exogenous proteins and plays an extremely important role in reducing costs and increasing efficiency for various protein products using CHO as a cell factory.

[0028] (II) This novel plasmid vector, which enhances the expression of exogenous proteins in CHO cells, belongs to a completely new plasmid system. After transfection into CHO cells, it can greatly increase the yield of exogenous proteins. The CHO cell's own bag6 gene (CHO-bag6) is added to this plasmid. By co-expressing the target exogenous protein gene and the CHO-bag6 gene, the yield of the target exogenous protein can be increased by 1-3 times.

[0029] (III) This novel plasmid vector, which enhances the expression of exogenous CHO proteins, has the following advantages:

[0030] ① Universality in enhancing exogenous protein expression; ② Experimental verification of the enhancement levels of exogenous protein expression by different Bag6 isoforms, facilitating more precise selection and construction of plasmid vectors; ③ The plasmid contains the glutamine synthase (GS) gene, compatible with GS-deficient cells, allowing for cloning and screening using glutamine-free media; ④ The Bag6 gene is followed by a ribosome-mediated introduction of a blastcin resistance gene, enabling cloning and screening by adding Basticidin antibiotic; ⑤ This plasmid can simultaneously express up to two exogenous genes, such as the heavy and light chain genes of antibodies. Attached Figure Description

[0031] Figure 1 The pCHO6GS plasmid constructed in Example 1 is shown in the image.

[0032] Figure 2 The pRRL-GPI-EGFP plasmid constructed in Example 2 is shown in the image.

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

[0034] Figure 4 The pLV-bla-H5HA plasmid constructed in Example 3 is shown in the image.

[0035] Figure 5 This is a comparison of the relative yields of H5HA trimer protein by different plasmids in Example 3.

[0036] Figure 6 This is a comparison of the expression levels of recombinant H5HA trimer in Example 4;

[0037] Figure 7 This is a comparison of the expression levels of recombinant IVD antibodies in Example 5. Detailed Implementation

[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 for illustrating the present invention and should not be regarded as limiting the scope of the present invention. Specific conditions not specified in the examples shall be carried out according to conventional conditions or conditions recommended by the manufacturer. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased commercially.

[0039] One specific embodiment of the present invention is as follows:

[0040] The technical solution of this application involves genomic and proteomic analysis of a CHO cell clone expressing a monoclonal antibody at a level of 6 g / L. Preliminary results were obtained of 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 study identified Bag6, a host factor protein native to CHO cells, as being highly expressed in this high-yield clone. A systematic study of Bag6 was then conducted, demonstrating that overexpression of Bag6 protein in CHO cells can effectively enhance the expression levels of various recombinant proteins.

[0042] Therefore, a novel recombinant protein expression vector, pCHO6GS, was constructed. It was found that this plasmid could increase the expression levels of common monoclonal antibodies and the difficult-to-express recombinant protein H5HA trimer in CHO by at least 1-fold.

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

[0044] Example 1: Construction of plasmid vector pCHO6GS

[0045] like Figure 1 The image shown is a map of the constructed pCHO6GS plasmid.

[0046] pCHO6GS is composed of the replication origin (ori), ampicillin resistance gene (AmpR), SV40 early promoter (SV40E), glutamine synthase gene (GS cDNA), SV40 intron and transcription tailing signal (SV40 intron+poly-A), mCMV promoter (mCMV), target exogenous protein gene fragment 1 (GOI1), SV40 tailing signal (SV40 poly-A), mCMV promoter (mCMV), target exogenous protein gene fragment 2 (GOI2), SV40 tailing signal (SV40 poly-A), SV40 early promoter (SV40E), different isoforms of Bag6 protein (Bag6X1-X13), ribosome intervention site (IRES), blastin resistance gene (Blasticidin), and SV40 tailing signal (SV40 poly-A) in sequence.

[0047] The pCHO6GS plasmid has the following characteristics: 1) The plasmid carries the glutamine synthase (GS) gene, making it compatible with GS-deficient cells and allowing for cloning and screening using glutamine-free culture media; 2) A blastcin resistance gene is introduced after the Bag6 gene via a ribosome entry site, allowing for cloning and screening by adding the antibiotic Basticidin; 3) This plasmid can simultaneously express up to two exogenous genes, such as the heavy and light chain genes of antibodies.

[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 NO. 1-9.

[0050] Different isoforms of the Bag6 protein: nucleotide sequences of Bag6X1-X13, 5'-3': 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 levels 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. Then, the EGFP-GPI gene was inserted between ClaI and NdeI, and the final names were pCHO6GS-X1-GPI-EGFP to pCHO6GS-X13-GPI-EGFP.

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

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

[0057] The above 14 plasmids (13 Bag6 isoform plasmids and 1 pRRL-GPI-EGFP control plasmid) were transiently transfected into CHO cells. 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 As shown, this illustrates 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, with isoforms 2, 3, 4, 5, 8, and 9 being the preferred isoforms.

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

[0061] To verify the universality of bag6 in enhancing the expression of exogenous proteins, the hemagglutinin protein H5HA of the H5N1 influenza A virus was selected and its trimer form was constructed. This protein has a low expression level and belongs to the category of "difficult-to-express" proteins.

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

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

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

[0065] Figure 5 The results showed that after transfecting CHO cells with Lipo2000 and culturing them for 3 days, the supernatant was collected and the target protein content was detected by FortebioOctet. The results showed that the protein expression level was significantly increased (approximately 1.8 times) compared with the control.

[0066] Example 4: Analysis of differences in recombinant H5HA trimer protein expression levels in stable transfected strains constructed using pCHO6GS and pLV-bla lentiviral vectors.

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

[0068] like Figure 6 As shown, this is a comparison of the expression levels of recombinant H5HA trimer.

[0069] Figure 6 The results showed that after culturing the stable cells in fed-batch culture for 14 days, the target protein content was detected by Fortebio Octet on the cells. Compared with the control, the protein expression level was significantly increased (approximately 2.66 times).

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

[0071] To verify the generalizability 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 pCHO6GS-X2-Ab1 and pCHO6GS-X2-Ab2 vectors were constructed, respectively. pLV-bla-Ab1 and pLV-bla-Ab2 lentiviral vectors were used as controls.

[0073] like Figure 7 As shown, this is a comparison of the expression levels of recombinant IVD antibodies.

[0074] Stable cell lines were constructed using the same method as in Example 4.

[0075] Figure 7 The results showed that after culturing stable cells in fed-batch culture for 14 days, the content of target proteins on the cells was detected by Fortebio Octet. Compared with the control, the expression levels of the two antibody proteins were significantly increased.

[0076] Finally, it should be noted that the above description is merely 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 should be included within the scope of protection of the present invention.

Claims

1. A novel plasmid vector for improving the expression of CHO exogenous proteins, characterized in that, The application relates to a novel recombinant protein expression plasmid vector pCHO6GS and a construction method thereof. The novel recombinant protein expression plasmid vector pCHO6GS is sequentially connected in series by a replication initiation 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, Bag6 isomer X2, a ribosome entry site, a blasticidin resistance gene and an SV40 tailing signal. The target exogenous protein gene fragment is secreted. The nucleotide sequences of the elements of the pCHO6GS plasmid are as follows: 5'-3': replication initiation site, ampicillin resistance gene, SV40 early promoter, glutamine synthetase gene, SV40 intron and transcription tailing signal, mCMV promoter, SV40 tailing signal, ribosome entry site, nucleotide sequences of the blasticidin resistance gene are shown in SEQ ID NO. 1-9. The nucleotide sequence of Bag6 isomer X2 is SEQ ID NO.

11. The replication initiation site and the ampicillin resistance gene fragment are obtained by amplification of a pRRL-GPI-EGFP plasmid, and the nucleotide sequence is shown in SEQ ID NO.

23. The glutamine synthetase gene and the blasticidin resistance gene are obtained by amplification of a pLV-bla-H5HA plasmid, and the nucleotide sequence is shown in SEQ ID NO.

24. The target exogenous protein gene fragment includes any one or more of an antibody, an antibody fragment and a difficult-to-express recombinant protein. The insertion sequence of 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 is as follows: replication initiation site, 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 isomers of Bag6 protein, ribosome entry site, blasticidin resistance gene and SV40 tailing signal.

2. The novel plasmid vector for improving the expression of exogenous proteins in CHO according to claim 1, wherein, The application further relates to a construction method of the novel recombinant protein expression plasmid vector pCHO6GS.

3. The novel plasmid vector for improving the expression of exogenous proteins in CHO according to claim 1, wherein, The replication initiation site and the ampicillin resistance gene fragment are obtained by amplification from a vector pRRL-GPI-EGFP; The glutamine synthetase gene and the blasticidin resistance gene fragment are obtained by amplification from a vector pLV-bla-H5HA; 4. A method for constructing a novel plasmid vector for improving the expression of an exogenous protein in CHO cells according to any one of claims 1 to 3, characterized in that, And the gene fragments such as the mCMV promoter, the intron and the SV40 tailing signal are connected by a seamless cloning technology; Bag6 protein different isomers are connected between XbaI / XmaI, and finally the pCHO6GS vector is formed. ​ ​ ​ The exogenous protein gene fragment 1 is connected between ClaI / NdeI; The exogenous protein gene fragment 2 is connected between MulI / SpeI.

5. The use of the novel plasmid vector with improved expression of CHO exogenous protein according to any one of claims 1-3 in improving the expression of secreted exogenous protein.

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