Novel dual vector expression system for protein expression and construction method and application thereof

Abstract

The application discloses a novel double-carrier expression system for protein expression and a construction method and application thereof. The novel double-carrier expression system for protein expression comprises a first carrier containing a nucleotide sequence for coding a first functional domain of glutamine synthetase and a nucleotide sequence of light chain and heavy chain of a protein to be expressed; and a second carrier containing a nucleotide sequence for coding a second functional domain of glutamine synthetase and a nucleotide sequence of a ScFv sequence of the protein to be expressed, and an amino acid sequence of the glutamine synthetase is composed of the first functional domain and the second functional domain. By using the expression system, the transfection efficiency and screening efficiency are greatly improved, and the expression amount of the target protein is obviously increased.

Description

Technical Field

[0001] This invention relates to the field of vector expression system technology, specifically to a novel dual-vector expression system for protein expression, its construction method, and its applications. Background Technology

[0002] Currently, when expressing bispecific or polyclonal antibodies, the main methods for screening expression cells include the following two: using the same screening pressure, such as single GS screening pressure. This screening method has a low screening efficiency for the target antibody; and using different screening pressures, such as GS and Zeocin (bleomycin or fumonisin D1) for combined screening. Although this method can improve the screening efficiency, it results in a higher molecular weight of the vector, which affects the transfection efficiency, and the screening efficiency is still not high. Summary of the Invention

[0003] To overcome the aforementioned shortcomings, the present invention aims to provide a novel dual-vector expression system for protein expression, its construction method, and its applications. This invention splits the GS gene into two functional domains with different functions, then constructs them into different vectors. Simultaneously, the target protein sequence is also split and constructed into the corresponding two vectors. Only by co-transfecting these two expression vectors can glutamine synthase be synthesized. This achieves rapid screening of cells fused with the target protein gene sequence, greatly improving the screening efficiency for cells capable of expressing the target protein.

[0004] One objective of this invention is to provide a novel dual-vector expression system for protein expression, comprising: a first vector containing a nucleotide sequence encoding a first functional domain of glutamine synthase and nucleotide sequences of the light and heavy chains of the protein to be expressed; and a second vector containing a nucleotide sequence encoding a second functional domain of glutamine synthase and a nucleotide sequence of the ScFv sequence of the protein to be expressed, wherein the amino acid sequence of the glutamine synthase is composed of the first and second functional domains. This invention has the following beneficial effects:

[0005] ① By splitting the glutamine synthase gene into two functional domains and correspondingly mapping them to the target protein... By constructing the gene into two vectors and then using these two vectors to co-transfect cells, cells that co-express the target gene can be screened out. This method is highly specific and greatly improves the screening efficiency.

[0006] ② Utilizing the dual-vector expression system of this invention, it is possible to simultaneously ensure high-throughput screening while also possessing… It has extremely high transfection efficiency, up to 80%, especially in the expression of bispecific or polyclonal antibodies, and has a greater advantage in transfection efficiency of CHO cells, which can increase the transfection efficiency by 60%.

[0007] ③ The expression level of cells obtained using the expression vector of the present invention is significantly increased, reaching 10.23 g / L, which is 80.1% higher than the expression level of the monoclonal antibody genome (5.68 g / L).

[0008] Furthermore, the amino acid sequence of the first functional domain of the glutamine synthase is shown in SEQ ID NO: 1, and the amino acid sequence of the second functional domain of the glutamine synthase is shown in SEQ ID NO: 2.

[0009] Furthermore, the amino acid sequence of the first functional domain of the glutamine synthase is shown in SEQ ID NO: 3, and the amino acid sequence of the second functional domain of the glutamine synthase is shown in SEQ ID NO: 4.

[0010] Furthermore, protein motif structures are added at the front ends of the first and second functional domains.

[0011] Furthermore, the protein motif structure is a leucine zipper, and the first and second carriers respectively contain nucleotide sequences for encoding the leucine zipper. The amino acid sequence for encoding the leucine zipper contained on the first carrier is shown in SEQ ID NO: 9; the amino acid sequence for encoding the leucine zipper contained on the second carrier is shown in SEQ ID NO: 10.

[0012] Furthermore, the protein to be expressed is a single protein or the co-expression of at least two proteins.

[0013] This invention also provides a method for constructing a novel dual-vector expression system for protein expression, comprising the steps of constructing a first vector and a second vector: STEP 11: Construct the nucleotide sequences encoding the first and second functional domains of glutamine synthase into two different empty expression vectors to obtain the first vector precursor and the second vector precursor, respectively. STEP 12: The light chain gene L of the protein to be expressed is digested with EcoRI and HindIII. The digested target fragment is then constructed into the first vector precursor digested with EcoRI and HindIII to form an intermediate vector. STEP 13: The heavy chain gene H of the protein to be expressed is digested with XbaI and NotI. The digested target fragment is then constructed into the intermediate vector digested with XbaI and NdeI to form the first vector. STEP 14: Digest the ScFv gene of the protein to be expressed using EcoRI and HindIII. Construct the target fragment after digestion into the second vector precursor digested with EcoRI and HindIII to form the second vector.

[0014] Furthermore, it also includes the transfection step: STEP 21: One day before stable transformation, CHO-K1Q host cells were seeded into CHOCD04 medium supplemented with 4 mM glutamine and cultured on a shaker. STEP 22: 24 hours before transfection, sample and count CHO cells, and passage them. STEP 23: On the day of transfection, count the cells, calculate the required cell suspension volume based on the cell density, centrifuge for 5 minutes, discard the supernatant after centrifugation, resuspend the cells in PBS in each tube, add the vector plasmid to the PBS, mix the vector plasmid with the above cell suspension, and transfer to an electroporation cuvette; place the electroporation cuvette in an electroporator and perform electroporation; after electroporation, transfer the cells in the electroporation cuvette to a T25 culture flask pre-filled with CD04 medium supplemented with glutamine for culture.

[0015] Furthermore, the empty expression vector is selected from at least one of PKS001, pEE series, and pCG series.

[0016] This invention also provides an application of the aforementioned novel dual-vector expression system for protein expression. For example, it can be used for high-throughput screening of target protein expression lines and efficient expression of target proteins. The target protein can be a bispecific antibody or a multi-antibody. Attached Figure Description

[0017] Figure 1 The effect of transfection of vectors containing different first and second functional domains on the density of CHO cells; Figure 2 This is a comparison of the gene co-expression transfection efficiency of the vector of the present invention with that of dual-resistance gene expression vectors and single-resistance gene expression vectors; Figure 3 This is a comparison of the expression levels of the vector of the present invention with those of dual-resistance gene expression vectors and single-resistance gene expression vectors. Detailed Implementation

[0018] The preferred embodiments of the present invention will now be described in detail with reference to the accompanying drawings, so that the advantages and features of the present invention can be more easily understood by those skilled in the art, thereby providing a clearer and more explicit definition of the scope of protection of the present invention.

[0019] The sources and types of reagents used in this application are shown in Table 1: Table 1. Sources of reagents in this invention

[0020] Example: 1. Construction of the carrier: The empty vectors pGS1+1-GS1, pGS1+1-GS2, pGS, and pZeocin used in this invention; the heavy chain gene H, light chain gene L, and ScFv of the antibody were all synthesized by a biotechnology company.

[0021] 1.1 Construction of the carrier of the present invention In this invention, the GS gene is split into a first functional domain and a second functional domain, and these domains are constructed into two different empty expression vectors. The gene sequence of the target protein is also constructed into the aforementioned empty expression vectors to obtain the first vector and the second vector. Only cells co-transfected with the first vector and the second vector can synthesize glutamine synthase and maintain growth under glutamine-free culture conditions, indicating that the obtained product also contains the gene sequence of the target antibody.

[0022] The specific construction steps are as follows: STEP 11: Construct the nucleotide sequences encoding the first and second functional domains of glutamine synthase into two different empty expression vectors to obtain the first vector precursor and the second vector precursor, respectively. STEP 12: The light chain gene L of the protein to be expressed is digested with EcoRI and HindIII. The digested target fragment is then constructed into the first vector precursor digested with EcoRI and HindIII to form an intermediate vector. STEP 13: The heavy chain gene H of the protein to be expressed is digested with XbaI and NotI. The digested target fragment is then constructed into the intermediate vector digested with XbaI and NdeI to form the first vector. STEP 14: Digest the ScFv gene of the protein to be expressed using EcoRI and HindIII. Construct the target fragment after digestion into the second vector precursor digested with EcoRI and HindIII to form the second vector.

[0023] In some implementations, a protein motif structure is added to the front end of each functional domain, such as a zinc finger structure, a β hairpin loop, or a leucine zipper.

[0024] 1.2 Construction of monoclonal antibody gene expression vector The light chain gene L was digested with EcoRI and HindIII. The digested target fragment was then constructed into the empty vector pGS, which was digested with EcoRI and HindIII, to form the intermediate vector pGS-L.

[0025] The heavy chain gene H was double-digested with XbaI and NotI. The digested target fragment was then constructed into the pGS-L double-digested with XbaI and NdeI to form the final vector 3: pGS-LH (monoclonal antibody gene expression vector 3).

[0026] The ScFv gene was digested with EcoRI and HindIII. The digested target fragment was then constructed into the empty expression vector pGS, which was digested with EcoRI and HindIII, to form the final vector 4: pGS-ScFv (monoclonal antibody gene expression vector 4).

[0027] In some implementations, the antibody is a PD1 antibody or a B7H3 antibody.

[0028] 1.3 Construction of the expression vector for dual resistance genes The construction process of the dual resistance gene expression vector is the same as that of the single resistance gene expression vector in 1.2.

[0029] The ScFv gene was double-digested with EcoRI and HindIII. The resulting target fragment was then constructed into the empty expression vector pZeocin, which was also double-digested with EcoRI and HindIII, forming the final vector 5: pZeocin-ScFv (bispecific antibody gene expression vector 5). In some embodiments, the antibody is a PD1 and B7H3 bispecific antibody.

[0030] 2. Transfection ① One day before stable transformation, CHO-K1Q host cells were seeded into CHOCD04 (+4mM Glutamine) medium and cultured at 36.5℃, 5% CO2, and 220rpm in a shaker. The host cell density can be 1E5-1E6 cells / ml, for example, 1E5 cells / ml, 5E5 cells / ml, or 1E6 cells / ml.

[0031] ② 24 hours before transfection, CHO-K1Q samples were taken and counted, and the cells were passaged. The density of passaged cells can be 0.5E6-1.5E6 cells / ml, for example, 0.8E6 cells / ml, 1.0E6 cells / ml or 1.2E6 cells / ml.

[0032] ③ On the day of transfection, count the cells, calculate the required cell suspension volume based on the cell density, centrifuge for 5 minutes, discard the supernatant after centrifugation, add 300 μL of PBS to each tube to resuspend the cells, add the vector plasmid to 300 μL of PBS, mix the vector plasmid with the above cell suspension, and transfer to an electroporation cuvette; place the electroporation cuvette in an electroporator and perform electroporation; after electroporation, transfer the cells in the electroporation cuvette to a T25 culture flask pre-filled with 8 mL of CD04 medium supplemented with 4 mM glutamine for culture. In some embodiments, the specific electroporation conditions are: voltage 1.8-2.5 kV; capacitance: 25 μF; resistance: 200-400 Ω; pulse duration: 5 ms; number of pulses: 1.

[0033] In some embodiments, the vector plasmids are added in a 1:1 mass ratio. For example, when transfecting using the vector in Example 1.1, the mass ratio of the first vector to the second vector is set to 1:1; when transfecting using the monoclonal antibody gene expression vector in Example 1.2, the mass ratio of vector 3 to vector 4 is set to 1:1. In other possible embodiments, the vector plasmids may also be added in other mass ratios, such as a mass ratio of 1:2, 2:1, 1.5:1, or 1:1.5, etc.

[0034] 3. Construction of Bulkpool After 24 hours of stable cell growth, cells were collected and counted. The cells were centrifuged at 200g for 5 minutes, the supernatant was discarded, and the cells were seeded into TPP tubes at a density of 0.5E6 using CD04 (+25 μM MSX) medium. The tubes were incubated at 36.5℃ in a 5% CO2 shaker. Cells were passaged every 2-3 days using CD04 (+25 μM MSX) medium until cell viability was >95%, at which point further MSX stress tolerance assessment or Fed-batch assessment was performed.

[0035] 4. MSX pressure tolerance assessment MSX (methylene sulfoxide imine) is an inhibitor of GS (glutamine synthase). By removing glutamine from the culture medium and adding MSX, the selection pressure is increased, so that only cells that integrate and express the exogenous GS gene can survive and proliferate under restrictive conditions, thereby achieving the selection effect.

[0036] Once the cell viability recovered to over 95%, cells from different experimental groups were seeded into CD04 medium with different MSX concentrations of 25μM, 35μM, 50μM, and 75μM. Cell density was observed, and groups with high cell density were selected for further study.

[0037] 5. Comparison of transfection efficiency and protein expression effect 5.1 Transfection efficiency test Add MSX-Alexa Fluor 488 conjugate to the recovered CHO transfected cells and incubate overnight. The following morning, centrifuge to collect the cells and carefully discard the supernatant. Resuspend the cells in growth medium, incubate for 1 hour, centrifuge to collect the cells and carefully discard the supernatant. Resuspend the cells again in growth medium and incubate for 1 hour, centrifuge to collect the cells and discard the supernatant. The precipitated cells are then resuspended in growth medium and ready for flow cytometry analysis.

[0038] 5.2 Fed-batch Evaluation To further verify the effectiveness of the expression vector of this invention, a Fed-batch evaluation was conducted.

[0039] Cells were seeded at a density of 0.5E6 cells / ml in 302plus medium and fed in a Fed-batch experiment. The working volume was 20 mL. During culture, Feed03A and Feed03B were used as feed additives. The feed ratio was adjusted according to the growth and metabolism of each MiniPool, and glucose was added as needed during culture. The Fed-batch cell flasks were placed in a shaker and cultured under the following conditions: temperature 36.5℃, 5% CO2, 220 rpm.

[0040] Fed-batch cultures are collected on day 14; if cell viability is below 70%, samples are collected earlier.

[0041] Experimental example: 1. Construction of the carrier: 1.1 Construction of the carrier of the present invention This invention splits the GS gene into a first functional domain and a second functional domain, and constructs the two functional domains into two different vectors. Simultaneously, the gene sequence of the target protein is split into two parts and constructed into the aforementioned two vectors, thus obtaining the vector of this invention.

[0042] In some embodiments, GS can be split into a first functional domain 1.1 and a second functional domain 1.2. The amino acid sequence of the first functional domain 1.1 is shown in SEQ ID NO: 1, specifically as follows: MATSASSHLNKNIKQMYLCLPQGEKVQAMYIWVDGTGEGLRCKTRTLDCEPKCVEELPEWNFDGSSTFQSEGSNSDMYLSPVAMFRDPFRRDPNKLVFCEVFKYNR.

[0043] The amino acid sequence of the second functional domain 1.2 is shown in SEQ ID NO: 2, specifically as follows: KPAETNLRHSCKRIMDMVSNQHPWFGMEQEYTLMGTDGHPFGWPSNGFPGPQGPYYCGVGADKAYGRDIVEAHYRACLYAGVKITGTNAEVMPAQWEFQIGPCEGIRMGDHLWVARFILHRVCEDFGVIATFDP KPIPGNWNGAGCHTNFSTKAMREENGLKHIEEAIEKLSKRHRYHIRAYDPKGGLDNARRLTGFHETSNINDFSAGVANRSASIRIPRTVGQEKKGYFEDRRPSANCDPFAVTEAIVRTCLLNETGDEPFQYKN.

[0044] In some embodiments, GS can be split into a first functional domain 2.1 and a second functional domain 2.2. The amino acid sequence of the first functional domain 2.1 is shown in SEQ ID NO: 3, specifically as follows: MATSASSHLNKNIKQMYLCLPQGEKVQAMYIWVDGTGEGLRCKTRTLDCEPKCVEELPEWNFDGSSTFQSEGSNSDMYLSPVAMFRDPFRRDPNKLVFCEVFKYNRKPAETN.

[0045] The amino acid sequence of the second functional domain 2.2 is shown in SEQ ID NO: 4, specifically as follows: LRHSCKRIMDMVSNQHPWFGMEQEYTLMGTDGHPFGWPSNGFPGPQGPYYCGVGADKAYGRDIVEAHYRACLYAGVKITGTNAEVMPAQWEFQIGPCEGIRMGDHLWVARFILHRVCEDFGVIATFDPKPI PGNWNGAGCHTNFSTKAMREENGLKHIEEAIEKLSKRHRYHIRAYDPKGGLDNARRLTGFHETSNINDFSAGVANRSASIRIPRTVGQEKKGYFEDRRPSANCDPFAVTEAIVRTCLLNETGDEPFQYKN.

[0046] This embodiment uses a protein motif with a leucine zipper added to the front of each functional domain as an example. Leucine zipper sequences are added to the 3' end of the first functional domain and the 5' end of the second functional domain. The amino acid sequence encoding the leucine zipper contained on the first vector is SEQ ID NO: 9, and its specific sequence is: VAALEKVKALEQLKALEQLKALEQ.

[0047] The amino acid sequence containing the second vector for encoding the leucine zipper is SEQ ID NO: 10, and its specific sequence is: VAALDKVDALKQLKALDK LKALDK.

[0048] The specific sequences constructed on the two vectors include those shown in group 1 (SEQ ID NO: 5 and SEQ ID NO: 6) and group 2 (SEQ ID NO: 7 and SEQ ID NO: 8), respectively.

[0049] The sequence of SEQ ID NO: 5 is shown below, where the underlined amino acid sequence is the leucine zipper sequence, and the rest are the amino acid sequences of the first functional domain 1.1 of GS: VAALEKVKALEQLKALEQLKALEQ MATSASSHLNKNIKQMYLCLPQGEKVQAMYIWVDGTGEGLRCKTRTLDCEPKCVEELPEWNFDGSSTFQSEGSNSDMYLSPVAMFRDPFRRDPNKLVFCEVFKYNR.

[0050] The sequence of SEQ ID NO: 6 is shown below, where the underlined part is the amino acid sequence of the leucine zipper, and the remaining part is the amino acid sequence of GS second functional domain 1.2: VAALDKVDALKQLKALDKLKALDK KPAETNLRHSCKRIMDMVSNQHPWFGMEQEYTLMGTDGHPFGWPSNGFPGPQGPYYCGVGADKAYGRDIVEAHYRACLYAGVKITGTNAEVMPAQWEFQIGPCEGIRMGDHLWVARFILHRVCEDFGVIATFDP KPIPGNWNGAGCHTNFSTKAMREENGLKHIEEAIEKLSKRHRYHIRAYDPKGGLDNARRLTGFHETSNINDFSAGVANRSASIRIPRTVGQEKKGYFEDRRPSANCDPFAVTEAIVRTCLLNETGDEPFQYKN.

[0051] The sequences in SEQ ID NO: 5 and SEQ ID NO: 6, excluding the leucine zipper, together constitute the complete amino acid sequence of GS.

[0052] The sequence of SEQ ID NO: 7 is shown below, where the underlined part is the amino acid sequence of the leucine zipper, and the remaining part is the amino acid sequence of the first functional domain 2.1 of GS: VAALEKVKALEQLKALEQLKALEQMATSASSHLNKNIKQMYLCLPQGEKVQAMYIWVDGTGEGLRCKTRTLDCEPKCVEELPEWNFDGSSTFQSEGSNSDMYLSPVAMFRDPFRRDPNKLVFCEVFKYNRKPAETN.

[0053] The sequence of SEQ ID NO: 8 is shown below, where the underlined part is the amino acid sequence of the leucine zipper, and the remaining part is the amino acid sequence of the second functional domain 2.2 of GS: VAALDKVDALKQLKALDKLKALDK LRHSCKRIMDMVSNQHPWFGMEQEYTLMGTDGHPFGWPSNGFPGPQGPYYCGVGADKAYGRDIVEAHYRACLYAGVKITGTNAEVMPAQWEFQIGPCEGIRMGDHLWVARFILHRVCEDFGVIATFDPKPI PGNWNGAGCHTNFSTKAMREENGLKHIEEAIEKLSKRHRYHIRAYDPKGGLDNARRLTGFHETSNINDFSAGVANRSASIRIPRTVGQEKKGYFEDRRPSANCDPFAVTEAIVRTCLLNETGDEPFQYKN.

[0054] The sequences in SEQ ID NO: 7 and SEQ ID NO: 8, excluding the leucine zipper, together constitute the complete amino acid sequence of GS.

[0055] Sequences SEQ ID NO: 5 and SEQ ID NO: 6 of group 1 were respectively constructed onto an empty expression vector to obtain the first and second vector precursors (pGS1+1-GS1 and pGS1+1-GS2). Similarly, Sequences SEQ ID NO: 7 and SEQ ID NO: 8 of group 2 were respectively constructed onto the first and second vector precursors (pGS1+1-GS1 and pGS1+1-GS2).

[0056] The light chain gene L was digested with EcoRI and HindIII. The digested target fragment was then constructed into the empty vector pGS1+1-GS1, which was digested with EcoRI and HindIII, to form the intermediate vector PL.

[0057] The heavy chain gene H was double-digested with XbaI and NotI. The digested target fragment was then constructed into the PL, which was double-digested with XbaI and NdeI, to form the final first vector: P-LH.

[0058] The ScFv gene was double-digested with EcoRI and HindIII. The digested target fragment was then constructed into the empty expression vector pGS1+1-GS2, which was double-digested with EcoRI and HindIII, to form the final second vector: P-ScFv.

[0059] 1.2 Construction of monoclonal antibody gene expression vector Same as step 1.2 in the previous embodiment. The antibody used in this embodiment is a PD1 antibody.

[0060] 1.3 Construction of the expression vector for dual resistance genes Same as step 1.3 of the previous embodiment. The antibody in this embodiment is a bispecific antibody against PD1 and B7H3.

[0061] 2. Transfection ① One day before stable transformation, host cells CHO-K1Q were seeded into CHOCD04 (+4mM Glutamine) medium at a cell density of 1E6 cells / ml and cultured in a shaker at 36.5℃, 5% CO2, and 220rpm.

[0062] ② 24 hours before transfection, CHO-K1Q samples were taken and counted, and cells were passaged at a density of 0.8E6-1E6.

[0063] ③ On the day of transfection, count the cells and calculate the required cell suspension volume for 1.5E7 cells based on the cell density. Centrifuge at 220g for 5 minutes, discard the supernatant, and resuspend the cells in 300μL PBS in each tube. Add 25μg of the vector plasmid to 300μL PBS, mix the vector plasmid with the cell suspension, and transfer the cells to an electroporation cuvette. Place the cuvette in an electroporator and perform electroporation. After electroporation, transfer the cells from the cuvette to a T25 culture flask pre-filled with 8mClCO4 + 4mMGln medium. In some embodiments, the specific electroporation conditions are: voltage 1.8-2.5kV; capacitance: 25μF; resistance: 200-400Ω; pulse duration: 5ms; number of pulses: 1. The first and second vector plasmids are added at a 1:1 mass ratio.

[0064] 3. Construction of Bulkpool After 24 hours of stable cell growth, cells were collected and counted. The cells were centrifuged at 200g for 5 minutes, the supernatant was discarded, and the cells were seeded into TPP tubes at a density of 0.5E6 using CD04 (+25 μM MSX) medium. The tubes were incubated at 36.5℃ in a 5% CO2 shaker. Cells were passaged every 2-3 days using CD04 (+25 μM MSX) medium until cell viability was >95%, at which point further MSX stress tolerance assessment or Fed-batch assessment was performed.

[0065] 4. MSX pressure tolerance assessment MSX (methylene sulfoxide imine) is an inhibitor of GS (glutamine synthase). By removing glutamine from the culture medium and adding MSX, the selection pressure is increased, so that only cells that integrate and express the exogenous GS gene can survive and proliferate under restrictive conditions, thereby achieving the selection effect.

[0066] Once the cell viability recovered to over 95%, cells from different experimental groups were seeded at a density of 0.5E6 cells / ml into CD04 medium with different MSX concentrations of 25μM, 35μM, 50μM, and 75μM. Cell density was observed, and the results are as follows: Figure 1 As shown, the results of MSX stress tolerance tests on cells obtained after transfection and Bulkpool evaluation using the two vector combinations constructed using Group 1 and Group 2 in Example 1.1 are as follows. Figure 1 As shown, the vector system constructed using group 1 can achieve a higher cell density. Therefore, the vector from group 1 was selected for subsequent experiments.

[0067] 5. Comparison of transfection efficiency and protein expression effect 5.1 Transfection efficiency test Add 100 μL of MSX-Alexa Fluor 488 conjugate to the recovered CHO transfected cells and incubate overnight. The following morning, centrifuge to collect the cells and carefully discard the supernatant. Resuspend the cells in 25 mL of growth medium, incubate for 1 hour, centrifuge to collect the cells, and carefully discard the supernatant. Resuspend the cells again in growth medium and incubate for 1 hour, centrifuge to collect the cells, and discard the supernatant. The precipitated cells are then resuspended in growth medium and ready for flow cytometry analysis. Specific results are shown below. Figure 2 .

[0068] Depend on Figure 2 The results show that the transfection efficiency of the vector of the present invention (80%) is higher than that of the dual-resistance gene expression vector (50%) and the single-resistance gene expression vector (72%).

[0069] 5.2 Fed-batch Evaluation To further verify the effectiveness of the expression vector of this invention, a Fed-batch evaluation was conducted.

[0070] Cells were seeded at a density of 0.5E6 cells / ml in 302plus medium and fed in a Fed-batch experiment. The working volume was 20 mL. During culture, Feed03A and Feed03B were used as feed additives. The feed ratio was adjusted according to the growth and metabolism of each MiniPool, and glucose was added as needed during culture. The Fed-batch cell flasks were placed in a shaker and cultured under the following conditions: temperature 36.5℃, 5% CO2, 220 rpm.

[0071] Fed-batch cultures are collected on day 14; if cell viability is below 70%, samples are collected earlier.

[0072] The expression level of the target protein was measured, and the results are as follows: Figure 3 As shown in the figure, the target protein expression level using the vector of the present invention (10.23 g / L) is higher than that using the dual resistance gene expression vector of Experimental Example 1.3 (8.29 g / L) and the target protein expression level using the single resistance gene expression vector of Experimental Example 1.2 (5.68 g / L).

[0073] The above embodiments are only for illustrating the technical concept and features of the present invention. Their purpose is to enable those skilled in the art to understand the content of the present invention and implement it. They should not be used to limit the scope of protection of the present invention. All equivalent changes or modifications made in accordance with the spirit and essence of the present invention should be covered within the scope of protection of the present invention.

Claims

1. A novel dual-vector expression system for protein expression, characterized in that, include: The first vector contains a nucleotide sequence encoding a first functional domain of glutamine synthase and nucleotide sequences of the light and heavy chains of the protein to be expressed. The second vector contains the nucleotide sequence encoding the second functional domain of glutamine synthase and the nucleotide sequence of the ScFv sequence of the protein to be expressed. The amino acid sequence of glutamine synthase is composed of the first functional domain and the second functional domain.

2. The novel dual-vector expression system for protein expression according to claim 1, characterized in that, The amino acid sequence of the first functional domain of the glutamine synthase is shown in SEQ ID NO: 1, and the amino acid sequence of the second functional domain of the glutamine synthase is shown in SEQ ID NO:

2.

3. The novel dual-vector expression system for protein expression according to claim 1, characterized in that, The amino acid sequence of the first functional domain of the glutamine synthase is shown in SEQ ID NO: 3, and the amino acid sequence of the second functional domain of the glutamine synthase is shown in SEQ ID NO:

4.

4. The novel dual-vector expression system for protein expression according to any one of claims 1-3, characterized in that, Protein motif structures are added at the front ends of the first and second functional domains.

5. The novel dual-vector expression system for protein expression according to claim 4, characterized in that, The protein motif structure is a leucine zipper. The first and second carriers also contain nucleotide sequences for encoding the leucine zipper. The amino acid sequence for encoding the leucine zipper contained on the first carrier is shown in SEQ ID NO: 9; the amino acid sequence for encoding the leucine zipper contained on the second carrier is shown in SEQ ID NO:

10.

6. The novel dual-vector expression system for protein expression according to any one of claims 1-3, characterized in that, The protein to be expressed is a single protein or the co-expression of at least two proteins.

7. The method for constructing the novel dual-vector expression system for protein expression according to any one of claims 1-6, characterized in that, The construction steps include the first and second carriers: STEP 11: Construct the nucleotide sequences encoding the first and second functional domains of glutamine synthase into two different empty expression vectors to obtain the first vector precursor and the second vector precursor, respectively. STEP 12: The light chain gene L of the protein to be expressed is digested with EcoRI and HindIII. The digested target fragment is then constructed into the first vector precursor digested with EcoRI and HindIII to form an intermediate vector. STEP 13: The heavy chain gene H of the protein to be expressed is digested with XbaI and NotI. The digested target fragment is then constructed into the intermediate vector digested with XbaI and NdeI to form the first vector. STEP 14: Digest the ScFv gene of the protein to be expressed using EcoRI and HindIII. Construct the target fragment after digestion into the second vector precursor digested with EcoRI and HindIII to form the second vector.

8. The method for constructing a novel dual-vector expression system for protein expression according to claim 7, characterized in that, It also includes the transfection step: STEP 21: One day before steady-state transformation, inoculate host cells CHO into CHO4 medium supplemented with glutamine and culture them on a shaker. STEP 22: 24 hours before transfection, sample and count CHO cells, and passage them. STEP 23: On the day of transfection, count the cells, calculate the required volume of cell suspension based on the cell density, centrifuge for 5 minutes, discard the supernatant after centrifugation, resuspend the cells in PBS in each tube, add the vector plasmid to the PBS, mix the vector plasmid with the above cell suspension, and then transfer it to an electroporation cuvette; place the electroporation cuvette in an electroporator and perform electroporation; after electroporation, transfer the cells in the electroporation cuvette to a T25 culture flask pre-filled with CD04 medium supplemented with glutamine for culture.

9. The method for constructing a novel dual-vector expression system for protein expression according to claim 7 or 8, characterized in that, The empty expression vector is selected from at least one of PKS001, pEE series and pCG series.

10. The application of the novel dual-vector expression system for protein expression according to any one of claims 1-9.

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