AU2021100307A4 - The preparation method and application of combined expression vector and expression system in mammalian cells - Google Patents

Abstract

The invention belongs to the technical field of genetic engineering, and it discloses the preparation method and application of combined expression vector and expression system in mammalian cells. The expression vector is inserted with an ARE sequence, an IRES sequence, a WPRE sequence, and a selection marker gene sequence. And the ARE sequence is shown in SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 3. The IRES sequence is shown in SEQ ID NO: 4, SEQ ID NO: 5 or SEQ ID NO: 6, and the structure of the combined expression vector is as follows: ARE-promoter-GOI of interest-WPRE-IRES-BSR-PolyA. The anti-repressor element was uesed to optimize the combination of different elements of the vector, on the one hand, it overcomes transgene silencing, on the other hand, it enables the gene of interest (GOI) to be efficiently, persistently, and stably expressed in the host cell.

Description

The preparation method and application of combined expression vector and

expression system in mammalian cells

TECHNICAL FIELD

[01] The present invention relates to a combined expression vector for mammalian cells, as well as an expression system, a preparation method and its application containing the expression vector, which belongs to the technical field of genetic engineering.

BACKGROUND

[02] Recombinant protein is produced by adopting genetic engineering technology to insert GOI into an expression vector to achieve the expression of GOI in heterologous cells. The productions of recombinant protein mainly include four major expression systems: prokaryotic expression system, yeast expression system, insect cell expression, and mammalian cell expression system. Proteins with complex structures or glycosylation, are very important for their activities. Mammalian cell expression system are currently the most commonly used expression system in the productions of recombinant therapeutic proteins. Since the E.coli prokaryotic expression system and lower eukaryotic expression systems such as yeast lack complex post-translational modification functions, thus recombinant therapeutic proteins need to be expressed in mammalian cells to have the activity. At present, nearly 70% of recombinant therapeutic proteins are produced in Chinese hamster ovary (CHO) cell expression system. However, the low expression level of GOI caused by transgene silencing has become a bottleneck technology in the CHO cells expression system. In addition, there are defects such as a long selection cycle for high-yield stable cells and high costs, which severely restrict the production of recombinant protein drugs.

[03] In recent years, researchers have made many significant attempts to improve the expression of recombinant proteins in mammalian cells, of which vector optimization is an effective strategy. For example, using strong promoters, enhancers, and nuclear matrix attachment regions to construct expression vectors, or perform demethylation during transgenic operations. However, most of the current vector optimization focuses on the functional identification and research of a certain element.

For example, the invention patent with publication number CN104975018A discloses A new type of enhancer and its application, of which the enhancer is particularly suitable for CHO cells. The expression of exogenous proteins in animal cells has been greatly increased by about 2 to 3 times. For example, the invention patent with publication number 201410129979.2 discloses A new type of animal cell expression vector containingMAR corefragments. Plasmids containing MAR core fragments with artificial transcription factor binding sites at both ends are particularly suitable for CHO cells. Compared with plasmids without containing MAR core fragments with artificial transcription factor binding sites at both ends, the expression level of exogenous protein in animal cells is greatly increased by 10-17 times. However, these elements are still not ideal in improving the expression level of transgenes. In addition, there is compatibility between different elements of expression vectors, such as the combination of MAR sequences and different promoters. There are differences in the level of expression of GOIs in CHO cells (Ho SC, Mariati, Yeo JH, Fang SG, Yang Y. Impact of using different promoters and matrix attachment regions on recombinant protein expression level and stability in stably transfected CHO cells.Mol Biotechnol. 2015; 57(2):138-44), therefore, it is necessary to optimize the combination of different elements of the vector to screen out an efficient expression vector.

SUMMARY

[04] The purpose of the present invention is to provide a mammalian cell combination expression vector, which can enable the GOI to be expressed efficiently and stably in host cells for a long-term expression.

[05] Another object of the present invention is to provide a method for preparing a mammalian cell combination expression vector.

[06] Another object of the present invention is to provide a eukaryotic expression system, preparation method and application of a mammalian cell combined expression vector.

[07] In order to achieve the above objectives, the present invention adopts the following technical solutions:

[08] The present invention provides a mammalian cell combined expression vector into which the anti-repressor element sequence (anti-repressor elements, as shown in SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 3) is inserted. ARE), as shown in SEQ ID NO: 4, SEQ ID NO: 5 or SEQ ID NO: 6 (internal ribosome entry site, IRES), the woodchuck hepatitis virus posttranscriptional regulatory element sequence (woodchuck hepatitis virus posttranscriptional regulatory element, WPRE) as shown in SEQ ID NO: 7 and the selection marker gene sequence. the ARE sequence is located upstream of the promoter, The WPRE sequence is located downstream of the GOI, the selection marker gene is located downstream of the IRES sequence, and the GOI is located between the promoter and the IRES sequence.

[09] Further, the selection marker gene sequence is selected from the blasticidin resistance gene sequence (blasticidin resistance, BSR) as shown in SEQ ID NO: 8.

[010] In addition, the original vector of the expression vector is pIRES-neo2 or pIRES-neo3.

[011] The present invention provides a method for preparing the above mentioned mammalian cell combination expression vector, which includes the following steps:

[012] Step a: Carrying out PCR amplification of EGFP, double digestion of EGFP reporter gene amplification product and original vector, recovery of digested fragments, identification after ligation and transformation, so as to produce expression vector I.

[013] Step b: The ARE sequence and expression vector I are double enzyme digested, respectively, and the digested fragments are recovered, ligated, transformed and identified to produce expression vector II..

[014] Step c: Seamlessly cloning the IRES sequence into expression vector II to produce expression vector III.

[015] Step d: The WPRE sequence and expression vector III are double enzyme digested, and the enzyme digested fragments are recovered, ligated, transformed and identified to produce expression vector IV..

[016] Step e: Performing seamless clone of the selection marker gene sequence into the expression vector IV to produce the mammalian cell combination expression vector V.

[017] The present invention provides a eukaryotic expression system that comprises the above-mentioned mammalian combination expression vector.

[018] The present invention also provides a preparation method of the eukaryotic expression system, including the following steps:

[019] Step a: Inserting the GOI between the promoter of the aforementioned combined expression vector and the IRES sequence to construct a recombinant expression vector.

[020] Step b: Transfecting the recombinant expression vector into the host cell, in order to obtain the expression system after screening.

[021] The above-mentioned host cell is selected from any one of DG44, DXB11, CHO-K, CHO-S and the like. Preferably, selecting CHO-S cells. The above mentioned transfection methods include calcium phosphate method, electrotransfection method, liposome transfection method, etc., preferably electrotransfection method.

[022] Application of the above-mentioned mammalian cell combined expression vector and eukaryotic cell expression system in the preparation of a drug containing the protein of interest.

[023] Compared with the prior art, the present invention has the following beneficial effects:

[024] The combined expression vector of the present invention is inserted with an anti-repressor element sequence, a ribosome entry site sequence, a woodchuck hepatitis virus post-transcriptional regulatory element and a blasticidin resistance gene (selection marker gene). The structure of the expression vector is: ARE-promoter-GOI-WPRE IRES-BSR-poly A. The vector uses the anti-repressor element to optimize the combination of different elements of the vector, on the one hand, it can overcome transgene silencing. In addition, it can make the GOI be efficiently, persistently and stably expressed in host cells. Under the same conditions, compared with the expression vector without ARE sequence and before optimization, the combined expression vector of the present invention can significantly increase the expression level of foreign genes, and it can be used for the production of recombinant protein.

BRIEF DESCRIPTION OF THE FIGURES

[025] FIG. 1 is the plasmid map of the original vector pIRES-neo2 in Example 1.

[026] FIG. 2 is the plasmid map of the combined expression vector of mammalian cell pIRES-4 in Example 1.

[027] FIG. 3 shows the redsults of the flow cytometry to detect the stable expression level of EGFP in different expression vectors.

[028] FIG. 4 is the results of the flow cytometry to detect the long-term expression level of EGFP in different expression vectors.

[029] FIG. 5 is the results of an ELISA to detect the expression level of Hepatitis B Antigen in different expression vectors.

DESCRIPTION OF THE INVENTION

[030] The following examples are used to illustrate the present invention, but not to limit the scope of protection of the present invention. Unless otherwise specified, the technical means used in the embodiments are conventional means well known to those skilled in the art. The test methods in the following examples are conventional methods unless otherwise specified.

[031] The plasmid mini-extraction kit used in the examples and test examples is purchased from Kangwei Century Biotechnology Co., Ltd. (article number: CW0500S). the seamless cloning kit is purchased from Biyuntian Biological Company (article number: D7010S). Tool enzymes, culture media, etc. are all commercially available products. pIRES-neo2, pEGFP-C1 plasmids are purchased from Clontech Biological Company, USA. The cell lines, plasmid vectors, reagents, tool enzymes, etc. used in the examples and test examples are all commercially available products.

[032] Example 1 Construction of Mammalian Cell Combination Expression Vector

[033] This example is the construction of a combined expression vector containing artificially synthesized ARE-1, IRES-1, WPRE, and BSR sequences, including the following steps:

[034] 1.1 Construction of the expression vector pIRES-EGFP containing the EGFP reporter gene

[035] (1) EGFP reporter gene amplification

[036] According to the enhanced EGFP gene sequence of pEGFP-C1 vector (GenBank: U55763.1, bases 613~1410), the upstream primer PF and downstream PR is designed, and introduce Not I and EcoRI enzymes into the 5' end of the primers. Cut site, the primer sequence is as follows (the underline is the restriction site):

[037] P-F: 5'-CCGGCGGCCGCATGGTGAGCAAGGGCGAGGAG-3'.

[038] P-R: 5'-CTAGAATTCTTACTAGATCCGGTGGATCC-3'.

[039] Using the pEGFP-C1 vector as a template, the EGFP gene is amplified by primers P-F and P-R. The PCR reaction system is shown in Table 1.

[040] Table 1 PCR reaction system PCR reaction system Reagent concentration Final concentration Volume (pL) 10 xPCR buffer lox 1x 2.5 Primer P-F 10ptmol/L 0.4tmol/L 1.0 Primer P-R 10ptmol/L 0.4tmol/L 1.0 dNTP 25prmol/L 200tmol/L 2.0 Template DNA 100ng/pL 4.Ong/ptL 1.0 Taq enzyme 5U/pL 0.lU/pL 0.5 ddH20 / / 17

[041] Reaction procedure: 95°C pre-denaturation for 3min. 94°C denaturation for s, annealing at 58°C for 30s, extension at 72°C for 40s, 4 cycles per annealing temperature. finally, annealing at 55°C for 1min, with total 30 cycles, extension at 72°C for 3min, which is kept at 4°C.

[042] Performing Agarose gel electrophoresis to recover the PCR amplification product, and sent it to the biological company for sequencing verification after purification. The results show that the amplified DNA fragment is completely consistent with the EGFP sequence published by GenBank.

[043] (2) Construction of the expression vector pIRES-EGFP containing the EGFP sequence and the original vector pIRES-neo2

[044] The PCR amplification products of EGFP are digested with Not I and EcoRI (the correct sequence is verified by sequencing). At the same time, the DNA of the original vector pIRES-neo2 is digested by Not I and EcoRI (refer to Figure 1 for pIRES-neo2 plasmid profile), and the enzyme is identified by agarose gel electrophoresis.

[045] The enzymatic system of EGFP sequence: 1OxNE Buffer 3.1 3[L, 1.0 L each of Not I/Bam HI (10U/L), 30 L of water. The enzymatic conditions includes that: reacting at 37°C, with enzymatic digestion for 3h..

[046] The enzyme digestion system of the original vector pIRES-neo2 is as follows: 10xNE Buffer 3.1 2L, Not I/BamH I (10U/L)enzyme 0.5tL each, pIRES neo2 DNA 0.81[ g/Ll 1.23 [L, and water to 20 L. After fully mixing, it is incubated at 37°C for 3 h.

[047] The digested EGFP sequence fragment and the original vector pIRES-neo2 are ligated with T4 ligase.

[048] The ligation system for pIRES-neo2 is as follows: 10 L of 2xQuick Ligation Buffer, 200ng of linear DNA of pIRES-neo2, 87.2ng of EGFP fragment after enzymatic excision, 1 L of T4 ligase at 350U/[L, 20 L of water supplemented with 350U/[L of T4 ligase, and performing overnight ligation at 16°C.

[049] The ligation product is added to the E.coli JM109 competent bacterial suspension for transformation, 100 L of the transformed bacteria solution is inoculated on an LB solid culture plate containing ampicillin, and cultured overnight at 37°C, pick a single colony of shake bacteria for subculture. The bacterial plasmid is extracted and digested with Not I/Bam HI. The digestion system is: 1OxNE Buffer 3.1 2L, Not I/BamH I (1OU/4L) each 0.5[tL, 0.81jg/4L pIRES-neo2 1.23 L of DNA, make up to Lwater. After mixing well, incubated at 37°C for 3h. Nucleic acid electrophoresis verification showed that the size of the band with the correct restriction digestion is 720 bp. The correct plasmid identified by restriction digestion is selected and verified by sequencing. Finally, the correct expression vector I is obtained, which is named pIRES EGFP in this example.

[050] 1.2 Construction of expression vector pIRES-1 containing ARE-1 sequence

[051] The ARE-i sequence(shown in SEQ ID NO:1), is designed and artificially synthesized by General Biological Gene (Anhui) Co., Ltd. To facilitate cloning and ensure sequence integrity, when synthesizing the ARE-1 sequence, GTCTCGCGA sequence is introduced at the 5'end, where GTC is the protective base, and TCGCGA is the NruI restriction site. the 3'end is introduced with AGCACGCGT, where AGC is the restriction protected bases, ACGCGT is the MluI restriction site.

[052] The synthesized ARE-1 sequence is digested with NruI/MluI, and the pIRES-EGFP vector is digested with NruI/MluI at the same time. The result of restriction digestion is identified by agarose gel electrophoresis, and the digested ARE 1 sequence fragment and pIRES-EGFP linear plasmid DNA are recovered from the gel.

[053] The double digestion system of ARE-1 sequence: ARE-1 sequence 10 L (1pg/pL), 10xNE Buffer 3.1 3[L, NruI/MluI (I0U/pL) each 1.0[L, added water to

[tL. The enzyme digestion conditions are: 37°C, digestion for 3 min.

[054] The double digestion system of pIRES-EGFP vector is as follows: pIRES EGFP plasmid 5 L (1 g/4L), 0 xNE Buffer3.1 2L,NruI/MluI (I0U/pL) each 0.5 L, added water to 20[L. enzyme The enzyme digestion conditions are: 37°C, digestion for 3 min.

[055] The digested ARE-1 sequence fragment and pIRES-EGFP linear plasmid DNA (molar ratio 5:1) is taken, and ligated for 5 min at 25°C by NEB TM ligationkit,. The ligation product is added to the competent cell suspension of E.coli JM109 strain for transformation, 150 L of the transformed bacteria solution is inoculated on an LB plate containing ampicillin, incubated at 37°C overnight, and a single colony is picked for subculture. The recombinant plasmid is extracted and verified by double restriction digestion (NruI/MluI), and the plasmid verified by restriction digestion is taken for sequencing verification, and a correctly constructed expression vector II is obtained, which is named pIRES- Iin this example.

[056] 1.3 Construction of the expression vector pIRES-2 containing the IRES-1 sequence (replace the IRES sequence on the pIRES-1 vector)

[057] (1) Synthesis of IRES-1 sequence

[058] The IRES-1 sequence (as shown in SEQ ID NO: 4) is designed and artificially synthesized by General Biological Gene (Anhui) Co., Ltd.

[059] (2) Construction of expression vector pIRES-2

[060] A seamless cloning kit is used to replace the nucleotide sequence of the IRES sequence of the pIRES-1 vector with IRES-1 to form a new expression vector. the new expression vector is verified by double enzyme digestion with SmaI/PacI enzymes. The vector verified by restriction enzyme digestion is perform sequencing verification to obtain the correctly constructed expression vector III, which is named pIRES-2 in this example.

[061] 1.4 Construction of expression vector pIRES-3 containing WPRE sequence

[062] (1) Synthesize WPRE sequence

[063] The WPRE sequence (as shown in SEQ ID NO: 7) is designed and artificially synthesized by General Biological Gene (Anhui) Co., Ltd. To facilitate cloning and ensure sequence integrity, when synthesizing the WPRE sequence, CCGGAATTC sequence is introduced at the 5'end, where CCG is the protective base, and GAATTC is the EcoRI restriction site. the3'end is introduced with CTAGGATCC, where CTA is the protective base for restriction digestion base, GGATCC is the BamHI restriction site.

[064] (2) Construction of expression vector pIRES-3

[065] The synthesized WPRE sequence is digested with EcoRI/BamHI, and the pIRES-2 vector DNA is digested with EcoR/BamHI. Agarose gel electrophoresis is used to identify the digestion results, and the digested WPRE sequence fragment and pIRES-2 vector DNA are recovered by the gel digestion.

[066] The restriction digestion system of WPRE sequence is: 10xM buffer 2L, 1OU/4L EcoRI, BamHI enzymes each 0.5 L, 1.289 g/L EGFP amplification product 0.78[tL, and add water to 20jL. After mixing throughly, it is incubated at 37°C for 6h.

[067] The digestion system of pIRES-2 vector is: 10xK buffer 2L, 1OU/L EcoRI and BamHI enzymes each 0.5 L, 0.81 g/4L pIRES-2 DNA 1.23 L, and added the water to 20jL. After mixing well, incubated at 37°C for 3h.

[068] Using T4 ligase to ligate the digested WPRE sequence fragment and pIRES-2 vector.

[069] The ligation system of pIRES-2 vector is: 10 L of 2xQuick Ligation Buffer, 200 ng of linear DNA of pIRES-2, 87.2ng of digested EGFP sequence fragment, 350U/[L of T4 ligase 1 L, supplemented with water to 20[L, and ligated overnight at 16 0 C

[070] The ligation product is added to the E.coli JM109 competent bacterial suspension for transformation, 100 L of the transformed bacteria solution is inoculated on an LB solid culture plate containing ampicillin, and incubated overnight at 37C, picked a single colony of shake bacteria for subculture. Extract the plasmid and digest the bacterial plasmid with EcoRI and BamHI. The digestion system is: 10xK buffer 24L, 1OU/L EcoR, BamHI enzyme each 0.5[tL, 0.81tg/4L DNA connected to the vector 1.23[tL, supplemented with water to 20jL. After mixing well, incubate at 370 C for 3h.

[071] As for nucleic acid electrophoresis verification, the size of the band with the correct restriction enzyme digestion is 720 bp, the correct plasmid identified by restriction restriction enzyme digestion is selected, and the sequencing verification is performed, and finally the correct expression vector IV is obtained, which is named pIRES-3 in this example.

[072] 1.5 Construction of expression vector pIRES-4 containing BSR sequence

[073] (1) Synthesize BSR sequence

[074] The BSR sequence (as shown in SEQ ID NO: 8) is designed and artificially synthesized by General Biological Gene (Anhui) Co., Ltd.

[075] (2) Construction of mammalian cell combination expression vector pIRES 4

[076] The artificially synthesized BSR sequence is inserted into the pIRES-3 vector using a seamless cloning kit, replaced the selection marker gene sequence on the pIRES-3 vector with the BSR sequence of the blasticidin resistance gene. The new expression vector is double-enzyme digested using SmaI/PacI enzyme for verification, the correct vector is taken for the restriction enzyme digestion, and then perform sequencing verification. The correctly constructed expression vector V is obtained, which is named pIRES-4 in this example (see Figure 2 for the plasmid map). The structure of the mammalian cell combination expression vector obtained by inserting the GOI between the promoter and IRES-1 sequence in the combination expression vector pIRES-4 is: (ARE-)-promoter-GOI-WPRE-(IRES-1 )-BSR-PolyA.

[077] Example 2 Construction of eukaryotic cell expression system

[078] CHO cells are cultured in DMEM medium containing 10% inactivated fetal bovine serum at 37°C and 5% C02. CHO cells (3x10 6/well) is inoculated in a 6-well plate. Transfection is performed when the cells reached about 90% confluence after plating for 24 h. Using Lipo 3000 (Lipofectamine@ 3000) as the transfection reagent, the combined expression vector pIRES-4 obtained in Example 1 is transfected into CHO cells. After 24h of transfection, G418 at a concentration of 800 g/mL is used to screen for positive clones for 2 weeks, and then G418 at a concentration of 800 g/mL is replaced by 400 g/mL for 1 month to obtain mammalian host cell CHO.

[079] Example 3 Method for expression of mammalian protein

[080] 3.1 Construction of the combined expression vector pIRES-4 containing the EGFP gene sequence and artificially synthesized nucleotide sequences of ARE-1, IRES-1, WPRE, and BSR, and the construction method is the same as in Example 1.

[081] 3.2 Transfection of the combined expression vector pIRES-4 into mammalian host cells CHO, the transfection method is the same as the transfection method in the second embodiment of the mammalian host cells.

[082] 3.3 Culture CHO cells to express the interest protein.

[083] Test Example 1

[084] The ARE-2 sequence (shown in SEQ ID NO: 2) and the ARE-3 sequence (shown in SEQ ID NO: 3) is designed and artificially synthesized by General Biological Gene (Anhui) Co., Ltd.

[085] The IRES-2 sequence (shown in SEQ ID NO: 5) and the IRES-3 sequence (shown in SEQ ID NO: 6) are designed and artificially synthesized by General Biological Gene (Anhui) Co., Ltd.

[086] Comparative example 1: Mammalian cell combined expression vector: referring to step 1.2 in Example 1, using NruI/MluI double enzyme digestion, replaced the ARE-i sequence of the pIRES-1 vector with ARE-2 (as shown in SEQ ID NO: 2) to construct a new expression vector. The NruI/MluI enzyme is used for double enzyme digestion verification of the new expression vector, and the vector with the correct enzymatic verification is sequenced to construct the correct expression vector II, named pIRES-1A. the rest is the same as Example 1.

[087] Comparative example 2: Mammalian cell combined expression vector: referring to step 1.2 in Example 1, using NruI/MluI double enzyme digestion, replaced the ARE-i sequence of the pIRES-1 vector with ARE-3 (as shown in SEQ ID NO: 3) to form a new expression vector. use NruI/MluI enzymes to perform double enzyme digestion for verification the new expression vector, and taken the enzyme digestion to verify the correct vector and then perform sequencing verification, the correct expression vector II is constructed and named pIRES-1B. the others are the same as in Example 1.

[088] Comparative example 3 of mammalian cell combination expression vector: referring to step 1.3 in example 1, the IRES-1 sequence of pIRES-2 vector is replaced with IRES-2 (as shown in seq id no: 5) by seamless cloning kit to form a new expression vector. The new expression vector is verified by double digestions with SmaI/PacI enzyme, and the correct expression vector III is constructed and named pIRES-2A;

Other things are the same as embodiment 1.

[089] Comparative Example 4: Mammalian Cell Combination Expression Vector: Refer to step 1.3 in Example 1, use the seamless cloning kit to replace the IRES-

1 nucleotide sequence of the pIRES-2 vector with IRES-3 (as shown in SEQ ID NO: 6) to form a new expression vector. The new expression vector is double digested using SmaI/Pac enzymes, the vector verified by restriction enzyme digestion is taken and then perform sequencing verification to construct the correct expression vector III, named pIRES-2B. the others are the same as in Example 1.

[090] Comparative examples 1 of mammalian host cell : the expression vector pIRES-1 in Example 2 for constructing a mammalian host cell are replaced with the expression vector pIRES-1A in comparative examples 1 for constructing a mammalian cell combination expression vector, otherwise as in Example 2. The structure of the resulting mammalian cell combination expression vector after inserting the GOI between the promoter and IRES-1 sequence is: (ARE-2)-promoter-GOI-WPRE-(IRES 1)-BSR-PolyA.

[091] Comparative Example 2 of Mammalian Host Cells: the expression vector pIRES-1 in Example 2 for constructing mammalian host cells is replaced with the expression vector pIRES-B in Comparative Example 1 of the mammalian cell combination expression vector, others are the same as in the Example 2. After inserting the GOI between the promoter and the IRES-1 sequence, the resulting mammalian cell combined expression vector structure is as follows: (ARE-3)-promoter-GOI-WPRE (IRES-1)-BSR-PolyA.

[092] Comparative Example 3 of Mammalian Host Cells: The expression vector pIRES-2 in Example 2 for constructing mammalian host cells is replaced with the expression vector pIRES-2A in Comparative Example 1 of the mammalian cell combination expression vector, others are the same as in the Example 2. After inserting the GOI between the promoter and the IRES-2 sequence, the structure of the mammalian cell combination expression vector is as follows: (ARE-1)-promoter-GOI WPRE-(IRES-2)-BSR-PolyA.

[093] Comparative Example 4 of Mammalian Host Cells: The expression vector pIRES-2 in Example 2 for constructing mammalian host cells is replaced with the expression vector pIRES-2B in Comparative Example 1 of the mammalian cell combination expression vector, others are the same as in the Example 2. After inserting the GOI between the promoter and the IRES-3 sequence, the structure of the mammalian cell combined expression vector is as follows: (ARE-1)-promoter-GOI WPRE-(IRES-3)-BSR-PolyA.

[094] The expression vectors pIRES-1, pIRES-2, pIRES-3, pIRES-4 obtained in Example 1 and the expression vectors pIRES-1A, pIRES-1B, pIRES- 2A and pIRES 2B are respectively transfected into CHO cells, the specific steps are as follows:

[095] (1) Cell transfection

[096] CHO cells are cultured in DMEM medium containing 10% inactivated fetal bovine serum at 37°C and 5% of C02. Inoculate CHO cells (3x106 /well) in a 6-well plate. The cells reached about 90% confluence after 24 h.

[097] The experiment is divided into 10 groups: 0 Normal CHO cell group.© Control group-transfection-control vector pIRES-EGFP. Transfection expression vector pIRES-1. Transfection expression vector pIRES-1A. @ Transfection expression Vector pIRES-1B. ®Transfection expression vector pIRES-2. OTransfection expression vector pIRES-2A. ®Transfection expression vector pIRES-2B. ®Transfection expression vector pIRES-3. @Transfection expression vector pIRES-4. Using Lipofectamine@ 3000 as the transfection reagent, the vectors of each test group are transfected into CHO cells.

[098] (2) Screening of stably transfected cell lines

[099] After 24 h of transfection, the vectors of each test group are transfected into CHO cells cultured in a 24-well plate (500 L/well) (the number of CHO cells is about 2x10), the concentration of @~@ group is 800kg /mL of G418 screening for 2 weeks when positive clones appear, G418 with a concentration of 800 g/mL is replaced with 400 g/mL and cultured stably for 1 month, and then cells of each test group are collected for flow cytometry. The @-@ group used 15 g/mL blasticidin to screen positive clones after 1 week (the screening time is shortened by 1 week). 15[tg/mL blasticidin is replaced with 10 g/mL and cultured stably for 1 month, and then the cells of each test group are collected for flow cytometry.

[0100] (3) Influence on the stable expression of EGFP gene

[0101] The ()~@group used the medium containing 500g/mL G418, and the ~-@ group used the polyclonal CHO cells obtained by subculturing with the concentration of 10Og/mL blasticidin after 30 days. The cells of each test group are collected for flow cytometry, and the results w shown in Figure 3. It can be seen from 3 that under the same conditions, compared with the average expression level of the control vector pIRES-EGFP, pIRES-1, pIRES-1A, pIRES-1B, pIRES-2, pIRES-2A, pIRES-2B, pIRES-3 , PIRES-4, increased 2.87, 2.15, 1.76, 3.24, 2.65, 2.31, 3.74, 4.58 times respectively. The expression trend is pIRES-4>pIRES-3>pIRES-2A>pIRES 2B>pIRES-1>pIRES-1A>pIRES-1B, that is, the levels of pIRES-4 mediated protein expressions is the highest, which is 4.58 times higher than the original vector pIRES EGFP.All data are repeated for three times.

[0102] (4) Impact on long-term expression of EGFP

[0103] After 30 days of screening, the polyclonal CHO cells are cultured for up to 180 days. Cells in each test group are collected for flow cytometry, and the effects of different expression vectors on the long-term expression of recombinant GOIs are analyzed. The results are shown in Figure 4.

[0104] As can be seen from Figure 4, compared with the control vector pIRES EGFP, pIRES-1, pIRES-1A, pIRES-1B, pIRES-2, pIRES-2A, pIRES-2B, pIRES-3, pIRES-4 vector, only the two expression vectors pIRES-1B and pIRES-2A have lower expression stability than the control vector pIRES-EGFP. The remaining six vectors can improve the stable expression of EGFP to a certain extent. Among them, the expression of EGFP mediated by pIRES-4 vector is the most stable, with a retention rate of 87.6%, which is 1.39 times higher than the stable expression of the control vector pIRES-EGFP. All data are statistical data of three repeated experiments.

[0105] Test Example 2

[0106] The synthesis of Hepatitis B Antigen gene (GenBank: V00867.1) in this test case is completed by General Biogene (Anhui) Co., Ltd.

[0107] (1) Construction of recombinant expression vector containing Hepatitis B Antigen GOI

[0108] The synthetic Hepatitis B Antigen gene sequence is digested with EcoRI/BamHI, and the expression vectors pIRES-EGFP, pIRES-1, pIRES-2, pIRES-3, pIRES-4 are digested with EcoR I/BamH I. Agarose gel electrophoresis is used to identify the digestion results, and the digested Hepatitis B Antigen gene sequence fragments and the linear plasmid DNA of pIRES-1, pIRES-2, pIRES-3, and pIRES-4 are recovered from the gel digestion.

[0109] The double enzyme digestion system of Hepatitis B Antigen gene sequence is: Hepatitis B Antigen sequence fragment 10 L (1 g/L), 10xNE Buffer 2.13[tL, EcoRI/BamHI enzyme (10U/4L) each 1.0 L, and makeup to 30 L with water. Enzyme digestion conditions are: 37°C, digestion for 3 min.

[0110] The double restriction digestion system of the plasmid is: plasmid 15 L (1pg/pL), 10xNE Buffer 2.12L, EcoR I/BamH I (10U/L)each 0.5[tL, make up water to 20[L. restriction conditions are as follows: 37°C , enzyme digestion for 3min.

[0111] Taking the digested Hepatitis B Antigen gene sequence fragments and linear plasmid DNA (molar ratio 5:1), use NEB TM ligation kit, and ligate for 5 min at °C. The ligation product is added to the E.coli strain JM109 competent cell suspension for transformation, 150 L of the transformed bacteria solution is inoculated on an LB plate containing ampicillin, incubated at 37°C overnight, and a single colony is picked for subculture. Extract the recombinant expression vector and perform double enzyme digestion (EcoR I/BamH I) verification, take the vector that is verified by the enzyme digestion and perform sequencing verification, and finally obtain the correct combined expression vector. The combined expression vectors containing the expression vectors pIRES-EGFP, pIRES-1, pIRES-2, pIRES-3, and pIRES-4 are named pIRES-EH, pIRES-1H, pIRES-2H, pIRES-3H, and pIRES-4H, respectively.

[0112] (2) The screening of stable transfected cell lines

[0113] 1) Cell transfection

[0114] CHO cells are cultured in DMEM medium containing 10% inactivated fetal bovine serum at 37°C and 5%CO2. Inoculate CHO cells (3x10 6 /well) in a 6-well plate. The cells reached about 90% confluence after 24 h of plating. The experiment is divided into 6 groups: 0 Normal CHO cell group. Control group-transfection control vector pIRES-EH. © Transfection with recombinant vector pIRES-1H. Transfection with recombinant vector pIRES-2H. Transfection with recombinant expression vector pIRES-3H. ®Transfect the recombinant expression vector pIRES 4H. Using Lip3000 (Lipofectamine@3000) as the transfection reagent, the vectors of each test group are co-transfected into CHO cells.

[0115] 2) Screening of stably transfected cell lines

[0116] The vectors are co-transfected into CHO cells cultured in a 24-well plate (500 L/well) (the number of CHO cells is about 2x106), and G418 is screened with a concentration of 800 g/mL for 2 weeks. When positive clones appeared, after changing the concentration of 400 g/mL G418 to stable culture for 2 weeks, the amount of inoculated cells per mL is 500,000 to 600,000, the speed of shaker is set to 110-120 rpm/min, and the culture supernatant is collected on the 7th day of suspension culture.

[0117] 3) Detection of Hepatitis B Antigen expression

[0118] ELISA kit is used to detect the expression level of Hepatitis B Antigen. The results are shown in Figure 5. As can be seen from Figure 5, the expression levels of the Hepatitis B Antigen containing the combined expression vectors pIRES-EH, pIRES-1H, pIRES-2H, pIRES-3H, and pIRES-4H are 9.21g/d/106 cells, 24.04[tg/d/10 6 cells, 28.73[tg/ d/10 6 cells, 33.62[tg/d/10 6 cells, 38.96[tg/d/10 6 cells. Compared with the control group pIRES-EH, the expression of Hepatitis B Antigen containing the combined expression vectors pIRES-1H, pIRES-2H, pIRES-3H, and pIRES-4 increased by 2.61, 3.12, 3.65, 4.23 times, That is, the expression vector of the present invention can significantly increase the expression level of foreign protein in animal cells.

[0119] Unless otherwise specified, the relevant operations in the embodiments and test examples are conventional technical means in the field. For example, refer to the molecular cloning experiment manual compiled by Sambrook et al. (Sambrook J &

Russell DW. Molecular cloning: a laboratory manual. 2001), or the instructions provided by the product manufacturer.

[0120] The above-mentioned embodiments are only preferred embodiments of the present invention. They are only used to explain the present invention and do not limit the scope of implementation of the present invention. For those skilled in the art, of course, other implementations can be easily made by substitution or modification according to the technical content disclosed in this specification, so all changes and improvements made on the principle of the present invention, etc., should be included in the scope of the patent application of the present invention.

[0121] Although the invention has been described with reference to specific examples, it will be appreciated by those skilled in the art that the invention may be embodied in many other forms, in keeping with the broad principles and the spirit of the invention described herein.

[0122] The present invention and the described embodiments specifically include the best method known to the applicant of performing the invention. The present invention and the described preferred embodiments specifically include at least one feature that is industrially applicable

Claims (7)

THE CLAIMS DEFINING THE INVENTION ARE AS FOLLOWS:

1. A combined expression vector for mammalian cells, characterized in that: The expression vector is inserted with the ARE sequence shown in SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 3, such as SEQ ID NO: 4, The IRES sequence shown in SEQ ID NO: 5 or SEQ ID NO: 6, the WPRE sequence shown in SEQ ID NO: 7 and the selection marker gene sequence. the ARE sequence is located upstream of the promoter, the WPRE sequence is located downstream of GOI, the selection marker gene is located downstream of the IRES sequence, and the GOI is located between the promoter and the IRES sequence.

2. The mammalian cell combined expression vector according to claim 1, wherein the selection marker gene sequence is selected from the BSR sequence shown in SEQ ID NO:8.

3. The mammalian cell combined expression vector of claim 1, wherein the original vector of the expression vector is pIRES-neo2 or pIRES-neo3.

4. The method for preparing a mammalian cell combined expression vector according to any one of claims 1 to 3, characterized in that it comprises the following steps:

Step a: PCR amplification of EGFP, double digestion of EGFP reporter gene amplification product and original vector, recovery of digested fragments, ligation, transformation and identification to obtain expression vector I.

Step b: Double-enzyme digestion of the ARE sequence and expression vector I, recover the digested fragments, ligate, and identify after transformation to obtain expression vector II.

Step c: Seamlessly clone the IRES sequence into expression vector II to obtain expression vector III.

Step d: Double digestion of the WPRE sequence and expression vector III respectively, recover the digested fragments, ligate, and identify after transformation to obtain expression vector IV.

Step e: Seamlessly clone the selection marker gene sequence into the expression vector IV to obtain the mammalian cell combination expression vector V.

5. A eukaryotic cell expression system comprising the mammalian cell combination expression vector of any one of claims 1 to 3.

6. The preparation method of the eukaryotic cell expression system of claim 5, characterized in that it comprises the following steps:

Step a: Insert the GOI between the promoter and IRES sequence of the combined expression vector of any one of claims 1 to 3 to construct a recombinant expression vector.

Step b: Transfect the recombinant expression vector into the host cell, and obtain the expression system after screening.

7. The use of the mammalian cell combination expression vector of any one of claims 1 to 3 and the eukaryotic cell expression system of claim 5 in the preparation of a drug containing the target protein.