Dual expression vector, and construction method and application thereof

Abstract

The invention discloses a dual expression vector, and a construction method and application thereof. The double expression vector comprises a transcription and translation element, a replication element and the like; wherein the transcription and translation element comprises an enhancer, an eukaryotic promoter, a prokaryotic promoter, a ribosome binding site, a spacer region, an initiation codon, a cloning site, a termination codon, an eukaryotic transcription termination sequence, a prokaryotic transcription termination sequence and the like which are sequentially connected from upstream to downstream. The same target protein can be efficiently expressed in a pronucleus-based cell-free protein expression system and a eukaryotic system by utilizing the double expression vector, the operation is simple and convenient, the efficiency is high, and the cost is low.

Description

Technical Field

[0001] The present invention relates to a protein expression system, in particular to a dual expression vector, a construction method and application thereof, and belongs to the field of biotechnology. Background Art

[0002] Recombinant proteins, produced using recombinant DNA or RNA technologies, are currently widely used in the research, development, and production of biopharmaceuticals, cellular immunotherapy, and diagnostic reagents. With the advancement of science and technology, cell-free protein expression has become a powerful protein expression method due to its unique open system characteristics, which allow for control of reaction conditions and reactant components, direct monitoring of reaction expression, and high-throughput screening. Currently, numerous systems capable of cell-free protein expression exist both domestically and internationally, including prokaryotic expression systems in Escherichia coli, eukaryotic expression systems in yeast, wheat germ, insect, and human-derived systems. Among these, the prokaryotic expression system in Escherichia coli is the most commonly used due to its relatively simple preparation, low cost, and high protein yield.

[0003] Although E. coli-based cell-free protein expression technology has many advantages as mentioned above, the directly expressed proteins are free of post-translational modifications, which has certain limitations for the study of the functions of some proteins that require glycosylation modification, such as antibodies. If you want to express the same protein in different expression systems, you need to construct different expression vectors, which is a time-consuming and cumbersome process. Therefore, if you want to take advantage of the convenience and high-throughput capabilities of the prokaryotic cell-free protein expression system and want to conduct functional studies of post-translational modifications, you need to develop a vector that can be expressed in both systems simultaneously.

[0004] Although numerous vectors are available for protein expression in both prokaryotic and eukaryotic expression systems, few can express the same protein simultaneously in both prokaryotes and eukaryotes, and even fewer can express the same protein in both prokaryotic and eukaryotic cell-free systems. In 1992, Alting-Mees, MA, designed a co-expression vector, pBK-CMV, for both prokaryotic and eukaryotic expression systems. However, target genes cloned into pBK-CMV were only minimally expressed in eukaryotic expression systems. In 1997, US20010016351A1 further optimized the pBK-CMV vector, incorporating the CMV enhancer and promoter regions necessary for target gene expression in mammalian cells. The lactose promoter and lacZ sequence were removed from pBK-CMV, and a prokaryotic expression sequence was inserted in its place. This prokaryotic expression sequence comprises a T7 promoter, a lac operator sequence, followed by a tandem ribosome binding site and a Kozak consensus sequence. Studies have demonstrated that this expression vector can provide high-level heterologous gene expression in mammalian cells and Escherichia coli. Although this expression vector can express high levels of protein in the cell body, its protein expression level in cell-free protein expression based on the prokaryotic system is very low. In 2022, Zhu Minghui and others constructed a dual expression vector that can be expressed simultaneously in Chinese hamster ovary (CHO) cells and Escherichia coli (E. coli) BL21. The study cloned the prokaryotic expression elements T7 promoter and T7 terminator into the eukaryotic expression vector pIRES-EGFP, and successfully expressed the expression vector in CHO cells and E. coli BL21. Although the expression vector successfully expressed GFP and HTrf in CHO cells and E. coli BL21, the protein expression level was still low. Although the above-mentioned vectors can be expressed in E. coli cells and eukaryotic cells, antibodies or antibody fragments are easily formed inclusion bodies in E. coli or the expression level is very low, so they are not suitable for the expression of antibodies or antibody fragments. Due to its open nature, cell-free protein expression systems can optimize the expression of antibodies or antibody fragments by adding molecular chaperones, changing redox potential, and testing the light-heavy chain ratio. Therefore, there is an urgent need to develop a vector suitable for cell-free and eukaryotic expression systems for the expression of antibodies or antibody fragments. Summary of the Invention

[0005] The main purpose of the present invention is to provide a dual expression vector, a construction method thereof, and its application in the expression of antibodies or antibody fragments. The dual expression vector can efficiently express the same target protein in a prokaryotic cell-free protein expression system and a eukaryotic system, eliminating the tedious operation of constructing different expression vectors in different expression systems to express the same target protein, thereby improving efficiency, saving time, and reducing costs, thereby overcoming the shortcomings of the prior art.

[0006] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0007] One aspect of the present invention provides a dual expression vector comprising:

[0008] Transcription and translation elements, at least for enabling the transcription and translation of the target gene in prokaryotic cell-free protein expression systems and eukaryotic cells;

[0009] a replication element, at least for enabling the vector to replicate in prokaryotic and eukaryotic cells;

[0010] The transcription and translation elements include enhancers, eukaryotic promoters, prokaryotic promoters, ribosome binding sites, spacers, start codons, cloning sites, stop codons, eukaryotic transcription termination sequences, and prokaryotic transcription termination sequences, which are sequentially connected from upstream to downstream.

[0011] Another aspect of the present invention provides a method for constructing a dual expression vector, which comprises: inserting a prokaryotic expression element into a eukaryotic expression vector, and making the insertion site of the prokaryotic transcription termination sequence in the prokaryotic expression element in the eukaryotic expression vector downstream of the eukaryotic transcription termination sequence, and the insertion sites of the remaining sequences in the eukaryotic expression vector downstream of the promoter and upstream of the start codon of the eukaryotic expression vector, thereby obtaining the dual expression vector.

[0012] Another aspect of the present invention provides a kit comprising the dual expression vector.

[0013] Another aspect of the present invention provides a protein expression method, comprising: inserting a gene encoding a target protein into the cloning site of the dual expression vector to obtain a recombinant vector;

[0014] The recombinant vector is used to express the target protein using a cell-free protein expression kit, and / or the recombinant vector is introduced into eukaryotic cells to express the target protein; wherein the target protein includes an antibody or an antibody fragment

[0015] Compared with the existing technology, the dual expression vector provided by the present invention can simultaneously express the same protein in a prokaryotic cell-free protein expression system and a eukaryotic system without repeating tedious vector construction steps, saving time and labor, improving efficiency and reducing costs. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 This is a schematic diagram of the screening of element sequences suitable for the prokaryotic cell-free protein expression system inserted into the pcDNA3.4 eukaryotic expression plasmid in Example 1.

[0017] Figure 2 The results show the effect of different expression element sequences on GFP protein expression levels screened using a cell-free protein expression system in Example 1.

[0018] Figure 3 The results show the effect of different expression element sequences on GFP protein expression levels screened using HEK293 cells in Example 1.

[0019] Figure 4 Schematic diagram of the transcription and translation elements of the dual expression vector P2 in Example 2.

[0020] Figure 5 The GFP protein expression level of the dual expression vector P2 was measured using a cell-free expression system in Example 2.

[0021] Figure 6 Schematic diagram of the transcription and translation elements of the dual expression vector P3 in Example 3.

[0022] Figure 7 The figure shows the effect of different expression-aiding tags on the GFP protein expression level of the dual expression vector P3 in Example 3.

[0023] Figure 8 This is a graph showing the affinity activity of bevacizumab expressed in HEK293 cells detected by the ELISA Kit in Example 4.

[0024] Figure 9 This is the spectrum of the commercial dual expression vector pdual-GFP in Comparative Example 1.

[0025] Figure 10 The GFP expression level of pdual-GFP in the cell-free protein expression system in Comparative Example 1 is shown. DETAILED DESCRIPTION

[0026] In view of the shortcomings of the existing technology, the present application aims to provide a dual expression vector in which the inserted target gene sequence can be expressed at high levels in both a prokaryotic cell-free protein expression system and a eukaryotic system. The dual expression vector, its construction method, and specific applications are described below.

[0027] As used herein, a "vector" is a nucleic acid molecule (DNA or RNA) capable of autonomous replication after introduction into a recipient cell. Examples of vectors include plasmids, viruses, and bacteriophages. The process of "expression" is well known and includes the use of cellular enzymes and the production of protein expression products from polynucleotides. As used herein, a polynucleotide is a DNA or RNA molecule. An "expression vector" is a vector capable of mediating the expression of a cloned polynucleotide.

[0028] The present invention relates to the design and use of vectors capable of allowing high-level transcription and translation of the aforementioned polynucleotides in eukaryotic organisms (particularly mammalian, such as human, mouse, ape, bovine, porcine or sheep cells) and prokaryotic cells (such as Escherichia coli, Escherichia coli, Enterobacter, Streptomyces). As used herein, expression vectors capable of mediating such high-level gene expression in both prokaryotic and eukaryotic cells are referred to as "dual" expression vectors.

[0029] Some embodiments of the present application provide a dual expression vector comprising:

[0030] Transcription and translation elements, at least for enabling the transcription and translation of the target gene in prokaryotic cell-free protein expression systems and eukaryotic cells;

[0031] a replication element, at least for enabling the vector to replicate in prokaryotic and eukaryotic cells;

[0032] The transcription and translation elements include enhancers, eukaryotic promoters, prokaryotic promoters, ribosome binding sites, spacers, start codons, cloning sites, stop codons, eukaryotic transcription termination sequences, and prokaryotic transcription termination sequences, which are sequentially connected from upstream to downstream.

[0033] In one embodiment, the transcription and translation elements include an enhancer, a eukaryotic promoter, a prokaryotic promoter, a ribosome binding site, a spacer, a kozak sequence, a second ATG sequence, a cloning site, a stop codon, a eukaryotic transcription termination sequence, and a prokaryotic transcription termination sequence connected sequentially from upstream to downstream.

[0034] In one embodiment, the transcription and translation elements include an enhancer, a eukaryotic promoter, a prokaryotic promoter, a ribosome binding site, a spacer, a first ATG sequence, a kozak sequence, a second ATG sequence, a cloning site, a stop codon, a eukaryotic transcription termination sequence, and a prokaryotic transcription termination sequence connected sequentially from upstream to downstream.

[0035] In one embodiment, the transcription and translation elements include an enhancer, a eukaryotic promoter, a prokaryotic promoter, a ribosome binding site, a spacer, a first ATG sequence, an expression-aiding sequence, a kozak sequence, a second ATG sequence, a cloning site, a stop codon, a eukaryotic transcription termination sequence, and a prokaryotic transcription termination sequence, connected sequentially from upstream to downstream.

[0036] In the above embodiments of the present invention, when prokaryotic expression is performed, the first ATG sequence serves as the start codon for protein expression, and the second ATG sequence serves as the common Met amino acid expression; and when eukaryotic expression is performed, the first ATG sequence serves as the spacer sequence, and the second ATG sequence serves as the start codon for protein expression.

[0037] In the above embodiments of the present invention, the auxiliary expression sequence can be selected from a variety of auxiliary expression sequences known in the art, for example, it can be an IKSK sequence, an IKRK sequence, a pelB sequence, an ompA sequence, a phoA sequence, a malE sequence, an ompC sequence, an ompT sequence, a His tag, a Sumo tag, a TrxA tag, a DsbA tag, a DsbC tag, a GST tag, etc., but is not limited thereto.

[0038] In the above embodiment of the present invention, the spacer is used to regulate the distance between the ribosome binding site and the start codon. Preferably, the length of the spacer is 5 to 11 bp. The sequence of the spacer can be arbitrary, for example, aagccacc, aatgccacc, aatagccacc, aatatgccacc, aaagccacc, aaaagccacc, aggccacc, atatacat, atatacc, but is not limited thereto.

[0039] In one embodiment, the prokaryotic promoter includes but is not limited to at least one of T7 promoter, lambda phage promoter, lactose promoter, arabinose promoter, tryptophan (trp) promoter, tac promoter and other known promoters, preferably T7 promoter.

[0040] In one embodiment, the eukaryotic promoter includes but is not limited to SV40 (green monkey vacuolating virus), CMV (cytomegalovirus), RSV (sarcoma virus), ADV (adenovirus), LTR (retroviral long terminal repeat) and at least one of other known promoters, preferably a CMV promoter.

[0041] In one embodiment, the enhancer includes but is not limited to at least one of SV40 enhancer, RSV enhancer, LTR enhancer and other known enhancers, preferably CMV enhancer.

[0042] In one embodiment, the dual expression vector further comprises:

[0043] A selectable marker element is used to enable selection of the vector in prokaryotic and eukaryotic cells.

[0044] In a typical implementation case, a dual expression vector comprises:

[0045] Transcription and translation elements, including:

[0046] a) Promoters suitable for prokaryotes, including T7 promoter, lambda phage promoter, lactose promoter, arabinose promoter, tryptophan (trp) promoter and tac promoter, etc.

[0047] b) ribosome binding site,

[0048] c) prokaryotic transcription termination sequence,

[0049] d) Suitable for eukaryotic promoters, including SV40, CMV, RSV, ADV, LTR and other promoters,

[0050] e) Enhancers, including SV40, CMV, RSV or LTR enhancers,

[0051] f) eukaryotic transcription termination sequences such as polynucleotide signals;

[0052] Replication elements include elements that enable the vector to replicate in prokaryotes and eukaryotes, such as the pUC replication region sequence, which is used to enable plasmid replication in E. coli.

[0053] Selectable marker elements include elements that allow the vector to be selected in prokaryotic and eukaryotic expression systems, such as a neomycin resistance gene in eukaryotic cells and an ampicillin resistance gene in prokaryotes.

[0054] In one embodiment, the sequence of the dual expression vector includes any one of SEQ ID NO.7 to SEQ ID NO.9 and SEQ ID NO.13 to SEQ ID NO.16, preferably any one of SEQ ID NO.13 to SEQ ID NO.16, and more preferably any one of SEQ ID NO.14 to SEQ ID NO.16.

[0055] Some embodiments of the present application also provide a method for constructing the dual expression vector, which comprises inserting a prokaryotic expression element into a eukaryotic expression vector, preferably inserting a prokaryotic expression element into a shuttle vector.

[0056] Specifically, the construction method includes: inserting a prokaryotic expression element into a eukaryotic expression vector, and making the insertion site of the prokaryotic transcription termination sequence in the prokaryotic expression element in the eukaryotic expression vector downstream of the eukaryotic transcription termination sequence, and the insertion sites of the remaining sequences in the eukaryotic expression vector downstream of the promoter and upstream of the ATG sequence of the eukaryotic expression vector, thereby obtaining the dual expression vector.

[0057] In one embodiment, the order of insertion of prokaryotic expression elements into a eukaryotic expression vector can be: enhancer + eukaryotic promoter + prokaryotic promoter and its flanking regulatory sequences + ribosome binding site + ATG sequence + target gene + stop codon + eukaryotic transcription termination sequence + prokaryotic transcription termination sequence, with the ribosome binding site and the ATG sequence being at an appropriate distance, controlled by a spacer disposed therebetween. The resulting sequence can be defined as the base sequence.

[0058] A more preferred order is: enhancer + eukaryotic promoter + prokaryotic promoter and its flanking regulatory sequences + ribosome binding site + Kozak sequence + ATG sequence + target gene + stop codon + eukaryotic transcription termination sequence + prokaryotic transcription termination sequence. The ribosome binding site and the ATG sequence are at an appropriate distance, which is controlled by the spacer region provided between the two. The sequence thus formed can be defined as the first preferred sequence. Compared to the aforementioned basic sequence, the first preferred sequence has an additional Kozak sequence. The Kozak sequence can bind to translation initiation factors to mediate mRNA translation initiation, which plays an important role in translation initiation and can enhance translation initiation efficiency, thereby increasing protein expression in eukaryotic systems.

[0059] A further preferred order is: enhancer + eukaryotic promoter + prokaryotic promoter and regulatory sequences on both sides thereof + ribosome binding site + first ATG sequence + Kozak sequence + second ATG sequence + target gene + stop codon + eukaryotic transcription termination sequence + prokaryotic transcription termination sequence, wherein the ribosome binding site and the first ATG sequence are at an appropriate distance, and the distance is controlled by the spacer region provided therebetween. The sequence thus formed can be defined as a second preferred sequence. Compared to the aforementioned first preferred sequence, the second preferred sequence uses the Kozak sequence as the amino acid for cell-free protein expression, which not only maintains the characteristic of Kozak enhancing protein expression in the eukaryotic system, but also because the sequence of the spacer region is not restricted by the Kozak sequence, the sequence with the best expression level screened out can be directly used, thereby also increasing the protein expression level of the cell-free protein expression system.

[0060] A further preferred order is: enhancer + eukaryotic promoter + prokaryotic promoter and regulatory sequences on both sides thereof + ribosome binding site + first ATG sequence + co-expression sequence + kozak sequence + second ATG sequence + target gene + stop codon + eukaryotic transcription termination sequence + prokaryotic transcription termination sequence, the ribosome binding site and the first ATG sequence are at an appropriate distance, and the distance is controlled by the spacer region provided between the two. The sequence thus formed can be defined as a third preferred sequence. Compared to the aforementioned second preferred sequence, the third preferred sequence selects the kozak sequence as the amino acid for cell-free protein expression, and by adding a co-expression sequence in front of it, the translation efficiency of the cell-free protein expression can be enhanced, and the protein expression amount of the cell-free protein expression system is further improved.

[0061] Some embodiments of the present invention further provide a kit comprising the dual expression vector.

[0062] In some cases, the kit may further include other components, such as restriction endonucleases for inserting the gene of interest into the cloning site of the dual expression vector.

[0063] Some embodiments of the present invention further provide a protein expression method comprising:

[0064] Inserting the coding gene of the target protein into the cloning site of the dual expression vector to obtain a recombinant vector;

[0065] After the recombinant vector is extracted, the target protein is expressed using a cell-free protein expression kit or introduced into eukaryotic cells. The target protein is an antibody or antibody fragment.

[0066] To make the purpose, technical solutions, and advantages of this application more clear, the technical solutions of this application will be clearly and completely described below in conjunction with the specific embodiments of this application and the corresponding drawings. Obviously, the embodiments described are only part of the embodiments of this application, not all of them. Based on the embodiments in this application, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of this application.

[0067] Unless otherwise specified, the various raw materials, reaction equipment, testing equipment and testing methods used in the following examples are all well known in the art.

[0068] Example 1 This example provides a method for constructing a dual expression vector using pcDNA3.4 as a template, which includes the following steps:

[0069] 1) Screening for transcription and translation element sequences for the cell-free protein expression system inserted into the pcDNA3.4 eukaryotic expression plasmid. The specific process includes:

[0070] a) Construction of linear template for target protein expression

[0071] In this example, in order to facilitate subsequent protein detection, GFP protein was first selected to screen the transcription and translation element sequences of the cell-free protein expression system.

[0072] The order of the transcription and translation elements of the eukaryotic system and cell-free protein expression system included in the linear template construction is as follows Figure 1 Based on the pcDNA3.4 eukaryotic expression element, a series of sequences SEQ ID NO.1 to SEQ ID NO.12 were designed through a combination of manual design and experimental verification.

[0073] b) Screening of prokaryotic expression element sequences

[0074] According to the instructions of the PLD cell-free protein expression kit, each component was added to a 48-well cell culture plate, as shown in Table 1 below, mixed evenly, and placed in a constant temperature mixer. After reacting for 3 hours at 30°C and 1000 rpm, the plate was removed and the protein expression level was measured using a microplate reader with 485 nm / 535 nm 0.1 s fluorescence.

[0075] Table 1 GFP protein expression based on different dual expression vectors screened

[0076]

[0077]

[0078] c) Experimental results

[0079] like Figure 2 As shown, SEQ ID NO. 12 has the best expression effect, but SEQ ID NO. 12 has the Kozak sequence removed, which may result in poor expression in eukaryotic cells; SEQ ID NO. 4, 7, 8, and 9 have the second best expression effects.

[0080] 2) Construction of GFP recombinant plasmids: Sequences SEQ ID NOs. 4, 7, 8, 9, and 12 screened above were inserted into plasmid pcDNA3.4 by homologous recombination. The resulting ligation products were then transformed into competent E. coli DH5α cells. After incubation at 37°C for 16 hours, positive clones were selected and sequenced to obtain expression plasmids pcDNA3.4-SEQ ID NO. 4, pcDNA3.4-SEQ ID NO. 7, pcDNA3.4-SEQ ID NO. 8, pcDNA3.4-SEQ ID NO. 9, and pcDNA3.4-SEQ ID NO. 12. Plasmids that were sequenced correctly were cultured and the plasmids were extracted.

[0081] 3) GFP protein expression in HEK293 cells:

[0082] ① Transfect the above expression plasmid according to the following steps. The reaction vessel is a 96-well cell culture plate. The brief operation is as follows: dilute 500ng of plasmid with 15μL Hybridoma medium, dilute the transfection reagent with Hybridoma medium, add the diluted transfection reagent to the diluted plasmid, and after 15 minutes, add 200μL of HEK293 cells at the third generation. Mix Hybridoma medium and DMEM in equal proportions, add 1.2% FBS, culture at 37℃, 5% CO2 for 4 days. Incubate at 4℃, 5000rpm. -1 Centrifuge for 10 minutes and harvest the cell culture pellet. Take an equal volume of the pellet and add the same volume of lysis buffer. Incubate on ice for 20 minutes at 4°C and 5000 rpm. -1 After centrifugation for 20 minutes, the lysate supernatant was harvested and the protein expression level was determined using a microplate reader at 485 nm / 535 nm with a 0.1 s fluorescence.

[0083] ② Experimental results

[0084] like Figure 3 As shown, vectors pcDNA3.4-SEQ ID NO.7, pcDNA3.4-SEQ ID NO.8, and pcDNA3.4-SEQID NO.9 can all be well expressed in HEK293 cells. Subsequently, pcDNA3.4-SEQ ID NO.8 was used as a vector to construct an antibody expression vector, which was named P1.

[0085] Example 2 To increase the yield of the target gene in the cell-free protein expression system, a dual expression vector named P2 was designed in this example, in which the Kozak sequence was used as the amino acid for cell-free protein expression. The template construction contained the transcription and translation elements and the element sequence as shown below: Figure 4As shown, its sequence is SEQ ID NO.13. When the vector expresses the target gene in a cell-free expression system, 3 more amino acids will be added, but when the target gene is expressed in a eukaryotic cell, this will not happen. Figure 5 It can be seen that the dual expression vector can effectively increase the yield of cell-free protein expression without affecting the expression yield in eukaryotes.

[0086] Example 3 In order to further improve the yield of the target gene in the cell-free protein expression system, this example adds an auxiliary expression sequence to the dual expression vector P2, thereby forming a dual expression vector P3. The template construction includes the transcription and translation elements and the element sequence as shown below: Figure 6 As shown, its sequence is SEQ ID NO.14 to SEQ ID NO.16. Figure 7 It can be seen that by adding different expression-aiding tags, the yield of cell-free protein expression can be effectively increased without affecting its expression yield in eukaryotes.

[0087] Example 4 Application of dual expression vectors in antibody expression

[0088] (1) Obtain the target protein coding sequence

[0089] Taking bevacizumab as an example, the amino acid sequences of the light chain and heavy chain of bevacizumab (DB00112) were queried from Drugbank and converted into nucleotide sequences based on mammalian codon preference, as shown in SEQ ID NO. 17 and SEQ ID NO. 18, respectively.

[0090] (2) Construction of bevacizumab expression plasmid

[0091] The target gene sequences SEQ ID NO.17 and SEQ ID NO.18 were inserted into the dual expression vector P1 described above in Example 1 by homologous recombination, and the GFP encoding gene sequences therein were replaced with SEQ ID NO.17 and SEQ ID NO.18, respectively. The resulting ligation products were then transformed into competent Escherichia coli DH5α cells. After culturing at 37°C for 16 hours, positive clones were picked and sequenced to obtain expression plasmids. The complete expression plasmids P1-SEQ ID NO.17 (hereinafter referred to as the light chain plasmid) and P1-SEQ ID NO.18 (hereinafter referred to as the heavy chain plasmid) were obtained. The correctly sequenced plasmids were cultured and the plasmids were extracted.

[0092] (3) Preparation of bevacizumab using the PLD cell-free protein expression kit

[0093] As shown in Table 2, each component was added to a 48-well cell culture plate according to the instructions of the PLD cell-free protein expression kit, mixed evenly as shown in Table 2, placed in a constant temperature mixer, reacted at 30°C and 600 rpm for 16 hours, and then taken out. The antibody concentration in the supernatant was determined using ELISA Kit. The results are shown in Table 3.

[0094] Table 2 Experimental conditions for preparing bevacizumab in this example

[0095]

[0096]

[0097] Table 3 Detection results of antibody concentration in cell-free reaction supernatant

[0098] Reaction number Protein name Antibody concentration in supernatant (μg / mL) 1 Negative control 0 2 IgG 5 3 IgG 3 4 IgG 1 5 IgG 0.3 6 IgG 0.1

[0099] (5) Bevacizumab protein expression in HEK293 cells:

[0100] The above expression plasmids were co-transfected into HEK293 cells using a 96-well cell culture plate. The procedure was briefly as follows: 500 ng of the light chain plasmid and 500 ng of the heavy chain plasmid were diluted with 15 μL of Hybridoma medium, with a light-heavy chain ratio of 1:1. The transfection reagent was diluted with Hybridoma medium and added to the diluted plasmids. After 15 minutes, 200 μL of HEK293 cells at the third generation were added. Hybridoma medium and DMEM were mixed in equal proportions, and cultured with 1.2% FBS at 37°C and 5% CO2 for 4 days. The cell culture supernatant was collected and the affinity activity of the expressed antibody was detected using an ELISA kit. The results are shown in the figure below. Figure 8 shown.

[0101] Table 4 Antibody concentration detection results in HEK293 cell expression supernatant

[0102] Protein name Antibody concentration in supernatant (μg / mL) IgG 15

[0103] Example 5 To further improve the yield of antibodies in a cell-free protein expression system, the target gene sequences SEQ ID NO. 17 and SEQ ID NO. 18 were inserted into the dual expression vector P3 described above in Example 3 by homologous recombination. The resulting ligation products were then transformed into competent E. coli DH5α cells. After culturing at 37°C for 16 hours, positive clones were selected and sequenced to obtain expression plasmids. Complete expression plasmids P3-SEQ ID NO. 17 (hereinafter referred to as the light chain plasmid) and P3-SEQ ID NO. 18 (hereinafter referred to as the heavy chain plasmid) were obtained. The plasmids with correct sequencing were expanded and the plasmids were extracted. According to the instructions of the PLD cell-free protein expression kit, the components were added to a 48-well cell culture plate, mixed evenly, placed in a thermomixer, reacted at 30°C and 600 rpm for 16 hours, and then the supernatant was measured using an ELISA kit for antibody concentration. The results are shown in Table 5. It can be seen that the expression sequence can significantly improve the yield of antibodies in cell-free protein expression.

[0104] Table 5 Detection results of antibody concentration in cell-free reaction supernatant

[0105] Reaction number Protein name Antibody concentration in supernatant (μg / mL) 1 Negative control 0 2 IgG 20

[0106] (2) Bevacizumab protein expression in HEK293 cells:

[0107] The above-mentioned expression plasmids were co-transfected into HEK293 cells using a 96-well cell culture plate according to the following steps: 500 ng of the light chain plasmid and 500 ng of the heavy chain plasmid were diluted with 15 μL of Hybridoma medium, with a light-heavy chain ratio of 1:1. The transfection reagent was diluted with Hybridoma medium and added to the diluted plasmids. After 15 minutes, 200 μL of HEK293 cells at the third passage were added. Hybridoma medium and DMEM were mixed in equal proportions, and cultured with 1.2% FBS at 37°C and 5% CO2 for 4 days. The cell culture supernatant was collected and the antibody concentration in the supernatant was measured using an ELISA kit. The results were consistent with the expression levels in Example 4.

[0108] Table 6 Antibody concentration detection results in HEK293 cell expression supernatant

[0109] Protein name Antibody concentration in supernatant (μg / mL) IgG 15

[0110] Comparative Example 1: Expression of GFP in a cell-free protein expression system using a commercial dual expression vector

[0111] (1) Construction of expression plasmid

[0112] The GFP target gene sequence was inserted into the commercial dual expression vector pdual (AF041247) by homologous recombination. The obtained ligation product was transformed into Escherichia coli competent cells DH5α. After culturing at 37°C for 16 hours, the positive clones were picked and sequenced to obtain the expression plasmid pdual-GFP (such as Figure 9 The plasmid solution with the correct sequencing result was expanded and then the plasmid was extracted.

[0113] (2) Expression of pdual-GFP in a cell-free protein expression system

[0114] According to the instructions of the PLD cell-free protein expression kit, each component was added to a 48-well cell culture plate, mixed evenly, and placed in a constant temperature mixer. After reacting for 3 hours at 30°C and 1000 rpm, the plate was taken out and the protein expression level was measured using a microplate reader with 485 nm / 535 nm 0.1 s fluorescence, as shown in Table 5 below.

[0115] Table 5 Expression of pdual-GFP in cell-free protein expression system

[0116]

[0117]

[0118] The results are as follows Figure 10 As shown, there is almost no difference between pdual-GFP and the negative control, and almost no expression in the cell-free protein expression system. This may be because the vector contains the lac operator sequence and lac I sequence. When LacI is present, the lac operator sequence will inhibit the activity of the T7 promoter and control the expression of the target gene at the basal level.

[0119] It should be understood that although the present invention is disclosed above in terms of preferred embodiments, it is not intended to limit the claims. Any person skilled in the art may make several possible changes and modifications without departing from the concept of the present invention. Therefore, the scope of protection of the present invention shall be based on the scope defined by the claims of the present invention.

Claims

1. A dual expression vector comprising: Transcription and translation elements, at least for enabling the transcription and translation of the target gene in prokaryotic cell-free protein expression systems and eukaryotic cells; a replication element, at least for enabling the vector to replicate in prokaryotic and eukaryotic cells; It is characterized in that the transcription and translation elements include an enhancer, a eukaryotic promoter, a prokaryotic promoter, a ribosome binding site, a spacer, a start codon, a cloning site, a stop codon, a eukaryotic transcription termination sequence and a prokaryotic transcription termination sequence connected in sequence from upstream to downstream.

2. The dual expression vector according to claim 1, characterized in that: The transcription and translation elements include an enhancer, a eukaryotic promoter, a prokaryotic promoter, a ribosome binding site, a spacer, a Kozak sequence, a second ATG sequence, a cloning site, a stop codon, a eukaryotic transcription termination sequence, and a prokaryotic transcription termination sequence, which are sequentially connected from upstream to downstream.

3. The dual expression vector according to claim 2, characterized in that: The transcription and translation elements include an enhancer, a eukaryotic promoter, a prokaryotic promoter, a ribosome binding site, a spacer, a first ATG sequence, a Kozak sequence, a second ATG sequence, a cloning site, a stop codon, a eukaryotic transcription termination sequence, and a prokaryotic transcription termination sequence, which are sequentially connected from upstream to downstream.

4. The dual expression vector according to claim 2, characterized in that: The transcription and translation elements include an enhancer, a eukaryotic promoter, a prokaryotic promoter, a ribosome binding site, a spacer, a first ATG sequence, an expression-aiding sequence, a Kozak sequence, a second ATG sequence, a cloning site, a stop codon, a eukaryotic transcription termination sequence, and a prokaryotic transcription termination sequence, which are sequentially connected from upstream to downstream.

5. The dual expression vector according to any one of claims 2 to 4, characterized in that: The length of the spacer region is 5 to 11 bp.

6. The dual expression vector according to claim 1, characterized in that: The prokaryotic promoter includes at least one of a T7 promoter, a lambda phage promoter, a lactose promoter, an arabinose promoter, a tryptophan promoter, and a tac promoter; And / or, the eukaryotic promoter includes at least one of SV40, CMV, RSV, ADV, and LTR; And / or, the enhancer includes at least one of SV40 enhancer, CMV enhancer, RSV enhancer, and LTR enhancer.

7. The dual expression vector according to claim 1, characterized in that Also includes: A selectable marker element is used to enable selection of the vector in prokaryotic and eukaryotic cells.

8. The dual expression vector according to claim 1, wherein: The sequence of the dual expression vector includes any one of SEQ ID NO.7 to SEQ ID NO.9 and SEQ ID NO.13 to SEQ ID NO.16, preferably any one of SEQ ID NO.13 to SEQ ID NO.16, and more preferably any one of SEQ ID NO.14 to SEQ ID NO.

16.

9. A method for constructing a dual expression vector, characterized in that: include: A prokaryotic expression element is inserted into a eukaryotic expression vector, and the insertion site of the prokaryotic transcription termination sequence in the prokaryotic expression element in the eukaryotic expression vector is downstream of the eukaryotic transcription termination sequence, and the insertion sites of the remaining sequences in the eukaryotic expression vector are located downstream of the promoter and upstream of the start codon of the eukaryotic expression vector, thereby obtaining the dual expression vector.

10. A kit, characterized in that The invention comprises the dual expression vector according to any one of claims 1 to 8.

11. A protein expression method, characterized in that: include: Inserting the gene encoding the target protein into the cloning site of the dual expression vector according to any one of claims 1 to 8 to obtain a recombinant vector; The recombinant vector is used to express the target protein using a cell-free protein expression kit, and / or the recombinant vector is introduced into eukaryotic cells to express the target protein; Wherein, the target protein includes an antibody or an antibody fragment.

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