A TRUNCATED INSULATOR AND ITS APPLICATION IN TRANSIENT EXPRESSION OF RECOMBINANT PROTEIN

Abstract

A truncated insulator, an expression system comprising the truncated insulator and a transposon (PiggyBac) element, and a method for transient expression and production of a recombinant protein by using the expression system.

Description

Truncated insulators and their use for transient expression of recombinant proteins

[0001] This application claims priority to the Chinese patent application filed with the China Patent Office on October 8, 2023, with application number 202311295717.9 and invention name “Truncated insulators and their use for transient expression of recombinant proteins”, the entire contents of which are incorporated by reference into this application. Technical Field

[0002] The present disclosure relates to the field of biotechnology, and in particular to a truncated insulator, an expression system comprising the truncated insulator and a transposon (PiggyBac) element, and a method for transient protein expression production using the same. Background Art

[0003] Currently, there are hundreds of approved antibody drugs worldwide, and the antibody market has reached $100 billion. Antibody drugs in various therapeutic areas are increasingly benefiting patients. Antibody drugs for therapeutic use must simultaneously meet multiple stringent criteria, including efficacy, stability, immunogenicity, efficacy, pharmacokinetics, ease of expression, and purification. These criteria typically require several rounds of engineering design and process optimization. Each round of design and optimization requires characterizing a large amount of protein, which creates the need for rapid production of large quantities of high-quality purified protein.

[0004] Studies have reported that insulators play a crucial role in regulating the spatiotemporal expression of eukaryotic genes. They protect target genes from the influence of surrounding regulatory factors, preventing them from being improperly activated or silenced. This, in turn, acts as an enhancer blocker and heterochromatin barrier, effectively suppressing the "position effect." HS4, a DNase I hypersensitive site (HS) located upstream of the 5' end of the chicken β-globin locus, is approximately 1.2 kp long and exhibits both of these functions. However, existing studies have shown that insertion of the complete sequence can result in an overly large vector, reducing transfection efficiency and thus affecting protein expression. Therefore, truncation studies of cHS4 are warranted. Research has shown that cHS4 relies on a 250-bp core region at its 5' end for its function, but this core region is incomplete and lacks sufficient expression enhancement. The 400-bp region at its 3' end also plays an important role, and cHS4-650 (a 250-bp core region and a 400-bp sequence at its 3' end) is more effective than cHS4-250. However, a 650-bp insulator still results in excessive vector size and reduced transfection efficiency. Therefore, there is a need to further optimize the insulator sequence length beyond the 650-bp length to improve transfection efficiency. However, current research on cHS4 insulator truncation has largely relied on combining different previously studied segments, primarily based on a 250-bp core region with other segments. Further optimization of the length and verification of the truncated insulators have not been performed. Furthermore, the construction of stable cell lines for antibody drug production has not been practically implemented, leaving a significant distance from industrial application.

[0005] Transient expression is a method for rapidly producing recombinant proteins at the milligram to gram level. Unlike stable expression, the gene expressing the target protein does not need to be stably integrated into the host cell genome. The target gene exists in the transfected cells in the form of plasmid DNA, and no screening pressure is required to maintain the plasmid DNA. Compared with stable expression, the main features of transient expression are: (1) rapid protein expression, generally only 1-3 weeks; (2) it can be applied to a variety of recombinant proteins, including proteins that are toxic to host cells. Due to these characteristics, transient expression is an ideal method for rapid protein production. However, the yield of transiently expressed proteins is usually relatively low, usually in the range of 60-80 mg / L.

[0006] To increase the yield of transient expression, researchers have developed a variety of methods, such as increasing host cell density, co-transfecting expression-promoting genes, and adding enhancing reagents. These methods require additional steps and increase costs. PiggyBac transposons can promote gene integration and protein expression, but conventional PiggyBac transposon expression systems typically require stress screening and cell recovery steps, which are time-consuming and have limited protein expression, making them unable to meet the demand for rapid protein production. Therefore, a transient expression system that is simple to operate, has low industrial costs, and is high-yielding is needed.

[0007] Summary of the Invention

[0008] On the one hand, the present disclosure provides an insulator for expressing a polypeptide or protein in a eukaryotic cell (see WO2024146587A1). The insulator comprises a nucleic acid sequence as shown in SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3 or SEQ ID NO.4, or comprises a nucleic acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to any of the nucleic acid sequences as shown in SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3 or SEQ ID NO.4; preferably, the insulator is a nucleic acid sequence as shown in SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3 or SEQ ID NO.4, or comprises a nucleic acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to any of the nucleic acid sequences as shown in SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3 or SEQ ID NO.4. A nucleic acid sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to any of the nucleic acid sequences shown in NO.4.

[0009] The present disclosure provides an expression vector comprising the insulator of the present disclosure. Preferably, the expression vector is a PiggyBac transposon transgenic vector; preferably, the expression vector does not contain a selection marker gene, or preferably, the expression vector comprises a selection marker gene.

[0010] The present disclosure also provides an expression system comprising the insulator of the present disclosure. Preferably, the expression system is a eukaryotic cell expression system, more preferably, it is a PiggyBac transposon expression system.

[0011] Preferably, the PiggyBac transposon expression system includes a PiggyBac transposon transgenic vector; preferably, the PiggyBac transposon expression system also includes a PiggyBac auxiliary vector or mRNA encoding PB transposase, and the PiggyBac auxiliary vector contains a nucleic acid encoding PB transposase. Preferably, the PiggyBac auxiliary vector is constructed by pcDNA3.4.

[0012] Preferably, the PiggyBac transposon transgenic vector comprises the insulator disclosed herein. Preferably, the PiggyBac transposon transgenic vector comprises a 5' terminal repeat sequence 5ITR and an inverted 3' terminal repeat sequence 3ITR, with the insulator positioned between the 5ITR and 3ITR; preferably, two insulators, one complementary in the forward and one complementary in the reverse direction, or both in the forward direction, are positioned between the 5ITR and 3ITR; preferably, a multiple cloning site is positioned between the insulators; preferably, other elements of the PiggyBac transposon transgenic vector are derived from the original P3 vector sequence.

[0013] Preferably, the PiggyBac transposon transgenic vector does not contain a selection marker gene; or preferably, the PiggyBac transposon transgenic vector contains a selection marker gene.

[0014] In another aspect, the present disclosure provides a method for using the expression system, in particular a method for performing transient expression of gene recombination, the method comprising the following steps:

[0015] 1) Construct an expression system containing the target protein encoding gene;

[0016] 2) introducing the expression system described in step 1) into host cells by transfection;

[0017] 3) After the transfection, using the transfected cell pool for expression production;

[0018] Wherein, the expression system comprises the insulator disclosed herein. Preferably, the expression system is a eukaryotic cell expression system. Preferably, the expression system is a PiggyBac transposon expression system; preferably, the PiggyBac transposon expression system comprises a PiggyBac transposon transgenic vector; preferably, the PiggyBac transposon transgenic vector comprises a 5' terminal repeat sequence 5ITR and a reverse 3' terminal repeat sequence 3ITR, and the insulator is provided between 5ITR and 3ITR; preferably, two insulators of forward and reverse complementarity or both of the forward direction are provided between the 5ITR and 3ITR in sequence; preferably, a multiple cloning site is provided between the insulators; preferably, the Other elements of the PiggyBac transposon transgenic vector are derived from the original sequence of the P3 vector; preferably, the PiggyBac transposon transgenic vector does not contain a selection marker gene, or preferably, the PiggyBac transposon transgenic vector contains a selection marker gene; preferably, the PiggyBac transposon expression system further comprises a PiggyBac helper vector or mRNA encoding a PB transposase, and the PiggyBac helper vector contains a nucleic acid encoding a PB transposase; preferably, the host cell is a eukaryotic host cell, more preferably, the host cell is a CHO cell.

[0019] Preferably, the target protein in step 1) can be an antibody, fusion protein, antigen, enzyme or other type of protein or polypeptide.

[0020] Preferably, in step 1), an in vitro transcription method is used, and the DNA sequence encoding the PB transposase is used as a template, and the mRNA encoding the PB transposase is obtained by in vitro transcription, capping and tailing modification.

[0021] Preferably, in step 1), an endotoxin-free plasmid extraction kit is used for vector construction.

[0022] Preferably, the transfection method in step 2) is electrofection, chemical transfection or other transfection methods.

[0023] Preferably, the time after transfection in step 3) is 1 day, 2 days, 3 days or other time after transfection.

[0024] Preferably, no pressure screening is required after transfection or before production in step 3). Preferably, the expression production in step 3) is transient expression production.

[0025] Preferably, the expression production mode in step 3) is fed-batch culture.

[0026] Compared with the prior art, the advantages of the present disclosure are: 250bp of the cHS4 core region is truncated, breaking the original core region setting. Compared with the 650bp cHS4 insulator sequence in the prior art, the insulator sequence length is significantly shortened and the protein expression amount is increased; when applied to protein transient expression, while achieving an increased protein transient expression yield, it also reduces production costs, and thus has broad application prospects in the field of antibody screening and production. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 is a map of the P3-5ITR-3ITR-cHS4-TT vector.

[0028] Figure 2 is a map of the pcDNA3.4-PiggyBac vector.

[0029] Figure 3 shows the protein yield within 7 days of transient transfection.

[0030] Figure 4 shows the time required for plasmid construction and protein production.

[0031] Figure 5 shows the cost of transient transfection.

[0032] Figure 6 is a map of the P3-AscI vector. DETAILED DESCRIPTION

[0033] the term

[0034] All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference.

[0035] Before describing the present disclosure in detail below, it should be understood that the present disclosure is not limited to the specific methodologies, protocols and reagents described herein, as these may vary. It should also be understood that the terms used herein are only for the purpose of describing specific embodiments and are not intended to limit the scope of the present disclosure. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which the present disclosure belongs.

[0036] Certain embodiments disclosed herein include numerical ranges, and certain aspects of the present disclosure may be described in terms of ranges. Unless otherwise stated, it should be understood that numerical ranges or descriptions in terms of ranges are intended for simplicity and convenience and should not be considered as strict limitations on the scope of the present disclosure. Therefore, descriptions in terms of ranges should be considered to specifically disclose all possible subranges and all possible specific numerical points within the range, as these subranges and numerical points have been clearly stated herein. Regardless of the width of the numerical value, the above principles apply equally. When describing in terms of ranges, the range includes the endpoints of the range.

[0037] When referring to a measurable value such as an amount, a temporal duration, etc., the term "about" is meant to include variations of ±20%, or in some cases ±10%, or in some cases ±5%, or in some cases ±1%, or in some cases ±0.1% of the specified value.

[0038] The term "antibody," as used herein, typically refers to a Y-shaped tetrameric protein comprising two heavy (H) polypeptide chains and two light (L) polypeptide chains held together by covalent disulfide bonds and non-covalent interactions. Natural IgG antibodies have this structure. Each light chain comprises a variable domain (VL) and a constant domain (CL). Each heavy chain comprises a variable domain (VH) and a constant domain (CH).

[0039] As used herein, the types of "antibodies" in a broad sense may include polyclonal antibodies, monoclonal antibodies, chimeric antibodies, humanized antibodies and primatized antibodies, CDR-grafted antibodies, human antibodies (including recombinantly produced human antibodies), recombinantly produced antibodies, intracellular antibodies, multispecific antibodies, bifunctional fusion proteins, monovalent antibodies, multivalent antibodies, anti-idiotypic antibodies, synthetic antibodies (including muteins and variants thereof), etc.

[0040] The term "PiggyBac transposon expression system" refers to the PiggyBac transposon vector system. The PiggyBac vector system primarily consists of a helper vector or plasmid encoding the PiggyBac (PB) transposase; a transposon vector (also called a donor vector) or plasmid containing optimized inverted terminal repeats (ITRs) at either end and a central transposable region, which is used to insert the desired gene sequence into the host genome. During the experiment, the helper plasmid and the transposon plasmid are co-transfected into target cells. The transposase encoded by the helper plasmid recognizes and cleaves the ITRs at both ends of the transposon plasmid, releasing the transposed region, which is then integrated into the host genome at a site containing a TTAA sequence. TTAA repeats appear at both ends of the transposed region. Alternatively, in vitro-transcribed transposase-encoding mRNA can be used in place of the helper plasmid and the transposon plasmid to co-transform the target cells for transposase expression.

[0041] The term "CHO ​​platform" refers to a CHO cell line screening technology platform. CHO (Chinese Hamster Ovary) cells are domesticated in the chemically defined medium CD CHO Fusion Medium and subcloned through screening to establish CHO cell lines. This platform also includes supporting reagents and processes, including the expression vector P3, culture medium for the clone construction phase, and fed-batch platform culture medium.

[0042] The term "vector" means a nucleic acid molecule that can carry another nucleic acid connected thereto, which can be single-stranded or double-stranded, including DNA and RNA. One type of vector is a "plasmid", which refers to a circular double-stranded DNA loop, wherein an additional DNA segment can be connected. Another type of vector is a viral vector, in which an additional DNA segment can be connected to the viral genome. Some vectors can replicate autonomously (for example, bacterial vectors and additional mammalian vectors with bacterial replication origin) in the host cell introducing them. Other vectors (for example, non-additional mammalian vectors) can be integrated into the genome of the host cell after introducing the host cell, thereby replicating with the host genome.

[0043] An expression vector is a vector that can express a target gene. Target genes include, but are not limited to, deoxynucleotide sequences encoding broadly defined products such as antibodies, antigens, fusion proteins, and polypeptides.

[0044] The term "insulator" or "insulator sequence" refers to a class of DNA sequences at the boundaries of chromatin domains that function as neutral barriers, preventing the influence of adjacent genetic elements or surrounding dense chromatin, allowing the protected genes to be expressed in their normal time and space. The effectiveness of an insulator is related to its location in the gene and the orientation of its own sequence. Exemplary insulator sequences include chicken hypersensitive site-4 (cHS4).

[0045] The term "multiple cloning site" refers to a synthetic DNA fragment contained within a vector that contains multiple unique restriction enzyme sites. This site serves as the insertion site for exogenous DNA. Also known as a multisite linker, it is a standard configuration sequence found in plasmids commonly used in genetic engineering. Each restriction enzyme site within a multiple cloning site is typically unique, meaning it occurs only once within a specific plasmid. Sites for different enzymes may overlap.

[0046] The term "recombinant expression" refers to an oligonucleotide or polynucleotide construct comprising a genetic modification, which allows the host cell to express an mRNA, protein, polypeptide or peptide when the construct is contacted with a host cell under conditions sufficient to allow expression of the mRNA, protein, polypeptide or peptide in the host cell, wherein the construct comprises a nucleotide sequence encoding the mRNA, protein, polypeptide or peptide.

[0047] The term "transfection" refers to a specialized technique for introducing exogenous genes into cells, including chemical transfection, biological transfection, and physical transfection.

[0048] The term "transient expression" refers to the introduction of a constructed plasmid into host cells through transfection. The exogenous gene on the plasmid does not integrate into the cell's own genome, but exists as a plasmid. As the cell grows and divides, the exogenous gene is gradually lost. During this time, the exogenous gene on the plasmid is transcribed and translated within the cell, producing protein.

[0049] Example

[0050] The present disclosure is further illustrated in detail by the following examples.

[0051] Specific embodiments are listed below to illustrate the present disclosure. However, it should be understood that these embodiments are only listed to illustrate the present disclosure, rather than to limit the scope of the present disclosure.

[0052] Materials and reagents:

[0053] The PB transposase coding sequence was synthesized by Suzhou Hongxun Biotechnology Co., Ltd., which optimized the sequence for CHO cells (added EcoRI restriction site and Kozak sequence at the 5' end flank, and HindIII at the 3' end flank) and cloned into the vector PUC57 to obtain PUC57-PiggyBac.

[0054] The PiggyBac transposase-encoding mRNA template DNA sequence was synthesized by Nanjing GenScript Biotechnology Co., Ltd.

[0055] The PiggyBac donor vector was synthesized and circularized by Nanjing GenScript Biotechnology Co., Ltd.

[0056] The blank expression vector pcDNA3.4 was provided by Nanjing GenScript Biotechnology Co., Ltd.

[0057] Example 1: Design of cHS4 insulator truncated sequence and vector construction

[0058] 1. Design of cHS4 insulator truncated sequence

[0059] The insulators used, cHS4-650, cHS4-400, and cHS4-250, are derived from the literature (for example, see CN102943092A, WO2018083274A1, and US20150315611A1). The cHS4-650 sequence (including the 250bp core region and the 400bp sequence at the 3' end) was input into the AnimalTFDB3.0 online prediction website to predict possible binding transcription factors. The results showed that there were 3897 TFBSs corresponding to 389 transcription factors. The STRING database was then used for enrichment analysis of the 389 transcription factors, and a total of 1993 GO terms were significantly enriched. 91 transcription factors corresponding to 85 GO terms related to chromatin structure regulation or insulator function were selected for PPI analysis. Then, transcription factors with high connectivity and three transcription factors reported to bind to the core region, CTCF, USF1 / 2, and VEZF1, were selected. According to the binding sequences predicted by AnimalTFDB3.0, these transcription factors were located on the cHS4-650 sequence. DNA regions with less or no binding by transcription factors were selected for truncation design, and single or multiple truncation regions were combined for truncation. Finally, four insulator sequences (SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, and SEQ ID NO.4) were obtained. NO.4. Its nucleic acid sequences are as follows:

[0060] SEQ ID NO.1

[0061] SEQ ID NO.2

[0062] SEQ ID NO.3

[0063] SEQ ID NO.4

[0064] 2. Donor Vector Construction

[0065] A donor vector was constructed using SEQ ID NO.1 as an exemplary truncated insulator. The PiggyBac donor vector element contains 5ITR-cHS4 (forward) and 3ITR-cHS4 (forward), wherein there are two forward cHS4s between 5ITR and 3ITR, and cHS4 contains the nucleic acid sequence shown in SEQ ID NO.1. Based on the P3-AscI vector (as shown in Figure 6, constructed by inserting the AscI restriction site recognition sequence GGCGCGCC into the P3 plasmid), the screening marker GS gene expression cassette and a target gene expression cassette were deleted, and the elements 5ITR and cHS4 were added by AscI single enzyme ligation or homologous recombination, and the elements 3ITR and cHS4 were added by SgrAI single enzyme ligation or homologous recombination, resulting in the PiggyBac donor vector sequence, which was named P3-5ITR-3ITR-cHS4-TT (Figure 1).

[0066] 3. Expression Vector Construction

[0067] The vector P3-5ITR-3ITR-cHS4-TT was double-digested and ligated using BstBI / PacI, and the nucleic acid sequences encoding the S1 and S2 chains of the QL01 protein (preserved by the present applicant) were respectively ligated to the multiple cloning site to construct the recombinant expression vectors P3-5ITR-3ITR-cHS4-TT-QL01-S1 and P3-5ITR-3ITR-cHS4-TT-QL02-S2; the nucleic acid sequence encoding the QL02 protein (preserved by the present applicant) was ligated to the multiple cloning site to construct the recombinant expression vector P3-5ITR-3ITR-cHS4-TT-QL02.

[0068] 4. Construction of auxiliary vector

[0069] The vector PUC57-PiggyBac and the blank vector pcDNA3.4 were double-digested with EcoRI / HindIII. The digested products were then purified and recovered using a NucleoSpin Gel and PCR Clean-up Kit and ligated to construct the helper plasmid pcDNA3.4-PiggyBac containing the PB transposase-encoding gene (Figure 2). Alternatively, in vitro transcription using the PB transposase-encoding DNA sequence as a template was performed, followed by in vitro transcription, capping, and tailing to generate PB transposase-encoding mRNA-PiggyBac.

[0070] Example 2: Transient transfection of cHS4 insulator truncated sequences for antibody expression

[0071] Transient transfection expression of expression vector containing cHS4 insulator truncated sequence: The host cell used was CHOZN (from Merck), and CHO cells were transfected by electroporation. The transfection plasmid amount was 60 μg expression vector + 6 μg auxiliary vector, the voltage was 300 V, 950 μF, the cell number was 1E7, and the electric shock was performed once. After cell transfection, the culture medium was used. Resuspend in CD CHO Fusion Medium (containing 6mM L-glutamine) and place in an incubator for static culture at 37°C and 5% CO2. Count the cells after 24 hours of culture, determine the viable cell density (VCD) and cell viability (Via), and centrifuge. Add appropriate amount of culture medium to 6×10 6 The cells were resuspended at a density of 10 cells / mL and fed-batch protein expression production was performed for 7 days.

[0072] As a control, ExpiCHO TM The protein was transiently expressed using the kit as recommended: 25 mL of expression system and 20 μg of plasmid for transfection. Transfection and feeding were performed according to the kit's instructions, and culture was continued until day 7.

[0073] The antibody expression levels were uniformly detected using a Cedex instrument. The test results are shown in Figure 3.

[0074] Comparing the transient transfection results of the two systems, the protein yield of the P3-5ITR-3ITR-cHS4-TT expression system was higher than that of the ExpiCHO TM The expression system disclosed herein can also achieve good expression results, with improvements of 488.2% (QL01) and 29.6% (QL02). QL01, a protein that is difficult to produce in existing expression systems, can also be expressed effectively using the expression system disclosed herein. Furthermore, the time required for plasmid construction and protein expression production is comparable (see Figure 4 ), while the cost is reduced by over 96% (see Figure 5 ).

[0075] Therefore, the present disclosure provides a transient expression system with simple operation, low industrial cost and high protein yield.

[0076] The embodiments of the present disclosure described above are merely exemplary and are not to be construed as limiting the embodiments of the present disclosure. Any person skilled in the art will recognize or be able to ascertain numerous equivalents to the specific compounds, materials, and procedures without undue experimentation. All such equivalents are within the scope of the present disclosure and are encompassed by the claims.

Claims

1. An insulator comprising a nucleic acid sequence as presented in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 or SEQ ID NO: 4, or comprising a nucleic acid sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to any of the nucleic acid sequences presented in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 and SEQ ID NO:

4.

2. The insulator of claim 1, which is a nucleic acid sequence as presented in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 or SEQ ID NO: 4, or which is a nucleic acid sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to any of the nucleic acid sequences presented in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 and SEQ ID NO:

4.

3. An expression vector containing an insulator according to claim 1 or 2.

4. The expression vector of claim 3, which is a PiggyBac transposon transgenic vector; wherein the expression vector preferably does not contain a marker gene for screening, or preferably the expression vector contains a marker gene for screening.

5. An expression system comprising an insulator according to claim 1 or 2.

6. The expression system according to claim 5, which is an expression system with the PiggyBac transposon.

7. The expression system of claim 6, wherein the expression system with the PiggyBac transposon comprises a PiggyBac transposon transgenic vector; preferably, the PiggyBac transposon transgenic vector comprises an insulator according to claim 1 or 2; preferably, the PiggyBac transposon transgenic vector comprises a marker gene for screening, or preferably, the PiggyBac transposon transgenic vector does not comprise a marker gene for screening.

8. The expression vector of claim 4 or the expression system of claim 7, wherein the transposon transgenic vector PiggyBac comprises a 5'-terminal repeat 5ITR and an inverted 3'-terminal repeat 3ITR, and an insulator is located between 5ITR and 3ITR; preferably, two insulators, which are inverted repeats or direct repeats, are sequentially located between 5ITR and 3ITR; preferably, a multiple cloning site is located between the insulators; preferably, other elements of the transposon transgenic vector PiggyBac are obtained from the original sequence of the P3 vector.

9. The expression system according to any one of claims 6-8, wherein the expression system with the PiggyBac transposon comprises a PiggyBac helper vector or mRNA encoding the PB transposase, and the PiggyBac helper vector comprises a nucleic acid encoding the PB transposase.

10. The expression system according to claim 9, wherein the PiggyBac helper vector is constructed from pcDNA3.

4.

11. A method for producing a protein by transient expression, comprising the following steps: 1) construction of an expression system containing a gene encoding the target protein; 2) introducing the expression system from step 1) into the host cell by transfection; and 3) use of a pool of transfected cells for production through expression after transfection; wherein the expression system comprises an insulator according to claim 1 or 2.

12. The method of claim 11, wherein the target protein is an antibody, a fusion protein, an antigen, an enzyme, or other types of protein or polypeptide.

13. The method according to claim 11 or 12, wherein the transfection method is electrotransfection, PEI or liposome transfection, or other transfection methods.

14. The method according to any one of paragraphs 11-13, which does not include a stress screening procedure.

15. The method according to any one of claims 11-14, wherein the host cell is a mammalian cell, preferably a CHO cell.

16. The method according to any one of claims 11-15, wherein said expression system is selected from the expression system according to any one of claims 5-10.

17. Use of an insulator according to claim 1 or 2, an expression vector according to any of claims 3, 4 and 8 or an expression system according to any of claims 5-10 in recombinant expression of a nucleic acid, where preferably said recombinant expression does not include a stress screening procedure.

18. The use according to claim 17, wherein said nucleic acid encodes an antibody, fusion protein, antigen, enzyme, or other types of protein or polypeptide.

19. Use of an insulator according to claim 1 or 2, an expression vector according to any of claims 3, 4 and 8, or an expression system according to any of claims 5-10 in the production of a protein or polypeptide.

20. The use according to claim 19, wherein said protein or polypeptide comprises an antibody, a fusion protein, an antigen and an enzyme.

21. Use of an insulator according to claim 1 or 2, an expression vector according to any of claims 3, 4 and 8, or an expression system according to any of claims 5-10 in the construction of a recombinant cell line.

22. The use according to claim 21, wherein said recombinant cell is a mammalian cell, preferably a CHO cell.