Truncated insulator and application thereof

Abstract

The invention provides a truncated insulator and an expression system comprising the insulator and applied to eukaryotic cells, the truncated insulator breaks the setting of an original core area, shortens the effective sequence of the insulator, and is more beneficial to the integration of the insulator and other elements of a carrier. The insulator can obviously improve the yield of protein in an expression system with transposon (such as PiggyBac) and obtain a recombinant cell strain with good hereditary stability.

Description

Truncated insulators and their applications

[0001] This application claims priority to the Chinese patent application filed with the China Patent Office on January 5, 2023, with application number 202310012420.0 and invention name “Truncated insulator and its application”, the entire contents of which are incorporated by reference into this application. Technical Field

[0002] The present application relates to the field of biotechnology, and in particular to a truncated insulator and applications thereof. Background Art

[0003] Currently, hundreds of antibody drugs have been approved worldwide, and they are increasingly benefiting patients across various therapeutic areas. With the development of antibody drugs, the market has reached hundreds of billions of dollars, with production scales reaching up to 10,000 liters per batch. Within the antibody production process, cell line construction, as the foundation of antibody production, has been the subject of extensive research and attention. The industry continues to strive to achieve rapid development and high production of antibody drugs through cell line construction.

[0004] During the construction of stable cell lines, the target gene is usually inserted into the host cell genome in a random integration manner, which has the following defects: the integration efficiency is usually less than one in ten thousand, and the construction of stable cell lines relies on a large amount of screening, which is labor-intensive and time-consuming; the target gene is easily affected by position effects and has low expression levels; the target gene is easily silenced by the host cell and has poor stability; the cell pool has low yield, making it difficult to quickly obtain sufficient protein in the early stages to meet the needs of new drug research and development.

[0005] Therefore, some site-specific and semi-site-specific integration technologies have emerged. For example, the PiggyBac transposon expression system consists of a donor plasmid and a helper plasmid or in vitro transcribed mRNA. The donor plasmid carries the transposase recognition sequences 5' inverted repeat (5ITR) and 3' inverted repeat (3ITR). The helper plasmid or in vitro transcribed mRNA encodes the PiggyBac transposase (PB transposase), which can recognize the 5ITR and 3ITR and can cut or copy them at the original position. After circularization and with the help of the transposase, it is inserted into a specific location in the host genome, achieving a precise integration with significant advantages such as high integration efficiency, good stability, and uniform copy form. However, transposon integration technology has the disadvantage of low expression, resulting in high production costs and is difficult to apply in large-scale production.

[0006] 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. Studies have shown that the function of cHS4 depends on the 250bp core region at the 5' end, but the function of this core region is incomplete and cannot effectively enhance expression. The 400bp at its 3' end also plays an important role, and the function of cHS4-650 (250bp core region and 400bp sequence at the 3' end) is better than that of cHS4-250. However, the 650bp insulator still causes the vector to be too large and reduces the transfection efficiency. Therefore, there is a technical need to further optimize the insulator sequence length and improve the transfection efficiency based on the 650bp length.

[0007] Literature reports also describe methods for early protein production using the PiggyBac transposon expression system, but the results indicate low protein expression levels, insufficient to meet the demands of industrial production. With advances in transposon research, literature reports indicate that the addition of truncated insulators flanking the target gene can significantly increase transgene expression and reduce the coefficient of variation in gene expression, thereby improving stability. However, current research on cHS4 insulator truncation relies heavily on combining previously studied segments, primarily based on a 250bp core region with other segments. However, no further length optimization has been performed, and the functionality of the truncated insulators has not been verified. None of these studies have been conducted in the construction of stable cell lines for antibody drug production, and industrial application is still a long way off.

[0008] Summary of the Invention

[0009] In a first aspect of the present application, an insulator for expressing a polypeptide or protein in a eukaryotic cell is provided, wherein the nucleic acid sequence of the insulator comprises the nucleic acid sequence 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 one of the nucleic acid sequences shown in SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3 or SEQ ID NO.4.

[0010] In some embodiments of the present application, 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 the insulator is 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 with any of the nucleic acid sequences shown in SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3 or SEQ ID NO.4.

[0011] The second aspect of the present application provides a eukaryotic cell expression system, which comprises the insulator described in the first aspect of the present application, and the insulator can play a role in increasing protein production and cell line stability.

[0012] In some embodiments of the present application, the eukaryotic cell expression system is a PiggyBac transposon expression system.

[0013] In some embodiments of the present application, the PiggyBac transposon expression system includes a PiggyBac transposon transgene vector; preferably, the PiggyBac transposon expression system further includes a PiggyBac helper vector or mRNA encoding a transposase, wherein the PiggyBac helper vector contains a transposase-encoding nucleic acid.

[0014] In some embodiments of the present application, the PiggyBac transposon transgenic vector contains the insulator described in the first aspect of the present application.

[0015] In some embodiments of the present application, the PiggyBac auxiliary vector is pcDNA3.4.

[0016] In some embodiments of the present application, 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, the insulators are positioned sequentially between the 5ITR and 3ITR in the forward and reverse directions, or two insulators in the forward direction. 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.

[0017] In some embodiments of the present application, the PiggyBac transposon transgenic vector further comprises a selection marker gene.

[0018] In some embodiments of the present application, the selection marker gene is a glutamine synthetase gene (GS gene).

[0019] The third aspect of the present application further provides the use of the insulator described in the first aspect of the present application in the construction of a PiggyBac transposon transgenic vector or a PiggyBac transposon expression system.

[0020] The fourth aspect of the present application further provides the application of the insulator described in the first aspect of the present application or the eukaryotic cell expression system described in the second aspect of the present application, in particular for the recombinant expression of nucleic acids.

[0021] In some embodiments of the present application, the nucleic acid can encode a polypeptide or protein, preferably an antibody, a fusion protein, an antigen, or an enzyme. Preferably, the recombinant expression is performed in a eukaryotic host cell. Preferably, the expression system is the PiggyBac transposon expression system. Preferably, the eukaryotic host cell is a CHO cell.

[0022] The fifth aspect of the present application further provides the use of the insulator described in the first aspect of the present application or the eukaryotic cell expression system described in the second aspect of the present application in constructing a recombinant cell line.

[0023] In some embodiments of the present application, the host cells used by the recombinant cell line are mammalian cells, preferably CHO cells.

[0024] In some embodiments of the present application, the step of constructing a recombinant cell line includes:

[0025] 1) Construct a Piggybac transposon transgene vector containing the target protein encoding gene and the glutamine synthetase gene, and a helper vector expressing the PB transposase or obtain mRNA encoding the PB transposase by in vitro transcription;

[0026] 2) introducing the transposon transgene vector and the helper vector, or the transposon transgene vector and mRNA encoding the PB transposase, from step 1) into a host cell by transfection;

[0027] 3) placing the cells obtained in step 2) in a culture flask to recover for a period of time;

[0028] 4) transferring the cells obtained in step 3) to a shake flask and screening them using GS;

[0029] 5) After the cells have fully recovered, perform an inoculation yield test to evaluate the antibody expression level.

[0030] In some embodiments of the present application, the target protein in step 1) may be an antibody, fusion protein, antigen, enzyme, or other type of protein or polypeptide.

[0031] In some embodiments of the present application, in step 1), an in vitro transcription method is used, and the PB transposase encoding DNA sequence is used as a template, and the PB transposase encoding mRNA is obtained through in vitro transcription, capping and tailing modification.

[0032] In some embodiments of the present application, in step 5), the cell state is completely restored to a cell viability of not less than 95%.

[0033] In some embodiments of the present application, the vector construction in step 1) uses an endotoxin-free plasmid extraction kit.

[0034] In some embodiments of the present application, the transfection method in step 2) is electrofection.

[0035] In some embodiments of the present application, the culture bottle in step 3) is a T bottle.

[0036] In some embodiments of the present application, the culture medium in step 3) is CD CHO Fusion Medium+6 mM glutamine, and the recovery time is 24 hours.

[0037] In some embodiments of the present application, the shake flask screening medium in step 4) is CD CHO Fusion Medium.

[0038] In some embodiments of the present application, the yield test in step 5) is a 7-day batch with an inoculation density of 5×10 5 cells / mL, the culture medium was BM2, the culture volume was 30 mL, 4 g / L sugar was added on the 4th day of culture, and the expression level in the supernatant was detected on the 7th day of culture.

[0039] The sixth aspect of the present application further provides a recombinant cell strain, wherein the recombinant cell strain comprises the insulator described in the first aspect of the present application or the eukaryotic cell expression system described in the second aspect of the present application; preferably, the host cell used by the recombinant cell strain is a mammalian cell, preferably a CHO cell.

[0040] Beneficial effects of the present application: This application truncated the cHS4 core region to 250bp for the first time, breaking the original core region setting. Compared with the 650bp cHS4 insulator sequence already available in the prior art, it significantly shortened the insulator sequence length while also increasing protein expression, shortening the construction time of stable cell lines, and the constructed cell lines have good genetic stability. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] The drawings described herein are used to provide further understanding of the present application and constitute a part of the present application. The illustrative embodiments of the present application and their descriptions are used to explain the present application and do not constitute improper limitations on the present application.

[0042] Figure 1 is a schematic structural diagram of the P3-AscI vector;

[0043] Figure 2 is a schematic structural diagram of the P3-5ITR-3ITR-cHS4 vector;

[0044] FIG3 is a schematic diagram of the structure of the pcDNA3.4-PiggyBac vector;

[0045] Figure 4 shows protein production within 6 days of transient transfection;

[0046] Figure 5 is a graph showing protein production within 14 days of stable transfection;

[0047] FIG6A is a schematic diagram of the structure of the P3-5ITR-R-3ITR-SEQ ID NO.1 reverse complement cHS4 vector;

[0048] FIG6B is a schematic diagram of the structure of the P3-5ITR-3ITR-SEQ ID NO.1 forward cHS4 vector;

[0049] FIG7 is a graph showing the effect of clone growth stability of the PiggyBac transposon expression system. DETAILED DESCRIPTION

[0050] To make the objectives, technical solutions, and advantages of this application more clearly understood, the present application is further described in detail below with reference to the accompanying drawings and examples. It is apparent that the described examples are only a portion of the embodiments of this application, rather than all of them. All other embodiments obtained by those skilled in the art based on this application are within the scope of protection of this application.

[0051] the term

[0052] 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.

[0053] Before describing the application in detail below, it should be understood that the application is not limited to the specific methodology, scheme and reagent described in the application, because these can change. It should also be understood that the terms used in the application are only for the purpose of describing specific embodiments, and are not intended to limit the scope of the application. Unless otherwise defined, all technical and scientific terms used in the application have the same meaning as those of ordinary skill in the art to which the application belongs.

[0054] Certain embodiments disclosed herein include numerical ranges, and certain aspects of the present application may be described in terms of ranges. Unless otherwise stated, it should be understood that numerical ranges or the use of range descriptions are intended solely for the purpose of brevity and convenience and should not be considered as strict limitations on the scope of the present application. Therefore, descriptions using 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 in the present application. 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.

[0055] 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.

[0056] 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 such a structure. Each light chain consists of a variable domain (VL) and a constant domain (CL). Each heavy chain comprises a variable domain (VH) and a constant region.

[0057] 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 mutant proteins and variants thereof), etc.

[0058] 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 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 accommodates the desired gene sequence to be transposed 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 to replace the helper plasmid and transposon plasmid to co-transform target cells for transposase expression.

[0059] The term "CHO ​​platform" refers to a CHO cell line screening technology platform. CHO (Chinese Hamster Ovary) cells are cultured in the chemically defined medium CD CHO Fusion Medium and then subcloned and screened 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.

[0060] The term "transgenic vector" refers to an expression vector that expresses a target gene. This refers to a vector that adds expression elements (such as a promoter, RBS, and terminator) to the basic cloning vector framework to enable the expression of the target gene. Target genes include, but are not limited to, deoxynucleotide sequences encoding products such as antibodies, antigens, fusion proteins, and polypeptides.

[0061] The term "insulator" or "insulator sequence" refers to a class of DNA sequences found at the boundaries of chromatin domains. These sequences 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 sequence. Exemplary insulator sequences include chicken hypersensitive site-4 (cHS4).

[0062] 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.

[0063] The term "transcription factors" (TF) refers to a group of protein molecules that can specifically bind to specific sequences upstream of the 5' end of a gene, thereby ensuring that the target gene is expressed with a specific intensity at a specific time and space.

[0064] The term "transcription factor binding site" (TFBS) refers to the region where a transcription factor binds to a gene template strand when regulating gene expression.

[0065] The Gene Ontology (GO) is a standardized functional classification system that provides a dynamically updated set of standardized vocabularies and describes the properties of genes and gene products in organisms in three aspects: biological process (BP), molecular function (MF), and cellular component (CC). GO terms are descriptive information of the Gene Ontology function.

[0066] The term "Protein Protein Interactions" (PPI) refers to the application of protein interaction databases, such as the STRING protein interaction database, to directly extract the interaction relationships of the target gene set (such as the differentially expressed gene list) from the database to construct a network for the species contained in the database.

[0067] The term "sequence identity" or "sequence similarity" or "sequence homology" refers to the percentage of nucleotides / amino acid residues in a candidate sequence that are identical to the nucleotides / amino acid residues in a reference sequence, after the sequences are aligned (and, if necessary, introducing gaps) to obtain maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity. Sequence alignment can be performed using various methods in the art to determine percent nucleotide / amino acid sequence identity, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN or MEGALIGN (DNASTAR) software. One skilled in the art can determine appropriate parameters for measuring alignment, including any algorithm required to achieve maximum alignment over the full length of the compared sequences.

[0068] 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.

[0069] The present invention is further described in detail by the following examples. Specific examples are listed below to illustrate the present invention. However, it should be understood that these examples are only listed to illustrate the present invention and are not intended to limit the scope of the present invention.

[0070] Materials and reagents:

[0071] P3-AscI (schematic structure shown in FIG1 ) has the AscI restriction site recognition sequence GGCGCGCC inserted into the P3 plasmid.

[0072] The PiggyBac transposase coding sequence was optimized for CHO cells by Suzhou Hongxun Biotechnology Co., Ltd. (with an EcoRI restriction site and a Kozak sequence added to the 5' end and a HindIII added to the 3' end), synthesized, and cloned into the vector PUC57 to obtain PUC57-PiggyBac.

[0073] The 5ITR-cHS4 sequence in the PiggyBac donor vector was synthesized by Suzhou Hongxun Biotechnology.

[0074] The 3ITR-cHS4 sequence in the PiggyBac donor vector element was synthesized by Suzhou Jinweizhi Biotechnology Co., Ltd.

[0075] The 3ITR-R-cHS4 sequence containing the reverse complementary insulator in the PiggyBac donor vector element was synthesized by Suzhou Jinweizhi Biotechnology Co., Ltd. and cloned into the PUC57 vector to obtain PUC57-3ITR-R-cHS4.

[0076] The PiggyBac mRNA template DNA sequence was synthesized by Nanjing GenScript Biotechnology Co., Ltd.

[0077] The blank expression vector pcDNA3.4 was provided by Nanjing GenScript Biotechnology Co., Ltd.

[0078] Example 1: Design of cHS4 insulator truncated sequence and vector construction

[0079] 1. Design and construction of truncated cHS4 insulators

[0080] The insulators used, cHS4-650, cHS4-400, and cHS4-250, are derived from literature (such as 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. Single or multiple truncation regions were combined and truncated. Finally, four insulators were obtained, with nucleic acid sequences of SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3 and SEQ ID NO.4, specifically as follows:

[0081] SEQ ID NO.1

[0082] SEQ ID NO.2

[0083] SEQ ID NO.3

[0084] SEQ ID NO.4

[0085] 2. Donor vector construction

[0086] The PiggyBac donor vector element contains 5ITR-cHS4 (forward) and 3ITR-cHS4 (forward), wherein there are two forward cHS4s between 5ITR and 3ITR, and the cHS4 sequence contains cHS4-650, cHS4-400, cHS4-250, the sequence shown in SEQ ID NO.1, the sequence shown in SEQ ID NO.2, the sequence shown in SEQ ID NO.3, or the sequence shown in SEQ ID NO.4. The 5ITR-cHS4 fragment was ligated to the P3-AscI plasmid by AscI single enzyme digestion and homologous recombination to construct the plasmid P3-5ITR-cHS4. Then, the 3ITR-cHS4 was integrated into the P3-5ITR-cHS4 by homologous recombination to construct the donor vector P3-5ITR-cHS4-3ITR-cHS4, which is abbreviated as P3-5ITR-3ITR-cHS4 below for convenience (the structural schematic is shown in Figure 2).

[0087] 3. Expression Vector Construction

[0088] The vector P3-5ITR-3ITR-cHS4 was double-digested and ligated using BstBI / PacI and HindIII / XhoI, respectively, and the nucleic acid sequences encoding the heavy and light chains of the QL01 monoclonal antibody were connected to the corresponding multiple cloning sites, respectively, to construct the recombinant expression vector P3-5ITR-3ITR-cHS4-QL01.

[0089] 4. Construction of auxiliary vector

[0090] The vector PUC57-PiggyBac and the blank vector pcDNA3.4 were double-digested with EcoRI / HindIII. The digested products of the vector PUC57-PiggyBac and the blank vector pcDNA3.4 were then purified and recovered using a NucleoSpin Gel and PCR Clean-up Kit and ligated to construct a helper plasmid pcDNA3.4-PiggyBac containing the transposase encoding gene (the schematic structure is shown in Figure 3). Alternatively, in vitro transcription can be used, using the PB transposase encoding DNA sequence as a template, to obtain PB transposase encoding mRNA-PiggyBac through in vitro transcription, capping, and tailing modifications.

[0091] Example 2: Transient transfection of cHS4 insulator truncated sequences for antibody expression

[0092] The host cells used were the ExpiCHO Expression System (Gibco), and transient protein expression was performed according to the recommended protocol for the host cells. The transient transfection system volume was 25 mL, and the amounts of plasmid used for transfection are shown in Table 1. Feeding was performed according to the recommended feeding protocol in the ExpiCHO Expression System kit, and culture was continued until day 6. Antibody expression was measured using a ForteBio instrument, as shown in Figure 4. The culture medium was harvested after 7 days of culture.

[0093] Comparing protein yields in ExpiCHO transient transfections, the PiggyBac transposon expression system supplemented with a 650bp cHS4 insulator (i.e., P3-5ITR-3ITR-650-QL01) showed significantly higher yields than the PiggyBac transposon systems supplemented with 400bp (i.e., P3-5ITR-3ITR-400-QL01) and 250bp (i.e., P3-5ITR-3ITR-250-QL01) insulators. Six days after transfection, the PiggyBac transposon expression system supplemented with a 650bp cHS4 insulator showed 23% and 28% higher protein yields than the PiggyBac transposon expression systems supplemented with 400bp and 250bp insulators, respectively. These results demonstrate that the addition of a 650bp cHS4 insulator to the PiggyBac transposon expression system is most beneficial for enhancing protein yield. Compared with the 650bp cHS4, the protein yield of the PiggyBac transposon expression system with the addition of the cHS4 insulator with the sequence shown in SEQ ID NO.1 was increased by 13%. The protein yield after transfection of the vectors P3-5ITR-3ITR-SEQ ID NO.2-QL01 (containing the cHS4 insulator with the sequence shown in SEQ ID NO.2), P3-5ITR-3ITR-SEQ ID NO.3-QL01 (containing the cHS4 insulator with the sequence shown in SEQ ID NO.3), and P3-5ITR-3ITR-SEQ ID NO.4-QL01 (containing the cHS4 insulator with the sequence shown in SEQ ID NO.4) was comparable to that of the PiggyBac transposon expression system with the addition of the cHS4 insulator with the sequence shown in SEQ ID NO.1.

[0094] Table 1 Transient transfection plasmids and dosage

[0095] Example 3: Stable transfection of cHS4 insulator truncated sequences for antibody expression

[0096] CHO cells were transfected by electroporation. The transfection conditions are shown in Table 2. After cell transfection, the cells were treated with culture medium. Resuspend the cells in CD CHO Fusion Medium (containing 6mM L-glutamine) and culture in an incubator at 37°C, 5% CO2. After 24 hours of culture, centrifuge the cells at 1000 rpm for 5 minutes, remove the supernatant, and resuspend the cells in an appropriate amount of EX-CELL CD CHO Fusion medium. Transfer all the cells to a 125 mL shake flask and culture on a shaker at 37°C, 5% CO2, 80% humidity, and 130 rpm. Count and passage the cells every 1-5 days until the cell viability of the pool recovers. 5 Yield testing was performed using a seeding density of 10 cells / mL. The results of the fed batch on day 14 of stable expression are shown in Figure 5. The results showed that all PiggyBac transposon expression systems containing insulators such as SEQ ID Nos. 1-4 achieved excellent expression capabilities. In particular, the expression levels of the PiggyBac transposon expression systems containing insulators such as SEQ ID Nos. 1, 3, and 4 were comparable to or higher than those of the PiggyBac transposon expression system containing a 650 bp insulator. The PiggyBac transposon expression system containing a truncated sequence such as SEQ ID No. 4 increased expression yield by 5%, or 134 mg / L. Compared to the expression system containing an insulator with a 250 bp core region, the PiggyBac transposon expression system containing the truncated sequence such as SEQ ID No. 4 increased expression yield by 28%, or 711 mg / L. Without being bound by theory, the inventors believe this may be because the truncated sequences not only retain the optimized expression capabilities of the 650 bp insulator sequence used in the control, but also, due to their shorter sequences and smaller vector size, improve expression efficiency. On the other hand, the PiggyBac transposon expression system comprising the insulator sequence shown in SEQ No. 1-4 also has significantly better expression capacity than the PiggyBac transposon expression system comprising the insulator core region of 250 bp.

[0097] Table 2 Stable transfection conditions

[0098] Example 4: Effect of insulator orientation on antibody expression in a transposon expression system

[0099] 1. Construction of donor vector P3-5ITR-3ITR-R-SEQ ID NO.1

[0100] Using the vector PUC57-3ITR-R-SEQ ID NO.1 (containing the reverse complementary sequence to the sequence shown in SEQ ID NO.1) as a template, PCR amplification of the target fragment 3ITR-R-SEQ ID NO.1 was performed. The P3-5ITR-SEQ ID NO.1 (forward) plasmid was digested with SgrAI, and the 3ITR-R-SEQ ID NO.1 gene fragment was ligated by homologous recombination to construct the vector P3-5ITR-SEQ ID NO.1-3ITR-R-SEQ ID NO.1, which contains a forward cHS4 and a reverse complementary cHS4 between the 5ITR and 3ITR, namely P3-5ITR-3ITR-R-SEQ ID NO.1 (schematic structure shown in Figure 6A).

[0101] 2. Construction of vectors P3-5ITR-3ITR-SEQ ID NO.1-QL02 and P3-5ITR-3ITR-R-SEQ ID NO.1-QL02

[0102] The Knob and Hole chain encoding nucleic acid sequences of the QL02 bispecific antibody were ligated to the corresponding multiple cloning sites of P3-5ITR-3ITR-SEQ ID NO.1 (schematic structure shown in FIG6B ) and P3-5ITR-3ITR-R-SEQ ID NO.1 using BstBI / PacI and HindIII / XhoI double enzyme digestion and ligation, respectively, to construct the recombinant expression vectors P3-5ITR-3ITR-SEQ ID NO.1-QL02 and P3-5ITR-3ITR-R-SEQ ID NO.1-QL02.

[0103] 3. Antibody Protein Expression

[0104] The host cells used were the ExpiCHO Expression System (Gibco), and transient protein expression was performed according to the recommended protocol for the host cells. The transient transfection system volume was 25 mL, and the amount of plasmid used for transfection is shown in Table 3. Feeding was performed according to the recommended feeding protocol for the ExpiCHO Expression System kit, and culture was continued until day 7. Antibody expression was measured using a ForteBio instrument, and the results are shown in Table 4.

[0105] Table 3 Transient transfection experimental groups

[0106] Table 4 Expression levels on day 7 of transient transfection

[0107] Comparing the expression levels of the PiggyBac transposon expression system containing 3ITR + reverse complementary insulator (vector P3-5ITR-3ITR-R-cHS4) and containing 3ITR + forward insulator (vector P3-5ITR-3ITR-cHS4), the vector containing 3ITR + reverse complementary insulator increased the expression level by 11.3% compared with that containing 3ITR + forward insulator, indicating that the combination of vector 3ITR + reverse complementary insulator has a more significant effect on improving yield.

[0108] Example 5: Application of Stable Transfection of cHS4 Insulator Truncated Sequences in the PiggyBac Transposon System for Antibody Expression

[0109] 1. Construction of donor vector P3-5ITR-3ITR

[0110] The insulator in the vector P3-5ITR-3ITR-cHS4 was deleted to construct the P3-5ITR-3ITR vector without an insulator.

[0111] 2. Expression Vector Construction

[0112] The light chain and heavy chain encoding nucleic acid sequences of antibody QL03 were respectively integrated into the light chain and heavy chain multiple cloning sites of P3-5ITR-3ITR-R-SEQ ID NO.1 and P3-5ITR-3ITR using HindIII / XhoI and BstBI / PacI enzyme digestion and ligation to construct expression vectors P3-5ITR-3ITR-R-SEQ ID NO.1-QL03 and P3-5ITR-3ITR-QL03.

[0113] 3. Transient expression of antibody protein

[0114] The host cells used were the ExpiCHO Expression System (Gibco), and transient protein expression was performed according to the recommended protocol for the host cells. The transient transfection system contained 50 mL of culture medium, with a transfection volume of 50 μg of donor vector plus 5 μg of helper vector. Feeding was performed according to the recommended feeding protocol for the ExpiCHO Expression System kit, and culture was continued until day 7. Antibody expression was measured using a ForteBio instrument. The results are shown in Table 5.

[0115] Comparing the yield seven days after transient transfection, the PiggyBac transposon expression system with the cHS4 insulator achieved a yield of 247 mg / L, a 1.5-fold increase compared to the PiggyBac transposon expression system without the insulator. These results demonstrate that the addition of the cHS4 insulator to the PiggyBac transposon expression system significantly increases cell line yield.

[0116] Table 5 Transient expression

[0117] 4. Stable expression of antibody protein

[0118] The amount of transfected plasmid was 50 μg donor vector + 5 μg auxiliary vector, voltage 300V, 950 μF, cell number 1E7, one electric shock. The cells were resuspended in CD CHO Fusion Medium (containing 6 mM L-glutamine) and cultured in an incubator at 37° C. and 5% CO 2 .

[0119] After 24 h of culture, the cells were counted to determine the viable cell density (VCD) and cell viability (Via), and minipools were plated. The cells were then screened and expanded in 96-well plates, 24-well plates, and TPP plates. When the cells were fully recovered (viability > 90%), 5 × 10 5 The yield test was conducted in a batch experiment with inoculation of cells / mL. The yield results of the top 14 minipools are shown in Table 6.

[0120] Comparing the yield results of the stable minipool yield test, the highest yield of the PiggyBac transposon expression system with the cHS4 insulator was 158 mg / L, a 15.3% increase compared to the highest yield of the PiggyBac transposon expression system without the insulator. The average expression level of the top three yielders was 150.6 mg / L, a 54.9% increase compared to the system without the insulator, and the average expression level was 112.3 mg / L, a 102.4% increase compared to the system without the insulator. These results indicate that the addition of the cHS4 insulator to the PiggyBac transposon expression system can increase the yield of cell lines.

[0121] Table 6 Expression levels of stable transfection minipool

[0122] Example 6: PiggyBac transposon expression system ensures cell line stability

[0123] Three stable cell lines constructed using the PiggyBac transposon expression system containing the insulator SEQ ID NO. 1 were randomly selected. The cell line construction method was as described in Example 5. The stability of the final monoclonal cells of the cell line was evaluated, including: (1) growth stability: the cell line was cultured continuously for 60 generations after recovery, and the cell density, viability, diameter, and growth rate of the cells during the passage process were evaluated; (2) production stability: the cells were subjected to yield tests after passage 30, 45, and 60 to evaluate the yield and quality of the product; and (3) genetic stability: the cell lines of passage 0 and passage 60 were tested for cDNA sequencing.

[0124] 1. Growth stability experiment

[0125] a) Resuscitating candidate clones;

[0126] b) Every 2-3 days, 2×10 5 -5×10 5 The cells were passaged at a density of cells / mL, and the culture conditions were temperature 37°C, humidity 80%, rotation speed 130 rpm, and CO2 concentration 5%;

[0127] c) Record cell density, viability, and diameter during subculture and calculate doubling time.

[0128] 2. Production stability test

[0129] a) Cryopreserved cells at PDL15, PDL30, PDL45, PDL60, or PDL70;

[0130] b) After the passage is completed, resuscitate PDL15, PDL30, PDL45, PDL60, and PDL70 cells and inoculate them for yield testing after the cells have recovered;

[0131] c) feeding as needed during the culture process;

[0132] d) After the cell culture is completed, the cell culture supernatant is harvested and 1 mL is taken for HPLC analysis. The CV value is calculated based on the expression level: CV = (nPDL expression level - 0PDL expression level) / 0PDL expression level × 100%, n = 30 / 45 / 60.

[0133] 3. Genetic stability experiment

[0134] After stable passage, cells of passages PDL0 and PDL60 were revived, total RNA was extracted and reverse transcribed into cDNA, and the target gene was amplified using the corresponding primers and then sequenced. The sequencing results were compared with the theoretical sequence to confirm whether the target gene DNA sequence had mutated.

[0135] Growth stability data are shown in Figure 7. After clones A, B, and C were passaged at the same density and cultured for the same time, there was no significant difference in cell density, cell viability remained above 95%, and diameter variation was within 8%, indicating that clones constructed using the PiggyBac transposon expression system maintained stable growth performance and cell morphology.

[0136] The cDNA sequencing results of clones A, B, and C all remained consistent with the theoretical sequences, showing no mutations. This demonstrates that the genetic properties of clones constructed using the PiggyBac transposon expression system remain unchanged, and that the target gene sequence is genetically stable after serial cell passage. The yield of the three project cell lines all varied within ±10% (see Table 7 for specific data), a range far less than the industry average standard of ±30%, demonstrating the system's excellent cell line genetic stability.

[0137] Table 7 Stability data of clone production constructed by PiggyBac transposon expression system

[0138] The above description is only a preferred embodiment of the present application and is not intended to limit the present application. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principles of the present application shall be included in the scope of protection of the present application.

Claims

1. An insulator, wherein: The nucleic acid sequence of the insulator comprises 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 one of the nucleic acid sequences shown in SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3 or SEQ ID NO.4, or the nucleic acid sequence of 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.

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

4.

3. A eukaryotic cell expression system, comprising the insulator according to claim 1 or 2.

4. The eukaryotic cell expression system according to claim 3, wherein The eukaryotic cell expression system is the PiggyBac transposon expression system.

5. The eukaryotic cell expression system according to claim 4, wherein The PiggyBac transposon expression system comprises a PiggyBac transposon transgenic vector; preferably, the PiggyBac transposon expression system further comprises a PiggyBac helper vector or mRNA encoding a transposase, wherein the PiggyBac helper vector comprises a nucleic acid sequence encoding a transposase.

6. The eukaryotic cell expression system according to claim 5, wherein The PiggyBac transposon transgenic vector comprises a 5' terminal repeat sequence 5ITR and an inverted 3' terminal repeat sequence 3ITR, and the insulator is arranged between the 5ITR and the 3ITR; preferably, the insulators complementary to each other in the forward and reverse directions or two insulators both in the forward direction are arranged between the 5ITR and the 3ITR in sequence; preferably, a multiple cloning site is arranged between the insulators.

7. The eukaryotic cell expression system according to claim 5 or 6, wherein The PiggyBac transposon transgenic vector comprises a selection marker gene.

8. The eukaryotic cell expression system according to claim 7, wherein The selection marker gene is the GS gene.

9. Use of the insulator according to claim 1 or 2 or the eukaryotic cell expression system according to any one of claims 3 to 8 in the recombinant expression of nucleic acid.

10. The use according to claim 9, wherein: The nucleic acid can encode an antibody, a fusion protein, an antigen or an enzyme.

11. Use of the insulator according to claim 1 or 2 or the eukaryotic cell expression system according to any one of claims 3 to 8 in preparing proteins or polypeptides.

12. The use according to claim 11, wherein: The proteins or polypeptides include antibodies, fusion proteins, antigens and enzymes.

13. Use of the insulator according to claim 1 or 2 or the eukaryotic cell expression system according to any one of claims 3 to 8 in constructing a recombinant cell line.

14. The use according to claim 13, wherein: The host cells used by the recombinant cell strain are mammalian cells, preferably CHO cells.

15. A recombinant cell strain, wherein: The recombinant cell strain comprises the insulator according to claim 1 or 2 or the eukaryotic cell expression system according to any one of claims 3 to 8; preferably, the host cell used by the recombinant cell strain is a mammalian cell, preferably a CHO cell.

Images