Cut insulator and its use

Abstract

This invention provides a cleaved insulator and an expression system for eukaryotic cells comprising the cleaved insulator. The cleaved insulator breaks away from conventional core region configurations, shortens the effective sequence of the insulator, and is advantageous for integration of the insulator with other elements of the vector. The insulator significantly improves protein yield and yields recombinant cell lines with good genetic stability in expression systems where transposons (e.g., PiggyBac) are present.

Description

Technical Field

[0001] This application claims the priority of a Chinese patent application filed with the China National Intellectual Property Administration on January 5, 2023, with an application number of 202310012420.0 and an invention title of "Truncated Insulator and Its Use", the entire content of which is incorporated herein by reference.

[0002] Technical Field The present invention relates to the field of biotechnology, and specifically, to truncated insulators and their use.

Background Art

[0003] Currently, there are more than 100 approved antibody drugs worldwide, bringing benefits to an increasing number of patients in various therapeutic fields. With the development of antibody drugs, the market scale of antibodies has already reached hundreds of billions of dollars, and the production scale can reach tens of thousands of liters per batch at most. In the production process of antibodies, the construction of cell lines, as the basis of production, has been widely studied and attracted attention. The method of realizing the rapid research and development and high yield of antibody drugs through the construction of cell lines is the goal that the industry has been pursuing consistently.

[0004] In the process of constructing stable transfection cell lines, the target gene is usually inserted into the host cell genome in a random integration manner, and there are the following defects. The integration efficiency is usually extremely low, less than 1 in 10,000, and the construction of stable transfection cell lines depends on a large amount of screening, with a large workload and a long construction time. The target gene is easily affected by the position effect and has a low expression level. The target gene is easily silenced by the host cell and has low stability. The yield of cell pools is low, and it is difficult to quickly obtain a sufficient amount of protein to meet the needs of early new drug development.

[0005] Therefore, several site-specific and semi-site-specific integration techniques have been developed. For example, the PiggyBac transposon expression system consists of a donor plasmid and a helper plasmid or in vitro transcribed mRNA. The donor plasmid has a 5' reverse repeat sequence (5ITR) and a 3' reverse repeat sequence (3ITR) as transposase recognition sequences. The helper plasmid or in vitro transcribed mRNA encodes the PiggyBac transposase (PB transposase). This PB transposase can recognize the 5ITR and 3ITR, and after cleaving or replicating at the original site, it is circularized and inserted into a specific site in the host genome with the assistance of the transposase. This results in accurate integration and has significant advantages such as high integration efficiency, high stability, and uniform copy format. However, at the same time, transposon integration techniques have the disadvantage of low expression levels, resulting in high production costs and making them difficult to apply to large-scale production.

[0006] According to research reports, insulators play a crucial role in regulating the spatiotemporal-specific expression of eukaryotic genes. Insulators can protect target genes from the influence of surrounding regulators and avoid erroneous activation or silencing of genes, possessing enhancer-blocking activity and heterochromatin barrier activity, thereby effectively suppressing the "position effect." HS4 is a DNase I hypersensitive site (HS), or cHS4, with insulator activity located upstream of the 5' end of the chicken β-globin locus, and its length is approximately 1.2 kp. Studies have shown that it is a previously... Although it has been shown to possess both of the aforementioned functions, previous studies have indicated that inserting the complete sequence results in an oversized vector, reducing its transfection efficiency and negatively impacting protein expression levels. Therefore, research into cHS4 cleavage is necessary. 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 insufficient on its own to enhance expression; the 400bp sequence at the 3' end of cHS4 also plays an important role. Furthermore, cHS4-650 (250bp core region and 400bp sequence at the 3' end) exhibits superior function compared to cHS4-250. However, the 650bp length insulator still suffers from the problem of being too large a vector, reducing transfection efficiency. Therefore, there is a technical need to further optimize the length of the insulator sequence based on the 650bp length to improve transfection efficiency.

[0007] While some literature reports methods for preparing initial proteins using the PiggyBac transposon expression system, the results show that the resulting protein expression levels are low and cannot meet the needs of industrial production. With advances in transposon research, the literature has reported that inserting cleaved insulators into the flanking sequence of target genes can significantly increase transgene expression, reduce the coefficient of variation of gene expression, and improve stability. However, most research on cleaving cHS4 insulators currently relies on strategies combining different regions as shown in existing studies, primarily using a 250 bp core region as a base and combining other different regions. However, the length of these combinations has not been further optimized, nor has the function of the cleaved insulators been verified. Furthermore, these studies have not yet been applied to the construction of stable transfection cell lines used in the production of antibody drugs, indicating a long road ahead to industrial application. [Overview of the project]

[0008] A first aspect of the present invention provides an insulator for the expression of polypeptides or proteins in eukaryotic cells. The nucleic acid sequence of the insulator includes a nucleic acid sequence shown in SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, or SEQ ID NO.4, or includes 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 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.

[0009] In some embodiments of the present invention, the insulator is a nucleic acid sequence represented by 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 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 one of the nucleic acid sequences represented by SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, or SEQ ID NO.4.

[0010] A second aspect of the present invention provides a eukaryotic cell expression system comprising the insulator described in the first aspect of the present invention, the insulator being capable of improving protein yield and cell line stability.

[0011] In some embodiments of the present invention, the eukaryotic cell expression system is a PiggyBac transposon expression system.

[0012] In some embodiments of the present invention, 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 contains nucleic acid encoding a transposase.

[0013] In some embodiments of the present invention, the PiggyBac transposon transgenic vector contains the insulator described in the first aspect of the present invention.

[0014] In some embodiments of the present invention, the PiggyBac helper vector is pcDNA3.4.

[0015] In some embodiments of the present invention, the PiggyBac transposon transgenic vector comprises a 5'-terminal repeat sequence 5ITR and a reverse 3'-terminal repeat sequence 3ITR, with the insulators positioned between 5ITR and 3ITR. Preferably, forward and reverse complementary insulators, or two forward insulators, are sequentially positioned between 5ITR and 3ITR. Preferably, a multi-cloning site is positioned between the insulators. Preferably, other elements of the PiggyBac transposon transgenic vector are derived from the original P3 vector sequence.

[0016] In some embodiments of the present invention, the PiggyBac transposon transgenic vector further comprises a screenable marker gene.

[0017] In some embodiments of the present invention, the screenable marker gene is the glutamine synthase gene (GS gene).

[0018] A third aspect of the present invention further provides the use of the insulator described in the first aspect of the present invention in the construction of a PiggyBac transposon transgenic vector or a PiggyBac transposon expression system.

[0019] A fourth aspect of the present invention further provides the use of the insulator described in the first aspect of the present invention or the eukaryotic cell expression system described in the second aspect of the present invention, in particular for recombinant expression of nucleic acids.

[0020] In some embodiments of the present invention, the nucleic acid can encode a polypeptide or a protein, and preferably an antibody, a fusion protein, an antigen, or an enzyme. Preferably, the recombinant expression is carried out in a eukaryotic host cell. Preferably, the expression system is a PiggyBac transposon expression system. Preferably, the eukaryotic host cell is a CHO cell.

[0021] A fifth aspect of the present invention further provides the use of the insulator described in the first aspect of the present invention or the eukaryotic cell expression system described in the second aspect of the present invention in the construction of recombinant cell lines.

[0022] In some embodiments of the present invention, the host cells used in the recombinant cell line are mammalian cells, preferably CHO cells.

[0023] In some embodiments of the present invention, the step of constructing a recombinant cell line is: 1) A step of constructing a Piggybac transposon transgenic vector containing a gene encoding a target protein and a glutamine synthase gene, and a helper vector expressing PB transposase, or obtaining mRNA encoding PB transposase by in vitro transcription, 2) introducing the transposon transgenic vector and the helper vector in step 1), or the transposon transgenic vector and the mRNA encoding PB transposase into the host cell by transfection; 3) placing the cells obtained in step 2) into a culture flask and allowing them to recover for a certain period of time; 4) transferring the cells obtained in step 3) into a shaking flask and screening them with GS; 5) after the cell state has fully recovered, performing an inoculation yield test and evaluating the antibody expression level.

[0024] In some embodiments of the present invention, the target protein in step 1) can be an antibody, a fusion protein, an antigen, an enzyme, or other types of proteins or polypeptides.

[0025] In some embodiments of the present invention, in step 1), the in vitro transcription method is used, with the DNA sequence encoding PB transposase as a template, and through in vitro transcription, capping, and tailing modifications, the mRNA encoding PB transposase is obtained.

[0026] In some embodiments of the present invention, in step 5), the complete recovery of the cell state means that the cell viability is 95% or more.

[0027] In some embodiments of the present invention, for the construction of the vector in step 1), an endotoxin-free plasmid extraction kit is used.

[0028] In some embodiments of the present invention, the transfection method in step 2) is electroporation.

[0029] In some embodiments of the present invention, the culture flask in step 3) is a T flask.

[0030] In some embodiments of the present invention, the culture medium in step 3) is CD CHO Fusion Medium + 6 mM glutamine, and the recovery time is 24 hours.

[0031] In some embodiments of the present invention, the screening medium in the shaking flask in step 4) is CD CHO Fusion Medium.

[0032] In some embodiments of the present invention, the yield test in step 5) employs a 7-day batch culture, and the inoculation density is 5 × 10 5 The cell density was 30 mL, the culture medium was BM2, the culture volume was 30 mL, 4 g / L of sugar was added on day 4 of culture, and the expression level of the supernatant was detected on day 7 of culture.

[0033] A sixth aspect of the present invention further provides a recombinant cell line comprising an insulator described in the first aspect of the present invention or a eukaryotic cell expression system described in the second aspect of the present invention, wherein the host cell used in the recombinant cell line is a mammalian cell, preferably a CHO cell.

[0034] The beneficial effects of the present invention are as follows: The present invention is the first to cleave the 250 bp cHS4 core region, breaking the original core region configuration. Compared to the existing 650 bp cHS4 insulator sequence in the prior art, the length of the insulator sequence is significantly shortened, protein expression levels are improved, and the time required to construct a stable cell line can be reduced. Furthermore, the constructed cell line exhibits good genetic stability.

[0035] Brief explanation of the drawing The drawings described herein are provided to further illustrate the present invention and constitute part of it. Exemplary embodiments and descriptions of the present invention are for interpretation purposes only and do not unduly limit the present invention. [Brief explanation of the drawing]

[0036] [Figure 1] Figure 1 is a schematic diagram of the P3-AscI vector structure. [Figure 2] Figure 2 is a schematic diagram of the P3-5ITR-3ITR-cHS4 vector structure. [Figure 3] Figure 3 is a schematic diagram of the pcDNA3.4-PiggyBac vector structure. [Figure 4] Figure 4 shows the protein yield 6 days after transient transfection. [Figure 5] Figure 5 shows the protein yield 14 days after stable transfection. [Figure 6A] Figure 6A is a schematic diagram of the structure of the P3-5ITR-R-3ITR-SEQ ID NO.1 reverse complementary cHS4 vector. [Figure 6B] Figure 6B is a schematic diagram of the structure of the P3-5ITR-3ITR-SEQ ID NO.1 forward cHS4 vector. [Figure 7] Figure 7 is a graph showing the effect of the PiggyBac transposon expression system on clonal proliferation stability. [Modes for carrying out the invention]

[0037] To further clarify the object, technical concept and advantages of the present invention, the present invention will be described in more detail below with reference to the drawings and examples. Clearly, the examples described below are only a selection of the embodiments of the present invention, not all embodiments. All other embodiments that those skilled in the art can obtain based on the present invention are all within the scope of protection of the present invention.

[0038] term All publications, patents, and patent applications referenced herein are incorporated herein by reference to the same extent as if each publication, patent, or patent application had been specifically and individually indicated to be incorporated by reference.

[0039] Before describing the present invention in detail below, it should be understood that the present invention is not limited to the specific methodologies, forms, and reagents described herein, and that these are modifiable. It should also be understood that the terms used in the present invention are intended solely to describe specific embodiments and are not intended to limit the scope of the invention. Unless otherwise defined, all technical and scientific terms used in the present invention have the same meaning as those generally understood by those skilled in the art to which the present invention pertains.

[0040] Some embodiments disclosed in this invention include numerical ranges, and some aspects of this invention can be described in range form. Unless otherwise specified, descriptions in numerical range form or in range form are for the sake of brevity and convenience only and should not be interpreted as strictly limiting the scope of this invention. Accordingly, descriptions in range form should be considered to specifically disclose all possible subranges and all possible specific numerical points within those ranges, as such subranges and numerical points are explicitly described in this invention. The above principle applies equally to the broadness of the numerical range. When described in range form, the range includes the endpoints of the range.

[0041] When referring to measurable values ​​such as quantity or temporary duration, the term "approximately" means that the specified value may include a variation of ±20%, possibly ±10%, possibly ±5%, possibly ±1%, or possibly ±0.1%.

[0042] As used in this invention, the term "antibody" 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 one variable domain (VL) and one constant domain (CL). Each heavy chain contains one variable domain (VH) and a constant region.

[0043] As used in the present invention, examples of "antibodies" in a broad sense include polyclonal antibodies, monoclonal antibodies, chimeric antibodies, humanized antibodies and primate-derived antibodies, CDR-grafted antibodies, human antibodies (including human antibodies produced by recombinant), recombinant antibodies, intracellular antibodies, multispecific antibodies, bifunctional fusion proteins, monovalent antibodies, multivalent antibodies, anti-idiotype antibodies, and synthetic antibodies (including mutant proteins and their variants).

[0044] The term "PiggyBac transposon expression system" refers to the PiggyBac transposon vector system. Members of the PiggyBac vector system primarily consist of one helper vector or plasmid encoding a transposase and one transposon vector (also called a donor vector) or plasmid. The transposon vector or plasmid contains optimized inverted terminal repeat sequences (ITRs) at both ends and a transposition region in the center into which a target gene sequence to be transposed into the host genome can be inserted. During experiments, the helper plasmid and transposon plasmid must be introduced into target cells simultaneously. The transposase expressed from the helper plasmid recognizes and cleaves the ITR sequences at both ends of the transposon plasmid. The released transposition region is incorporated by the transposase into a site in the host genome containing a TTAA sequence, resulting in the appearance of TTAA repeat sequences at both ends of the transposition region. Alternatively, instead of a helper plasmid, in vitro transcribed mRNA encoding the transposase can be used and introduced into target cells along with the transposon plasmid for transposase expression.

[0045] The term "CHO ​​platform" refers to the CHO cell line screening technology platform. CHO (Chinese hamster ovary cells) are screened in chemically restricted medium CD CHO After acclimatization to Fusion Medium, subclonal screening is performed to establish the CHO cell line. Furthermore, the process includes related reagents and processes such as the expression vector P3, culture medium for the clonal construction stage, and fed-batch process platform medium.

[0046] The term "transgenic vector" refers to an expression vector that expresses a target gene. An expression vector is a vector that has been modified to express a target gene by adding expression elements (e.g., promoter, RBS, terminator, etc.) to the basic framework of a cloning vector. Target genes include, but are not limited to, deoxyribonucleotide sequences that encode products such as antibodies, antigens, fusion proteins, and polypeptides in a broad sense.

[0047] The term "insulator" or "insulator sequence" refers to a DNA sequence located at the boundary of a chromatin domain. It acts as a neutral barrier, blocking the influence of adjacent gene elements or the surrounding dense chromatin. This ensures that the protected gene is properly positioned. This allows for expression in various time and space. The effect of an insulator is related to its position within the gene and the orientation of its own sequence. An exemplary insulator sequence is chicken hypersensitive site-4 (cHS4).

[0048] The term "multicloning site" refers to a single artificially synthesized DNA fragment contained within a vector, containing multiple single restriction enzyme cleavage sites. It is an insertion site for exogenous DNA, also known as a multisite linker, and is a standard sequence of vector plasmids commonly used in genetic engineering. In a multicloning site, each restriction enzyme cleavage site is usually unique, meaning it appears only once in a particular vector plasmid, and the enzyme cleavage sites of different enzymes may overlap.

[0049] The term "transcription factors" (TF) refers to a group of protein molecules that can specifically bind to particular sequences upstream of the 5' end of a gene, thereby ensuring that the target gene is expressed at a specific intensity at a specific time and space.

[0050] The term "transcription factor binding site" (TFBS) refers to the region on a gene template that a transcription factor binds to when it regulates gene expression.

[0051] The term "Gene Ontology" (GO) is a standardized functional classification system. This system provides a dynamically updated standardized vocabulary to describe the characteristics of genes and gene products within living organisms from three perspectives: Biological Process (BP), Molecular Function (MF), and Cellular Component (CC). GO terms are descriptive information regarding Gene Ontology function.

[0052] The term "protein-protein interaction network analysis" (PPI) refers to constructing a network by directly extracting interaction relationships of target gene sets (e.g., differential gene lists) from protein-protein interaction databases, such as the STRING protein-protein interaction database, for species included in the database.

[0053] The terms "sequence identity," "sequence similarity," or "sequence homology" refer to the percentage of nucleotide / amino acid residues in a candidate sequence that are identical to those in a reference sequence, when sequences are compared to obtain the maximum percentage of sequence identity (with gaps introduced as necessary), and no conserved substitutions are considered as part of the sequence identity. For example, sequence alignment is performed using various methods in the field, such as publicly available computer software like BLAST, BLAST-2, ALIGN, or MEGALIGN (DNASTAR), to obtain the percentage of nucleotide / amino acid sequence identity. The alignment can be determined. A person skilled in the art can determine appropriate parameters for measuring the alignment, including any algorithm necessary to achieve the maximum alignment over the entire length of the arrays being compared.

[0054] The term “recombinant expression” refers to an oligonucleotide or polynucleotide construct containing genetic modifications that, when brought into contact with host cells under conditions sufficient to enable the expression of mRNA, protein, polypeptide, or peptide within host cells, allows host cells to express mRNA, protein, polypeptide, or peptide. The construct contains nucleotide sequences encoding mRNA, protein, polypeptide, or peptide. include.

[0055] The present invention will be described in more detail by the following examples. While the present invention will be described below with reference to specific examples, it should be understood that these examples are merely for illustrative purposes and do not limit the scope of the present invention.

[0056] Materials and reagents: P3-AscI (a schematic diagram of its structure is shown in Figure 1) involves inserting the recognition sequence GGCGCGCC, which is the AscI enzyme cleavage site, into the P3 plasmid.

[0057] The PiggyBac transposase coding sequence was optimized for CHO cells by Synbio Technologies (by adding the EcoRI enzyme cleavage site and kozak sequence to the 5' flanking end and HindIII to the 3' flanking end), synthesized into the gene, and cloned into the vector PUC57 to obtain PUC57-PiggyBac.

[0058] The 5ITR-cHS4 sequence in the PiggyBac donor vector element was synthesized by Synbio Technologies.

[0059] The 3ITR-cHS4 sequence in the PiggyBac donor vector element was synthesized by Suzhou GeneWith Biotechnology Co., Ltd.

[0060] The 3ITR-R-cHS4 sequence, including the reverse complementary insulator in the PiggyBac donor vector element, was synthesized by Suzhou GeneWith Biotechnology Co., Ltd. and cloned into the PUC57 vector to obtain PUC57-3ITR-R-cHS4.

[0061] The PiggyBac mRNA template DNA sequence is from GenScript Biotech. Synthesized by Corporation.

[0062] The blank expression vector pcDNA3.4 was provided by GenScript Biotech Corporation.

[0063] Example 1: Design of cHS4 insulator-cut array and construction of vector 1. Design and construction of a cut cHS4 insulator The cHS4-650, cHS4-400, and cHS4-250 sequences used in the insulators were derived from literature (e.g., CN102943092A, WO2018083274A1, US20150315611A1). The cHS4-650 sequence (including a 250 bp core region and a 400 bp 3' terminal sequence) was entered into the AnimalTFDB3.0 online prediction website to predict bindable transcription factors. The results showed a total of 3897 TFBSs corresponding to 389 transcription factors. Subsequently, enrichment analysis was performed on the 389 transcription factors using the STRING database, resulting in significant enrichment of a total of 1993 GO terms. PPI analysis was performed on 91 transcription factors corresponding to 85 GO terms related to chromatin structure regulation or insulator function. Next, we selected three transcription factors with high binding affinity and those that bind to the reported core region: CTCF, USF1 / 2, and VEZF1. Based on the binding sequences predicted by AnimalTFDB3.0, we mapped these transcription factors onto the cHS4-650 sequence. We selected DNA regions with little or no transcription factor binding and designed cleavage sequences. By combining single or multiple cleavable regions, we obtained four insulators, with the nucleic acid sequences designated as SEQ ID NO.1 and SEQ ID NO. 2. These were shown in SEQ ID NO.3 and SEQ ID NO.4, and specifically as follows:

[0064] [array] [Table 1]

[0065] Construction of a 2-donor vector The PiggyBac donor vector element contains 5ITR-cHS4 (forward direction) and 3ITR-cHS4 (forward direction), with two forward-direction cHS4 sequences positioned between the 5ITR and 3ITR. The cHS4 sequences are cHS4-650, cHS4-400, cHS4-250, the sequence shown in SEQ ID NO.1, and SEQ ID NO.2 The sequence includes the sequence shown in , the sequence shown in SEQ ID NO.3, or the sequence shown in SEQ ID NO.4. Using the methods of ligation by single-enzyme cleavage of P3-AscI and homologous recombination, 5ITR-cHS4 fragments were ligated to the P3-AscI plasmid to construct plasmid P3-5ITR-cHS4. Next, 3ITR-cHS4 fragments were incorporated into P3-5ITR-cHS4 using homologous recombination to construct the donor vector P3-5ITR-cHS4-3ITR-cHS4. For simplicity of notation, it will be abbreviated as P3-5ITR-3ITR-cHS4 below (structural schematic diagram is shown in Figure 2).

[0066] 3. Construction of the expression vector For the vector P3-5ITR-3ITR-cHS4, the nucleic acid sequences encoding the heavy and light chains of the QL01 monoclonal antibody were linked to the corresponding multi-cloning sites using BstBI / PacI and HindIII / XhoI double enzyme cleavage, respectively, to construct the recombinant expression vector P3-5ITR-3ITR-cHS4-QL01.

[0067] 4. Construction of Helper Vectors The vector PUC57-PiggyBac and the blank vector pcDNA3.4 were double-enzymed with EcoRI / HindIII. Next, the enzymatic cleavage products of the vector PUC57-PiggyBac and the blank vector pcDNA3.4 were processed using NucleoSpin. After purification and recovery using a Gel and PCR Clean-up kit, the DNA was ligated to construct a helper plasmid pcDNA3.4-PiggyBac (structural schematic diagram shown in Figure 3) containing the gene encoding the transposase. Alternatively, in vitro transcription may be used. Specifically, using the DNA sequence encoding the PB transposase as a template, mRNA-PiggyBac encoding the PB transposase was obtained by in vitro transcription, capping, and tailing modification.

[0068] Example 2: Antibody expression by transient transfection of cHS4 insulator-cleaved sequences We used the ExpiCHO expression system (Gibco) as the host cell and performed transient protein expression according to the host cell's recommended method. The transient transfection system was 25 mL, and the amount of plasmid for transfection is shown in Table 1. We replenished the plasmids according to the recommended replenishment scheme for the ExpiCHO expression system kit, cultured them for 6 days, and then transferred them to the ForteBio instrument. Antibody expression levels were detected using a device, and the results are shown in Figure 4. The culture medium was collected after 7 days of incubation. .

[0069] Comparing the protein yields of transient ExpiCHO transfections, the PiggyBac transposon expression system with a 650 bp (i.e., P3-5ITR-3ITR-650-QL01) cHS4 insulator showed higher yields than the PiggyBac transposon expression systems with 400 bp (i.e., P3-5ITR-3ITR-400-QL01) and 250 bp (i.e., P3-5ITR-3ITR-250-QL01) insulators. Six days after transfection, the protein yield of the PiggyBac transposon expression system with the 650 bp cHS4 insulator increased by 23% and 28%, respectively, compared to the PiggyBac transposon expression systems with the 400 bp and 250 bp insulators. The above results indicate that adding a 650 bp cHS4 insulator is most advantageous for improving yield in the PiggyBac transposon expression system. Furthermore, compared to 650 bp cHS4, SEQ The yield of protein in the PiggyBac transposon expression system increased by 13% when the cHS4 insulator of the sequence shown in ID NO.1 was added. Furthermore, the vector P3-5ITR-3ITR-SEQ ID NO.2-QL01 (containing the cHS4 insulator of the sequence shown in SEQ ID NO.2), P3-5ITR-3ITR-SEQ ID The protein yields after transfection of NO.3-QL01 (containing a cHS4 insulator of the sequence shown in SEQ ID NO.3) and P3-5ITR-3ITR-SEQ ID NO.4-QL01 (containing a cHS4 insulator of the sequence shown in SEQ ID NO.4) were equivalent to the protein yield of the PiggyBac transposon expression system with the addition of a cHS4 insulator of the sequence shown in SEQ ID NO.1.

[0070] [Table 2]

[0071] Example 3: Antibody expression by stable transfection of cHS4 insulator-cleaved sequence CHO cells were transfected by electroporation, and the transfection conditions are shown in Table 2. After transfection, the cells were resuspended in EX-CELL® CD CHO Fusion Medium (containing 6 mM L-glutamine) and cultured in an incubator at 37°C and 5% CO2 for 24 hours. Subsequently, the cells were centrifuged at 1000 rpm for 5 minutes, the supernatant was removed, and the cells were resuspended in an appropriate amount of EX-CELL CD CHO Fusion Medium. All cells were then transferred to a 125 mL shaking flask and cultured in a shaker at 37°C, 5% CO2, 80% humidity, and 130 rpm. The cells were counted and subcultured every 1-5 days until the viability of the pool cells recovered, and then 5 × 10⁶ cells were passed through. 5 Yield tests were conducted by inoculating at a cell / mL density. The results of the fed batch on day 14 of stable transfection expression are shown in Figure 5. As can be seen from the results, the sequence was SEQ ID All PiggyBac transposon expression systems containing the insulators shown in Nos. 1-4 can achieve good expression capacity. In particular, the PiggyBac transposon expression systems containing the insulators shown in SEQ Nos. 1, 3, and 4 achieved expression levels equivalent to or higher than those of the PiggyBac transposon expression system containing the 650bp insulator. Here, the expression level of the PiggyBac transposon expression system containing the cleaved sequence shown in SEQ No. 4 increased by 5%, i.e., the yield increased by 134 mg / L. Compared to the expression system containing the 250bp core region insulator, the expression level of the PiggyBac transposon expression system containing the cleaved sequence shown in SEQ No. 4 increased by 28%, i.e., the yield increased by 711 mg / L. Although not bound by any theory, it is thought that the cleaved sequence not only retains the function of optimizing the expression of the 650bp insulator sequence used as a control, but also that the shorter sequence and smaller vector size improved expression efficiency. On the other hand, the PiggyBac transposon expression system containing insulators whose sequences are shown in SEQ No. 1-4 also showed significantly superior expression ability compared to the PiggyBac transposon expression system containing insulators with a 250 bp core region.

[0072] [Table 3]

[0073] Example 4: Effect of insulator orientation on antibody expression in a transposon expression system 1. Construction of donor vector P3-5ITR-3ITR-R-SEQ ID NO.1 Using the vector PUC57-3ITR-R-SEQ ID NO.1 (containing the sequence shown in SEQ ID NO.1 and a sequence reverse-complementary to it) as a template, the target fragment 3ITR-R-SEQ ID NO.1 was amplified by PCR. The P3-5ITR-SEQ ID NO.1 (forward direction) plasmid was single-enzyme cleavage 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 contained one forward cHS4 and one reverse-complementary cHS4 between 5TIR and 3ITR, i.e., P3-5ITR-3ITR-R-SEQ ID NO.1 (a schematic diagram of the structure is shown in Figure 6A).

[0074] 2. Construction of vectors P3-5ITR-3ITR-SEQ ID NO.1-QL02 and P3-5ITR-3ITR-R-SEQ ID NO.1-QL02 The nucleic acid sequences encoding the knob and hole chains of the QL02 biantibody were linked using BstBI / PacI and HindIII / XhoI double enzyme cleavage, respectively, to P3-5ITR-3ITR-SEQ ID NO.1 (structural schematic diagram is shown in Figure 6B) and P3-5ITR-3ITR-R-SEQ ID NO.1, respectively. The recombinant expression vectors P3-5ITR-3ITR-SEQ ID NO.1-QL02 and P3-5ITR-3ITR-R-SEQ ID NO.1-QL02 were constructed by linking them to the cloning site.

[0075] 3. Expression of antibody proteins We used the ExpiCHO expression system (Gibco) as the host cell and performed transient protein expression according to the host cell's recommended method. The transient transfection system was 25 mL, and the amount of transfection plasmid was as shown in Table 3. After replenishing according to the recommended replenishing scheme for the ExpiCHO expression system kit and culturing for 7 days, antibody expression levels were detected using a ForteBio instrument, and the results are shown in Table 4.

[0076] [Table 4]

[0077] [Table 5]

[0078] In the PiggyBac transposon expression system, when comparing the expression levels of a system containing 3ITR + reverse complementary insulator (vector P3-5ITR-3ITR-R-cHS4) and a system containing 3ITR + forward insulator (vector P3-5ITR-3ITR-cHS4), the vector containing 3ITR + reverse complementary insulator showed an 11.3% improvement compared to the vector containing 3ITR + forward insulator, indicating a more significant improvement in yield with the combination of vector 3ITR + reverse complementary insulator.

[0079] Example 5: Use of stable transfection antibody expression of cHS4 insulator-cleaved sequences in the PiggyBac transposon system 1. Construction of the donor vector P3-5ITR-3ITR The insulator was removed from the vector P3-5ITR-3ITR-cHS4 to construct a P3-5ITR-3ITR vector that does not contain the insulator.

[0080] 2. Construction of the expression vector Using ligation by HindIII / XhoI and BstBI / PacI enzymes, respectively, the nucleic acid sequences encoding the light and heavy chains of antibody QL03 were incorporated into the light and heavy chain multicloning sites of P3-5ITR-3ITR-R-SEQ ID NO.1 and P3-5ITR-3ITR, respectively, to construct the expression vectors P3-5ITR-3ITR-R-SEQ ID NO.1-QL03 and P3-5ITR-3ITR-QL03.

[0081] 3. Expression of antibody proteins by transient transfection We used the ExpiCHO expression system (Gibco) as the host cell and performed transient protein expression according to the host cell's recommended method. The transient transfection system was 50 mL, and the amount of plasmid for transfection was 50 μg donor vector + 5 μg helper vector. Replenishment was performed according to the recommended replenishment scheme of the ExpiCHO expression system kit, and after 7 days of culture, antibody expression levels were detected using a ForteBio instrument, and the results are shown in Table 5.

[0082] Comparing the yields 7 days after transient transfection, the PiggyBac transposon expression system with added cHS4 insulator yielded 247 mg / L, which was 1.5 times higher than the PiggyBac transposon expression system without the insulator. These results demonstrate that adding cHS4 insulator to the PiggyBac transposon expression system can significantly improve the yield of the cell line.

[0083] [Table 6]

[0084] 4. Expression of antibody proteins by stable transfection CHO cells were transfected using electroporation. The plasmid amount for transfection was 50 μg donor vector + 5 μg helper vector, the voltage was 300 V, 950 μF, and the cell volume was 1E7. One electroporation cycle was performed. After transfection, the cells were resuspended in EX-CELL® CD CHO Fusion Medium (containing 6 mM L-glutamine) and cultured in an incubator at 37°C and 5% CO2.

[0085] Cell counts were taken after 24 hours of culture to determine the viable cell density (VCD) and cell viability (Via). Minipools were plated, and stepwise screening and expansion cultures were performed in 96-well plates, 24-well plates, and TPPs. After the cell state was fully recovered (viability >90%), 5 × 10⁶ cells were cultured. 5 Batch experiments were conducted at an inoculation density of cells / mL, and the yield was measured. The yield results for the top 14 minipools are shown in Table 6.

[0086] Comparing the yields of stable transfections in minipool yield tests, the highest yield of the PiggyBac transposon expression system with cHS4 insulator added was 158 mg / L, a 15.3% improvement over the highest yield of the PiggyBac transposon expression system without insulator addition. Furthermore, the average expression level of the top three yielding systems was 150.6 mg / L, a 54.9% improvement over the system without insulator addition. Additionally, the average expression level was 112.3 mg / L, a 102.4% improvement over the system without insulator addition. These results demonstrate that adding cHS4 insulator to the PiggyBac transposon expression system can improve cell line yield.

[0087] [Table 7]

[0088] Example 6: Ensuring cell line stability using the PiggyBac transposon expression system. Three cell lines were randomly selected from stable transfection cell lines constructed using a PiggyBac transposon expression system containing the SEQ ID NO.1 insulator. The cell line construction method was the same as in Example 5, and the stability of the final monoclonal cells of the cell line was evaluated. The evaluation items were as follows: (1) Proliferation stability: After resuscitation, the cell line was continuously cultured for 60 passages, and cell density, viability, diameter, and proliferation rate were evaluated during the passage process. (2) Production stability: Yield tests were performed on cells after 30, 45, and 60 passages, and the yield and quality of the product were evaluated. (3) Genetic stability: cDNA sequencing was performed on cell lines of generation 0 and generation 60.

[0089] 1. Experiment on growth stability a) The candidate clone was revived.

[0090] b) 2 x 10 at intervals of 2-3 days 5 ~5×10 5 Cells were subcultured at a density of cells / mL under the following conditions: temperature 37°C, humidity 80%, rotation speed 130 rpm, and CO2 concentration 5%.

[0091] c) Cell density, viability, and diameter were recorded during the passage process, and the doubling time was calculated.

[0092] 2. Experiment on production stability a) Cells were cryopreserved at PDL15, PDL30, PDL45, PDL60, and PDL70.

[0093] b) After the passage was completed, PDL15, PDL30, PDL45, PDL60, and PDL70 cells were revived, and after the cells recovered, they were inoculated and yield tests were conducted.

[0094] c) The culture medium was replenished as needed during the culture process.

[0095] d) After cell culture was completed, the cell culture supernatant was collected, 1 mL was taken and analyzed by HPLC, and the CV value was calculated from the expression levels. CV = (nPDL expression level - 0PDL expression level) / 0PDL expression level × 100%, n = 30 / 45 / 60.

[0096] 3. Experiments on genetic stability After stable passage, PDL0 and PDL60 cells were revived, total RNA was extracted and reverse transcribed into cDNA, and the target gene was amplified using the corresponding primers before sequencing. The sequencing results were compared with the theoretical sequence to confirm the presence or absence of mutations in the target gene DNA sequence.

[0097] As shown in the proliferation stability data in Figure 7, clones A, B, and C were subculturified at the same density and cultured for the same amount of time. There was no significant difference in cell density, cell viability was maintained at over 95%, and the change in diameter was within 8%. Clones constructed using the PiggyBac transposon expression system were confirmed to have stable proliferation performance and cell morphology.

[0098] The cDNA sequencing results for clones A, B, and C all matched the theoretical sequences, and no mutations were found. The genetic characteristics of the clones constructed using the PiggyBac transposon expression system remained unchanged, and the target gene sequence was confirmed to be genetically stable after continuous cell passage. The yield changes for all three cell lines were within ±10% (see Table 7 for specific data), and the range of change was much smaller than the industry average standard change of ±30%, confirming that the system possesses good genetic stability for cell lines.

[0099] [Table 8]

[0100] The foregoing describes merely better embodiments of the present invention and does not limit it. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention are all protected within the scope of the present invention.

Claims

1. It is an insulator, The nucleic acid sequence of the insulator contains 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 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 includes the nucleic acid sequence shown in SEQ ID NO. 1, SEQ ID NO. 2, SEQ ID NO. 3, or SEQ ID NO.

4.

2. 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 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 insulator according to claim 1, wherein the nucleic acid sequence of the insulator is 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 described in claim 1 or 2.

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

5. The PiggyBac transposon expression system includes a PiggyBac transposon transgenic vector. Preferably, the PiggyBac transposon expression system further comprises mRNA encoding a PiggyBac helper vector or transposase, and the PiggyBac helper vector comprises a nucleic acid sequence encoding a transposase, according to claim 4.

6. The PiggyBac transposon transgenic vector comprises a 5'-terminal repeat sequence 5ITR and a reverse 3'-terminal repeat sequence 3ITR, with the insulator positioned between the 5ITR and the 3ITR. Preferably, between the 5ITR and the 3ITR, there are forward and inversely complementary insulators, or two forward insulators arranged sequentially. Preferably, the eukaryotic cell expression system according to claim 5, wherein multiple cloning sites are arranged between the insulators.

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

8. The eukaryotic cell expression system according to claim 7, wherein the screenable marker gene is a 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 recombinant nucleic acid expression.

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 the preparation of a protein or polypeptide.

12. The use according to claim 11, wherein the protein or polypeptide comprises an antibody, a fusion protein, an antigen, and an enzyme.

13. The 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 construction of a recombinant cell line.

14. The use according to claim 13, wherein the host cell used in the recombinant cell line is a mammalian cell, preferably a CHO cell.

15. Recombinant cell lines, The recombinant cell line comprises the insulator described in claim 1 or 2 or the eukaryotic cell expression system described in any one of claims 3 to 8. Preferably, the host cell used in the recombinant cell line is a mammalian cell, preferably a CHO cell.

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