Fusion protein, regulatory system and method for constructing transgenic cells using the system

Abstract

The application discloses a kind of fusion protein, regulation system and the method for constructing transgenic cell using the system, belong to biotechnology field.The unstable domain (Destabilizing Domain, DD) formed by FKBP12 mutant is fused with IFP2 transposase, and the activity of transposase is accurately regulated using small molecule ligand Shield-1.The application provides the construction method of the fusion protein formed by DD and IFP2, the regulation system and the method for constructing transgenic cell, has the advantages such as high efficiency of exogenous gene integration, good safety, can be accurately regulated, can also be widely applied in transgenic cell, transgenic animal model construction, cell therapy and other fields.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to a fusion protein, a regulatory system containing the fusion protein, and a method for constructing transgenic cells using the regulatory system. Background Technology

[0002] The technology of producing recombinant glycoproteins using mammalian cells has been deeply integrated into the biopharmaceutical industry chain, such as recombinant antibodies, recombinant vaccine antigens, and therapeutic drugs. Establishing stable and high-yield genetically engineered recombinant cell lines and multi-level cell banks is the prerequisite and foundation for the stable and large-scale production of biopharmaceuticals. However, under natural conditions, the efficiency of stable integration of exogenous genes into the host cell genome is low and highly random, making the screening of stable, high-expression monoclonal cell lines a time-consuming and labor-intensive task.

[0003] Transposases are enzymes that catalyze the movement and integration of transposon DNA fragments into the genome, and they have important applications in gene editing and transgenic technologies. IFP2 (also known as PiggyBac) transposase, isolated from the white armyworm, is a type of transposase that can more efficiently insert exogenous gene expression frames (transposons) carrying asymmetric terminal inverted repeat sequences (ITRs) recognized by IFP2 into the TTAA target site of the cell genome. More importantly, it can insert exogenous gene expression frames of a specific length intact into the cell genome, avoiding the random fragmentation of exogenous fragments during natural recombination and thus reducing the uncertainty of protein expression defects after the integration of exogenous genes into the genome.

[0004] The integration of IFP2 transposases is independent of host cell replication, and they can transpose efficiently in both dividing and non-dividing cells. IFP2's "cut and paste" mechanism is a classic example of class II transposons; the transposase recognizes ITRs and TTAA sites, achieving reversible cleavage and integration through transesterification. Therefore, the prolonged presence of IFP2 may lead to continuous transposon movement, resulting in genomic instability and the loss of exogenous genes. Furthermore, the replication of the genome during cell proliferation and the asymmetric distribution in daughter cells further complicate genetic diversity, which is detrimental to the selection and maintenance of genetically stable and homogeneous cell lines for bioproduct production. Currently, there is a lack of effective methods for precisely regulating IFP2 transposase activity.

[0005] Unstable domain (DD) technology is a tool for regulating protein stability using small molecule ligands. FKBP12 is a 12 kDa FK506-binding protein. DD12 formed by FKBP12 mutants (such as F36V and L106P) exposes a ubiquitination site degradation signal in the absence of a ligand, leading to rapid degradation by the ubiquitin-proteasome system (UPS). However, upon binding to a high-affinity ligand (such as Shield-1), the DD12 ubiquitination site is hidden, DD12 regains its stable conformation, is preserved, and continues to function.

[0006] Because Shield-1 is a specific, cell-penetrating, high-affinity DD12 ligand, it reverses the instability of DD12 by binding to it, thus allowing the protein fused with DD12 to remain stable without being degraded by the proteasome. The amount of DD12 protein can be modulated by changing the dosage of Shield-1. This technique has been applied to protein expression regulation, but not yet to the regulation of IFP2 transposase activity. Summary of the Invention

[0007] To overcome the problems existing in the prior art, the present invention proposes a fusion protein, a regulatory system containing the fusion protein, and a method for constructing transgenic cells using the regulatory system.

[0008] To achieve the above objectives, the first aspect of the present invention provides a DD-IFP2 fusion protein, characterized in that the fusion protein comprises an unstable domain formed by an FKBP12 mutant and an IFP2 transposase; the FKBP12 mutant is F36V or L100G, and its amino acid sequence is shown in SEQ ID NO.1; the IFP2 is a wild-type sequence or a mutated highly active HyPeggybac, and the amino acid sequence of the IFP2 transposase is shown in SEQ ID NO.2; the unstable domain is located at the N-terminus of the IFP2 protein and is connected therebetween by a flexible linker peptide, and the amino acid sequence of the DD-IFP2 fusion protein is shown in SEQ ID NO.3.

[0009] A second aspect of the present invention provides a system for regulating the activity of IFP2 transposase, the system comprising:

[0010] (a) A vector expressing the DD-IFP2 fusion protein;

[0011] (b) A donor plasmid containing the gene to be transposonized and the transposase recognition site;

[0012] (c) Small molecule ligands for stabilizing the DD-IFP2 fusion protein.

[0013] A third aspect of the present invention provides a method for constructing transgenic cells, the method comprising:

[0014] (a) The vector expressing the DD-IFP2 fusion protein and the donor plasmid are introduced into host cells, with the ratio of the plasmid expressing the fusion protein to the donor plasmid being 10:1 to 1:100;

[0015] (b) Add small molecule ligands to the cell culture medium to stabilize the DD-IFP2 fusion protein and enable it to exert transposase activity. The concentration of the small molecule ligands shall not be less than 100 nM.

[0016] (c) After transposition is complete, remove the small molecule ligand to degrade the DD-IFP2 fusion protein and terminate the transposase activity.

[0017] Furthermore, the small molecule ligand is Shield-1 or a derivative thereof.

[0018] Furthermore, the host cells include mammalian cells, stem cells, and / or immune cells.

[0019] The fourth aspect of the present invention provides the application of the fusion protein of the first aspect, the system of the second aspect, and / or the method of the third aspect in transgenic cells, transgenic animal model construction, and / or cell therapy.

[0020] Through the above technical solution, the present invention can achieve at least the following beneficial effects:

[0021] 1. Precisely regulate IFP2 transposase activity

[0022] While the "cutting and pasting" mechanism of IFP2 transposases is unique, the lack of effective regulatory mechanisms means that their prolonged presence can lead to genomic instability and off-target effects. The unstable domain technology, through the binding of the small molecule ligand Shield-1 to DD12, enables precise regulation of DD-IFP2 transposase activity. With the addition of Shield-1 ligand, the fusion protein is stable, allowing the transposase to function. In the absence of Shield-1 ligand, the DD-IFP2 transposase is rapidly degraded, inhibiting its activity and thus avoiding adverse consequences caused by the continuous action of the transposase.

[0023] 2. Improve the efficiency and accuracy of exogenous gene integration at the appropriate time.

[0024] The low efficiency and high randomness of stable integration of exogenous genes into the host cell genome is a major obstacle to the large-scale production of biopharmaceuticals. While IFP2 transposases can insert exogenous gene expression cassettes into the genome, their lack of activity regulation hinders the full realization of their high transposition efficiency. Under the regulation of the unstable domain technology, IFP2 transposases can be activated at the appropriate time, enabling them to more efficiently and precisely insert exogenous gene expression cassettes into the cell genome, preserving the complete coding sequence of the exogenous gene, reducing the uncertainty of protein expression, and significantly improving the efficiency and accuracy of complete exogenous gene integration.

[0025] 3. Accelerate the screening of genetically stable monoclonal recombinant cell lines

[0026] Screening for genetically stable and homogeneous production cell lines is crucial for ensuring the quality of biopharmaceuticals. Plasmids carrying transposases and the transposase proteins they express require a period of time to be completely degraded and metabolized within cells. For example, the peak expression of IFP2 transposase activity after transient transfection of plasmids is 24-48 hours, but transposase activity can persist for more than a week. As the genome of cells integrating foreign genes replicates with cell proliferation, transposases can continue to move transposons, leading to continuous changes in the cellular genetic background. It is necessary to wait for transposase activity to subside before the selection and expansion of stable monoclonal cells can proceed. The DD-IFP2 system, however, can switch transposase activity "on" and "off" within hours, shortening the period of genomic instability and enabling faster selection of genetically stable monoclonal cell lines, thus reducing development time.

[0027] 4. Differentiated regulation of exogenous gene integration efficiency

[0028] Based on the differences in stability, endoplasmic reticulum stress, and cytotoxicity of exogenous proteins, the concentration and duration of Shield-1 can be flexibly adjusted to regulate the transposase activity of DD-IFP2. For exogenous proteins with low cytotoxicity, a relatively aggressive regulatory strategy can be adopted, such as moderately increasing the probability and copy number of exogenous gene integration at higher concentrations of Shield-1 ligand to improve protein yield. For exogenous proteins with high cytotoxicity, the concentration of Shield-1 ligand can be carefully adjusted, using a low-concentration, gradient-increment approach to gradually increase the integration efficiency and expression level of exogenous genes, while monitoring cell status in real time. Therefore, by changing the dosage and duration of Shield-1, the goal of differentially regulating the integration efficiency of exogenous genes with different cytotoxicities can be achieved. Attached Figure Description

[0029] Figure 1 This is the structural formula of the small molecule ligand Shield-1 in this invention;

[0030] Figure 2 This is a schematic diagram of the DD-IFP2 fusion protein in this invention;

[0031] Figure 3 This is the pCW1430 plasmid map in this invention;

[0032] Figure 4 This is a diagram showing the expression of DLH-IFP2 protein at different shield-1 concentrations in this invention;

[0033] Figure 5 This is the pPIG-nCoV S1 plasmid map from the present invention. Detailed Implementation

[0034] Unless otherwise stated, all materials and reagents used in this invention are commercially available.

[0035] The amino acid sequences involved in this invention are shown in Table 1 below:

[0036] Table 1 Amino acid sequence listing

[0037] sequence name Serial Number sequence DD12 amino acid sequence SEQ ID NO.1 GVQVETISPGDGRTFPKRGQTCVVHYTGMLEDGKKVDSSRDRNKPFKFMLGKQEVIRGWEEGVAQMSVGQRAKLTISPDYAYGATGHPGIIPPHATLVFDVELLKPE IFP2 transposase amino acid sequence SEQ ID NO.2 MGSSLDDEHILSALLQSDDELVGEDSDSEISDHVSEDDVQSDTEEAFIDEVHEVQPTSSGSEILDEQNVIEQPGSSLASNRILTLPQRTIRGKNKHCWSTSKSTRRSRVSALNIVRSQRGPTMCRNIYDPLLCFKLFFTDEIISEIV KWTNAEISLKRRESMTGATFRDTNEDEIYAFFGILVMTAVRKDNHMSTDDLFDRSLSMVYVSVMSRDRFDFLIRCLRMDDKSIRPTLRENDVFTPVRKIWDLFIHQCIQNYTPGAHLTIDEQLLGFRGRCPFRMYIPNKPSKYGIKILM MCDSGTKYMINGMPYLGRGTQTNGVPLGEYYVKELSKPVHGSCRNITCDNWFTSIPLAKNLLQEPYKLTIVGTVRSNKREIPEVLKNSRSRPVGTSMFCFDGPLTLVSYKPKPAKMVYLLSSCDEDASINESTGKPQMVMYYNQTKGG VDTLDQMCSVMTCSRKTNRWPMALLYGMINIACINSFIIYSHNVSSKGEKVQSRKKFMRNLYMSLTSSFMRKRLEAPTLKRYLRDNISNILPNEVPGTSDDSTEEPVMKKRTYCTYCPSKIRRKANASCKKCKKVICREHNIDMCQSCF Amino acid sequence of DD-IFP2 fusion protein SEQ ID NO.3 MGVQVETISPGDGRTFPKRGQTCVVHYTGMLEDGKKVDSSRDRNKPFKFMLGKQEVIRGWEEGVAQMSVGQRAKLTISPDYAYGATGHPGIIPPHATLVFDVELLKPEGSGGSGGSGGSGGSGTSMYPYDVPDYAMGSSLDDEHILSALLQSDDELVGEDSDSEISDHVSEDDVQSDTEEAFIDEVHEVQPTSSGSEILDEQNVIEQPGSSLASNRILTLPQRTIRGKNKHCWSTSKSTRRSRVSALNIVRSQRGPTRMCRNIYDPLLCFKLFFTDEIISEIVKWTNAEISLKRRESMTGATFRDTNEDEIYAFFGILVMTAVRKDNHMSTDDLFDRSLSMVYVSVMSRDRFDFLIRCLRMDDKSIRPTLRENDVFTPVRKIWDLFIHQCIQNYTPGAHLTIDEQLLGFRGRCPFRMYIPNKPSKYGIKILMMCDSGTKYMINGMPYLGRGTQTNGVPLGEYYVKELSKPVHGSCRNITCDNWFTSIPLAKNLLQEPYKLTIVGTVRSNKREIPEVLKNSRSRPVGTSMFCFDGPLTLVSYKPKPAKMVYLLSSCDEDASINESTGKPQMVMYYNQTKGGVDTLDQMCSVMTCSRKTNRWPMALLYGMINIACINSFIIYSHNVSSKGEKVQSRKKFMRNLYMSLTSSFMRKRLEAPTLKRYLRDNISNILPNEVPGTSDDSTEEPVMKKRTYCTYCPSKIRRKANASCKKCKKVICREHNIDMCQSCF Flexible Linker Peptide SEQ ID NO.4 GSGGSGGSGGSGGSGTS

[0038] The nucleotide sequences involved in this invention are shown in Table 2 below:

[0039] Table 2 Nucleotide Sequence List

[0040] Primer Name Sequence Number 5´-3´ IFP2-1448F SEQ ID NO.5 TCATCTACAGCCACAACGTG IFP2-1585R SEQ ID NO.6 GCAGGTATCTCTTCAAGGTGG IFP2-1440PR SEQ ID NO.7

[0041] Example 1: Construction of the DD-IFP2 fusion gene

[0042] Materials and Methods:

[0043] Plasmid: pCISMA vector

[0044] Amino acid sequence: The IFP2 transposase protein sequence is shown in SEQ ID NO.2, and the amino acid sequence of the DD12 mutant containing the F36V&L106P mutation is shown in SEQ ID NO.1.

[0045] Build steps:

[0046] 1. The DD12 and IFP2 sequences were linked using the flexible linker GSGGSGGSGGSGGSGSTS. An HA tag was added to the N-terminus of IFP2 for protein expression detection. A schematic diagram of the DD-IFP2 fusion protein structure is attached. FAM-AGCTGGAGGTCAGGGACATATACAGG-BHQ1 As shown;

[0047] 2. Based on the hamster species Cricetulus griseus, the nucleotide sequence codons of the DD-IFP2 protein were optimized and converted into a 2190bp coding nucleic acid sequence containing a stop codon;

[0048] 3. Through gene synthesis, the fusion gene was cloned into the pCISMA eukaryotic expression vector via HindIII and BamHI restriction sites to construct the pCW1430 pCISMA-DIFP2 plasmid. The pCW1430 plasmid map is attached. Figure 2 As shown;

[0049] 4. Construct the donor plasmid pPIG-nCoV S1, which contains the S1 target gene of the SARS-CoV-2 Omeprone virus strain, the glutamine synthase (GS) selection gene, and IFP2 transposon recognition sites (ITRs). The pPIG-nCoV S1 plasmid map is attached. Figure 3 As shown.

[0050] Example 2: Validation of the regulation of DD-IFP2 expression levels

[0051] 1. Transfect CHO-K1 cells with pCISMA-DIFP2 plasmid;

[0052] 2. After culturing the cells in culture medium with different concentrations of Shield-1 (0, 0.0001, 0.001, 0.01, 0.1, 1 μM) for 48 hours, cell lysates were collected. The expression of the DD-IFP2 fusion protein was detected using HA tagging. The expression of DD-IFP2 protein at different Shield-1 concentrations is shown in the attached figure. Figure 5 Figure 4 As shown in the figure, the results indicate that DD-IFP2 protein is highly expressed at a concentration of 1 μM Shield-1, lowly expressed at a concentration of 0.1 μM Shield-1, and almost undetectable at concentrations of 1–0.01 μM Shield-1. The expression level of DD-IFP2 fusion protein is regulated by the concentration of the small molecule ligand Shield-1.

[0053] Example 3: Validation of DD-IFP2 system activity and its application in constructing stable transgenic cell lines

[0054] Experimental methods:

[0055] 1. 10 μg pCISMA-DIFP2 and 50 μg pPIG-nCoV S1 were electroporated at 1x10⁻¹⁰ mm². 7 CHO-K1 suspension cells;

[0056] 2. After transfection, the cells were divided into three portions and placed into three 250ml suspension cell culture flasks (Greiner). Each flask contained 12ml of culture medium. No shield-1 was added to the first flask. 0.1μM shield-1 was added to the second flask 24 hours after transfection. 0.1μM shield-1 was added directly to the third flask after transfection. All flasks were cultured in a carbon dioxide constant temperature incubator for 48 hours.

[0057] 3. After culturing for 48 hours, centrifuge each of the three flasks of cells at 300g for 10 minutes, discard the culture medium, and remove Shield-1.

[0058] 4. Add GS selective medium containing 25 μM MSX and adjust the cell concentration to 150,000 viable cells / ml;

[0059] 5. Add 100 μL of transfected cells to each well of a 96-well cell culture plate and incubate statically in a CO2 incubator for two weeks to observe the cell clone formation efficiency.

[0060] 6. The expression of S1 protein in the cell supernatant was detected using an ELISA kit.

[0061] result:

[0062] The first flask of cells, without the addition of shield-1, yielded 28 colony-forming units (CFU) after selection and culture. The second flask of cells, treated with 0.1 μM shield-1 for 24 hours, yielded 213 CFU after plating. The third flask of cells, treated with 0.1 μM shield-1 for 48 hours, yielded 432 CFU. This demonstrates that DD-IFP2 possesses transposase activity, and that the shield-1 treatment time can further regulate the efficiency of stable cell colony formation by controlling transposase activity.

[0063] After testing, less than one-third of the cell clones without Shield-1 stably expressed the SARS-CoV-2 S1 protein, while most of the cell clones treated with Shield-1 stably expressed the SARS-CoV-2 S1 protein, and cell lines expressing the SARS-CoV-2 S1 protein at higher levels could be screened from them.

[0064] Genome sequencing confirmed that most of the SARS-CoV-2 S1 protein gene after Shield-1 treatment was integrated into the host genome TTAA sequence via ITRs.

[0065] Real-time PCR detection (probe sequences are shown in Table 2) showed no residual DD-IFP2 gene in the cloned cells.

[0066] After long-term culture of cell clones (>60 generations), the expression and genetic stability of the SARS-CoV-2 S1 protein are stable.

[0067] Finally, it should be noted that the above preferred embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit it. Although the present invention has been described in detail through the above preferred embodiments, those skilled in the art should understand that various changes can be made to it in form and detail without departing from the scope defined by the claims of the present invention.

Claims

1. A DD-IFP2 fusion protein, characterized in that, The fusion protein comprises an unstable domain DD12 formed by FKBP12 mutant and IFP2 transposase; the amino acid sequence of the FKBP12 mutant is shown as SEQ ID NO. 1; the amino acid sequence of the IFP2 transposase is shown as SEQ ID NO. 2; the unstable domain is located at the N-terminus of the IFP2 protein, and is connected by a flexible connecting peptide, the amino acid sequence of the DD-IFP2 fusion protein is shown as SEQ ID NO. 3, and the amino acid sequence of the flexible connecting peptide is shown as SEQ ID NO.

4.

2. A system for modulating IFP2 transposase activity, comprising, The system comprises: (a) a vector expressing the DD-IFP2 fusion protein of claim 1; (b) a donor plasmid comprising a gene to be transposed and a transposase recognition site; (c) a small molecule ligand for stabilizing the DD-IFP2 fusion protein.

3. A method of constructing a transgenic cell, comprising, The method comprises: (a) introducing the vector expressing the DD-IFP2 fusion protein of claim 1 and the donor plasmid into cells; (b) adding a small molecule ligand in the cell culture medium to keep the DD-IFP2 fusion protein stable and exert transposase activity, and the concentration of the small molecule ligand is not less than 100 nM; (c) after the transposition is completed, removing the small molecule ligand, degrading the DD-IFP2 fusion protein, and terminating the transposase activity.

4. The system of claim 2 or the method of claim 3, wherein: The small molecule ligand is Shield-1.

5. The method of claim 3, wherein: The cells are mammalian cells.

6. Use of the DD-IFP2 fusion protein of claim 1, the system for regulating the activity of IFP2 transposase of claim 2, and / or the method of claim 3 in constructing transgenic cells and / or transgenic animal models.

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