Antibiotic resistance gene-free plasmid-containing production strain and use thereof

Abstract

Provided is an antibiotic resistance gene-free plasmid-containing production strain. The production strain is a gene-edited strain of the PIR strain, designated as PIR1-WN::0636 or PIR1-PR::0636. The production strain contains a nucleotide sequence encoding a toxin protein and an antibiotic resistance gene-free plasmid, wherein the antibiotic resistance-free plasmid contains a nucleotide sequence encoding an antitoxin protein; preferably, the replicon DNA element for the antibiotic resistance gene-free plasmid is R6K-γ. The toxin protein gene of the provided production strain can be stably maintained during strain passage, and has lethality upon induction, which can be used for plasmid screening. When the provided antibiotic resistance gene-free plasmid is transformed into the PIR1-WN::0636 strain, the positive rate reaches 80% or higher. Moreover, the antibiotic resistance gene-free plasmid can enable the stable production of plasmids with a high supercoiled proportion.

Description

Antibiotic-free plasmid production strain and application thereof

[0001] The present application claims priority to the Chinese application with the application number 202410952401.0, the title of which is "Antibiotic-free plasmid production strain and application thereof", filed on July 16, 2024, the content of which is incorporated herein by reference in its entirety. TECHNICAL FIELD

[0002] The present application belongs to the field of biological engineering, and relates to an antibiotic-free plasmid production strain and application thereof. BACKGROUND

[0003] Toxin-antitoxin (TA) systems were first discovered in 1983 on low-copy plasmids, which are important for increasing the stability and spread of plasmids in the population, and were once considered to be plasmid addiction systems. TA systems are widely present in the chromosomes and plasmids of prokaryotes and archaea. This system consists of two co-expressed genes, which encode stable toxin proteins and easily degradable antitoxins, respectively. The toxin usually exerts a toxic effect to inhibit bacterial growth, while the antitoxin can neutralize the toxicity. Some TA systems have been isolated and modified into killing systems, which have become a method for plasmid selection instead of antibiotics. The principle is that when the plasmid is lost in the progeny cells, the half-life of the antitoxin molecule is short, and the free toxin protein is released to exert molecular toxicity, which finally leads to the death of the progeny cells with plasmid loss, thereby ensuring the stability of the plasmid in the population.

[0004] The prior patent CN202310072956.1 provides an antibiotic-free microplasmid produced by using a toxin-antitoxin system, and the plasmid backbone is small. It is proved that it can be stably replicated and efficiently produced in GT115 strain. Different strains have different stability, efficiency of producing antibiotic-free plasmid, and supercoiled structure of antibiotic-free plasmid. During the production of antibiotic-free plasmid, the toxin protein gene needs to be inserted into the genome of the strain, and in the case of loss of antibiotic-free plasmid, strong lethality is required. The R6K replicon contained in the antibiotic-free plasmid also requires the presence of Pai protein in the strain. Based on the above factors, the present disclosure provides a new antibiotic-free plasmid production strain for efficient and high-quality production of antibiotic-free plasmid, to expand the production method of antibiotic-free plasmid and meet different demands for antibiotic-free plasmid. SUMMARY

[0005] The present disclosure provides a production strain without an anti-plasmid, the production strain being a genome-reconstructed strain PIR1-WN::0636 or PIR1-PR::0636 with PIR1 as the original strain, the production strain containing a nucleotide sequence encoding a toxin protein and an anti-plasmid without an antibiotic resistance gene.

[0006] The amino acid sequence of the toxin protein is shown as SEQ ID NO: 4, and the amino acid sequence of the anti-toxin protein contains an amino acid sequence shown as SEQ ID NO: 1, or an amino acid sequence with one or more mutations of E24D, I36V, V43I compared with SEQ ID NO: 1; or

[0007] The amino acid sequence of the toxin protein is shown as SEQ ID NO: 13, and the amino acid sequence of the anti-toxin protein contains an amino acid sequence shown as SEQ ID NO: 9, or an amino acid sequence with one or more mutations of T6I, T43A, K47E, A50S, E51D, G52A, N54K compared with SEQ ID NO: 9. The anti-plasmid does not contain an antibiotic resistance gene.

[0008] In some embodiments, the amino acid sequence of the toxin protein is shown as SEQ ID NO: 4, and the amino acid sequence of the anti-toxin protein is shown as SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3; or the amino acid sequence of the toxin protein is shown as SEQ ID NO: 13, and the amino acid sequence of the anti-toxin protein is shown as SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, or SEQ ID NO: 12.

[0009] In some embodiments, the nucleotide sequence encoding the toxin protein is integrated in the non-coding region between yjiP and yjiR or the non-coding region between thrW and ykfN.

[0010] In some embodiments, the nucleotide sequence encoding the toxin protein is integrated in the non-coding region between yjiP and yjiR or the non-coding region between thrW and ykfN.

[0011] In some embodiments, the production strain contains a gene expression cassette for inducible expression of the toxin protein, the gene expression cassette for inducible expression of the toxin protein containing an operon or a promoter for inducible expression of the toxin protein, preferably the operon or the promoter is selected from a Lac lactose operon, an L-arabinose-inducible pBAD promoter, an L-rhamnose-inducible rhapBAD promoter, or a lambda PL / PR-clts857 temperature-sensitive promoter.

[0012] In some embodiments, the nucleotide sequence encoding the toxin protein is set forth in SEQ ID NO: 6 or 15.

[0013] In some embodiments, the antibiotic-free plasmid is an antibiotic-free miniplasmid comprising a nucleotide sequence encoding an anti-toxin protein and a replicon, the replicon having a length of < 800 bp, preferably < 600 bp or < 400 bp.

[0014] In some embodiments, the replicon is selected from the group consisting of ColEl, ColE2, pMB1, pSC101, RSF, R6K, pUC57, RK2 and p15A; preferably R6K-gamma or pUC57.

[0015] In some embodiments, the plasmid backbone of the antibiotic-free miniplasmid has a length of < 1000 bp, preferably < 900 bp, < 800 bp or < 700 bp.

[0016] In a preferred embodiment, when the replicon in the plasmid backbone of the antibiotic-free miniplasmid is pUC57, ColEl, pMB1, the plasmid backbone has a length of < 1000 bp.

[0017] In a preferred embodiment, when the replicon in the plasmid backbone of the antibiotic-free miniplasmid is R6K-gamma replicon, the plasmid backbone has a length of < 700 bp.

[0018] In some embodiments, the nucleotide sequence of the replicon does not contain CpG motifs.

[0019] In some embodiments, the nucleotide sequence of the replicon is set forth in SEQ ID NO: 18 or 22, preferably in SEQ ID NO: 22.

[0020] In some embodiments, the nucleotide sequence encoding the anti-toxin protein is set forth in SEQ ID NO: 5 or SEQ ID NO: 14.

[0021] In some embodiments, the nucleotide sequence encoding the anti-toxin protein does not contain CpG motifs, preferably the nucleotide sequence encoding the anti-toxin protein is set forth in SEQ ID NO: 7, 16 or 23.

[0022] In some embodiments, the antibiotic-free miniplasmid comprises a structured DNA sequence selected from the group consisting of: polyA repeats, SV40 origin of replication, viral LTRs, lentiviral LTRs, retroviral LTRs, transposon IR / DR repeat sequences, AAV ITRs, transposon ITRs, CMV enhancer, and SV40 enhancer.

[0023] In some embodiments, the anti-microplasmid-free plasmid is selected from the group consisting of Sleeping Beauty transposon vector, PiggyBac transposon vector, ZB transposon vector, PS transposon vector, Tol2 transposon vector, and polyA-containing mRNA vector.

[0024] In some embodiments, the anti-microplasmid-free plasmid comprises a gene expression cassette of an exogenous gene, preferably, the exogenous gene encodes a chimeric antigen receptor and / or encodes an antibody.

[0025] In some embodiments, the anti-microplasmid-free plasmid further comprises a gene expression cassette encoding a recombinant peptide, polypeptide, or protein of interest.

[0026] In some embodiments, the gene expression cassette of the anti-microplasmid-free plasmid is used for preparing a gene therapy drug, preparing a cell therapy drug, preparing a DNA vaccine, virus production, or antibody production.

[0027] The present disclosure also provides a method for preparing a production strain of the anti-microplasmid-free plasmid of any one of the embodiments herein, comprising: integrating a nucleotide sequence encoding a toxin protein into the genome of a PIR1 strain; and then transforming the anti-microplasmid-free plasmid into the PIR1 strain.

[0028] In some embodiments, the nucleotide sequence encoding the toxin protein is homologously recombined into the genome of the PIR1 strain by a RED recombination system.

[0029] In some embodiments, the nucleotide sequence encoding the toxin protein is integrated in the non-coding region between yjiP and yjiR or the non-coding region between thrW and ykfN.

[0030] The present disclosure also provides an anti-microplasmid-free plasmid with a high proportion of supercoils, comprising a nucleotide sequence encoding an anti-toxin protein and a replicon, the amino acid sequence of the anti-toxin protein comprising: the amino acid sequence set forth in SEQ ID NO: 1, or an amino acid sequence having one or more mutations of E24D, I36V, V43I compared with SEQ ID NO: 1, the nucleotide sequence of the replicon being set forth in SEQ ID NO: 22;

[0031] In some embodiments, the amino acid sequence of the anti-toxin protein is set forth in any one of SEQ ID NOs: 1-3, more preferably, the amino acid sequence of the anti-toxin protein is set forth in SEQ ID NO: 1.

[0032] In some embodiments, the plasmid backbone of the anti-microplasmid comprises a nucleotide sequence encoding an antitoxin protein, a replicon and a promoter of the antitoxin protein gene; preferably, the nucleotide sequence encoding the antitoxin protein is shown as SEQ ID NO: 23; more preferably, the nucleotide sequence of the plasmid backbone is shown as SEQ ID NO: 24.

[0033] The present disclosure also provides a method for producing an anti-microplasmid, comprising: culturing the anti-microplasmid production strain of any of the embodiments herein, inducing expression of the toxin protein and expression of the antitoxin protein, obtaining the plasmid from the cultured production strain.

[0034] In a preferred embodiment, the expression of the gene encoding the toxin protein is induced by adding arabinose during the culturing.

[0035] The present disclosure also provides the use of the anti-microplasmid production strain of any of the embodiments herein in the preparation of a gene therapy drug, the preparation of a cell therapy drug, the preparation of a DNA vaccine, viral production or antibody production.

[0036] The present disclosure also provides a method for producing a recombinant peptide, polypeptide or protein of interest by the anti-microplasmid production strain of any of the embodiments herein, the plasmid further comprising a gene expression cassette encoding the recombinant peptide, polypeptide or protein of interest, the method comprising culturing the system and recovering the recombinant peptide, polypeptide or protein of interest.

[0037] The present disclosure provides an anti-microplasmid production strain produced by using a toxin-antitoxin system, a nucleotide sequence encoding a toxin protein is integrated into the genome of a PIR1 strain, the toxin protein gene is capable of stable passage and has lethality after induction and can be used for plasmid screening; the anti-microplasmid of the present disclosure is transformed into a PIR1 strain, the positive rate is more than 80%; and stable production of plasmids with high supercoiling can be achieved. BRIEF DESCRIPTION OF DRAWINGS

[0038] Figure 1 is a schematic diagram of the integration fragment Donor-FRT-Gam-FRT-0636 (yyPR).

[0039] Figure 2 is a schematic diagram of the integration fragment Donor-FRT-Gam-FRT-0636 (tyWN).

[0040] Figure 3 is the electrophoresis result of extracted plasmid in E. coli PIR1 electrocompetent cells containing plasmid pKD46.

[0041] Figure 4 is a schematic diagram of the detection of the integration of the toxin protein at the PIR1-PR, WN two sites.

[0042] Figure 5 is the result of plate streaking of PIR1-PR::0636, PIR1-WN::0636 clone removal tool plasmid.

[0043] Figure 6 is the result of plate colony of PIR1-PR::0636, PIR1-WN::0636 toxicity verification induced by arabinose.

[0044] Figure 7 is the result of PCR verification of PIR1-PR::0636, PIR1-WN::0636 toxin gene mutation.

[0045] Figure 8 is the plasmid map of pTini-JL(CIT)-1194NLA.

[0046] Figure 9 is the plate colony of PIR1-PR::0636, PIR1-WN::0636, GT115::0636 (left, middle, right) after introducing pTini-JL(CIT)-1194NLA.

[0047] Figure 10 is the positive rate of PIR1-PR::0636, PIR1-WN::0636, GT115::0636 transformation of pTini-JL(CIT)-1194NLA after introducing pTini-JL(CIT)-1194NLA.

[0048] Figure 11 is the result of PIR1-PR::0636, PIR1-WN::0636, GT115-0636 positive clone toxin gene test after introducing pTini-JL(CIT)-1194NLA.

[0049] Figure 12 is the bar chart of PIR1-PR::0636, PIR1-WN::0636 passage bacterial liquid OD 600 .

[0050] Figure 13 is the plate colony result of PIR1-PR::0636, PIR1-WN::0636 P2 generation, P4 generation toxicity verification in medium with different concentrations of glucose.

[0051] Figure 14 is the plate colony result of PIR1-PR::0636, PIR1-WN::0636 P4 generation, P8 generation toxicity verification in medium with different concentrations of glucose.

[0052] Figure 15 is the original plasmid backbone sequence map used by pTini-JL(CIT)-1194NLA.

[0053] Figure 16 is the sequence map of the improved plasmid backbone.

[0054] Fig. 17-19 are the results of supercoiled plasmid ratio electrophoresis of three different pTini plasmids pTini-PB(CES)-dCGEGFP, pTini-JL(CES)-dCGEGFP, pTini-PB(DTS)-1444-8E before and after improvement of the plasmid backbone.

[0055] Fig. 20 is the results of supercoiled plasmid ratio capillary electrophoresis of JL-DE-wt-CACC before and after improvement of the plasmid backbone. Fig. 21 is the results of pTini-JL(DTS)-E1 V2 plasmid transformation of PIR1-WN::0636 competent strain, first experiment, heat shock, 2h incubation, plated clones.

[0056] Fig. 22 is the results of pTini-JL(DTS)-E1 V2 plasmid transformation of PIR1-WN::0636 competent strain, second experiment, heat shock, 3h incubation, plated clones.

[0057] Fig. 23 is the results of JL-DE-WT-CACC Qa6 V3 fermentation intermediate sample detection.

[0058] Fig. 24 is the results of pTini-JL(DTS)-2396(R73)v3-PIR1, pTini-JL(DTS)-d1EGFP v3-PIR1 fermentation intermediate sample detection.

[0059] Fig. 25 is the plasmid map of pTini-JL(CES)-dCGEGFP using the improved plasmid backbone as shown in Fig. 25. DETAILED DESCRIPTION

[0060] The term "toxin-antitoxin" (TA) refers to a group of toxin proteins and antitoxin proteins, wherein the toxin proteins are toxic to the host cell, and the antitoxin proteins are capable of neutralizing the toxicity of the toxin proteins to the host cell. When there is an antitoxin gene, either chromosomally or plasmidically encoded, in the cell, the antitoxin proteins are continuously expressed, neutralizing the toxin proteins, and maintaining the survival of the cell. If the plasmid with the antitoxin gene is lost from the cell, the synthesized toxin proteins remain longer than the antitoxin proteins, and are capable of killing or inhibiting the growth of the cell. In this document, the terms "toxin protein" and "toxin" are used interchangeably to refer to the protein in the toxin-antitoxin system that is capable of killing or inhibiting the growth of the cell; and the terms "antitoxin protein" and "antitoxin" are used interchangeably to refer to the protein in the toxin-antitoxin system that forms a complex with the toxin protein, and neutralizes the toxicity of the toxin protein.

[0061] The term "codon optimization" refers to the technique of improving and maximizing protein expression in living organisms by converting / replacing the DNA sequence of nucleotides of one species with the nucleotide sequence of another species to increase the efficiency of translation of the gene of interest. Codon optimization includes replacing the wild-type DNA sequence and rare codons with more highly expressed species sequences and frequently occurring codons, without changing the protein.

[0062] CpG motifs refer to immunostimulatory CpG oligonucleotides, i.e. short single-stranded synthetic nucleic acid molecules, which comprise a cytosine triphosphate deoxynucleotide ("C") and a guanine triphosphate deoxynucleotide ("G"). "p" refers to a phosphodiester or phosphorothioate linkage between consecutive nucleotides. Unmethylated CpG motifs are considered as pathogen-associated molecular patterns (PAMPs) because they are abundant in microbial genomes but rare in vertebrate genomes. The present application removes CpG motifs from the nucleotide sequence encoding the antiviral protein by codon optimization.

[0063] The term "antibiotic-free plasmid" refers to a plasmid that does not contain an antibiotic resistance gene. The term "miniplasmid" or "mini plasmid" refers to small (less than 4 kb) circular plasmid derivatives that have been isolated from all prokaryotic vector parts (i.e. they do not contain bacterial DNA sequences) and are used as transgene vectors for genetic modification of mammalian cells. Miniplasmids and their use are described in Minicircle and Miniplasmid DNA Vectors: The Future of Nonviral and Viral Gene Transfer, Dr. Martin Schleef, May 2013, Wiley-Blackwell, page 258. The term "antibiotic-free miniplasmid" refers to a miniplasmid that does not contain an antibiotic resistance gene.

[0064] The term "plasmid backbone" refers to a DNA sequence comprising at least the elements necessary for the autonomous replication of a plasmid within a bacterial host. The plasmid backbone comprises at least a replicon for the replication of a copy of the plasmid. The plasmid backbone generally also comprises antagonistic elements that maintain the plasmid stable, such as antibiotic resistance genes, antitoxin protein genes, etc., which are expressed simultaneously, enabling the plasmid to be stable. For example, in the present application, the plasmid is stabilized by the expression of an antitoxin protein, thus also comprising the promoter of the antitoxin protein gene. In a particular embodiment, the plasmid backbone comprises the promoter EM2K, the antitoxin protein gene and the replicon.

[0065] The term "origin" refers to a piece of DNA sequence that enables autonomous replication and maintenance of normal copy number in bacteria or plasmids or vectors, also referred to as "origin of replication" in some literature.

[0066] The present disclosure provides a plasmid-free production strain, which utilizes a toxin-antitoxin system. The strain lacking the plasmid containing the gene encoding the toxin protein will die, and the strain containing the plasmid will survive and multiply in large quantities during the culture process. As the strain is cultured and proliferated, the plasmid is replicated and amplified, and the plasmid is efficiently produced.

[0067] The present disclosure takes the PIR1 strain as the original strain, and through genome modification, a plasmid-free production strain is obtained, which contains a nucleotide sequence encoding a toxin protein and a plasmid-free plasmid containing a nucleotide sequence encoding an antitoxin protein. The toxin protein and the antitoxin protein belong to the same toxin-antitoxin system, such as GF_0636 / GF_0637 or CcdA / CcdB (43009).

[0068] In some embodiments, the amino acid sequence of the toxin protein is as shown in SEQ ID NO: 4, and the amino acid sequence of the antitoxin protein contains: an amino acid sequence as shown in SEQ ID NO: 1, or an amino acid sequence having one or more mutations of E24D, I36V, V43I compared with SEQ ID NO: 1; or the amino acid sequence of the toxin protein is as shown in SEQ ID NO: 13, and the amino acid sequence of the antitoxin protein contains: an amino acid sequence as shown in SEQ ID NO: 9, or an amino acid sequence having one or more mutations of T6I, T43A, K47E, A50S, E51D, G52A, N54K compared with SEQ ID NO: 9. The E24D mutation refers to the 24th amino acid in the amino acid sequence being mutated from E to D, and other mutations are the same.

[0069] SEQ ID NO: 1 is a truncated sequence of the antitoxin protein GF_0637, which is the shortest sequence that can achieve the function of the antitoxin protein. The antitoxin protein GF_0637 is derived from the marine strain Roseivirga spongicola_GF047. The antitoxin protein can also produce natural mutations during the cultivation of the strain. Known natural mutations include E24D, I36V, and V43I. The antitoxin protein after mutation can still achieve the same antitoxin function. Therefore, the antitoxin protein GF_0637 and its truncated sequence can include one or more mutations of E24D, I36V, and V43I. Similarly, SEQ ID NO: 9 is a truncated sequence of the antitoxin protein CcdA (43009). Known natural mutations include T6I, T43A, K47E, A50S, E51D, G52A, and N54K. Therefore, the antitoxin protein and its truncated sequence can include one or more mutations of T6I, T43A, K47E, A50S, E51D, G52A, and N54K. In some embodiments, the amino acid sequence of the antitoxin protein is as set forth in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, or SEQ ID NO: 12.

[0070] In some embodiments, the production strain cells contain a gene expression cassette inducibly expressing a toxin protein, which expresses the toxin protein under inducing conditions. In some embodiments, the gene expression cassette expressing the toxin protein can be inserted into the genome of the production strain cells or located on a plasmid. The gene expression cassette expressing the toxin protein is inserted into the genome of the strain cells, which is stably present and expressed in the host cells, and is more preferred. In some embodiments, the nucleotide sequence encoding the toxin protein is integrated in the non-coding region between yjiP and yjiR or the non-coding region between thrW and ykfN. The gene expression cassette expressing the toxin protein can also be located on other plasmids other than the production antibiotic-free plasmid, which can also contain an antibiotic resistance gene to avoid the loss of the plasmid, and since the other plasmid is not the final product and is not used for gene therapy, it will not cause the misuse of the antibiotic resistance gene. The gene expression cassette inducibly expressing the toxin protein includes an operon or a promoter, which expresses the toxin protein under inducing conditions, and the operon or the promoter is selected from the group consisting of a Lac lactose operon, an L-arabinose-induced pBAD promoter, an L-rhamnose-induced rhapBAD promoter, and a lambda PL / PR-clts857 temperature-sensitive promoter. In some embodiments, depending on the type of the promoter or the operon, the culture conditions are adjusted to induce the expression of the toxin protein, for example, when the L-arabinose-induced pBAD promoter is used, arabinose can be added to the culture to induce the expression of the gene encoding the toxin protein. In some embodiments, the nucleotide sequence encoding the toxin protein is set forth in SEQ ID NO: 6 or 15.

[0071] In the present disclosure, the production of the antibiotic-free plasmid mainly depends on the toxin-antitoxin system, and is not related to the size of the antibiotic-free plasmid, thus being suitable for the production of various antibiotic-free plasmids. In some embodiments, the antibiotic-free plasmid is an antibiotic-free microplasmid, for example, the antibiotic-free microplasmid of any one of the embodiments of CN202310072956.1 (also referred to as "Tai plasmid"), the entire contents of which are incorporated herein by reference. The plasmid backbone of the existing microplasmid is generally less than 4000 bp, and the plasmid generally contains an antibiotic resistance gene with a length of 810 bp, thus the length of the plasmid backbone of the existing microplasmid is generally greater than 1200 bp, and the antibiotic-free microplasmid of the present disclosure uses an antitoxin protein instead of an antibiotic resistance gene, greatly reducing the length of the plasmid backbone. The length of the nucleotide sequence of the antitoxin protein of the present disclosure is ≤400 bp, and the length of the plasmid backbone of the antibiotic-free microplasmid of the present disclosure is ≤1000 bp, for example, ≤900 bp, 800 bp or 700 bp. In some embodiments, the antibiotic-free microplasmid comprises a nucleotide sequence encoding an antitoxin protein and a replicon, and the length of the replicon is ≤800 bp, preferably ≤600 bp or ≤400 bp. The replicon is selected from ColE1, ColE2, pMB1, pSC101, RSF, R6K, pUC57, RK2 and p15A, and the replicon is preferably R6K-γ or pUC57, considering the plasmid copy number, plasmid toxicity and plasmid backbone size. The antibiotic-free microplasmid is amplified and replicated with the culture of the bacterial host cell, and the copy number in the bacterial host cell increases, so that a large amount of antibiotic-free microplasmid can be obtained in a relatively short time. In some embodiments, the length of the plasmid backbone of the antibiotic-free microplasmid is ≤1000 bp, preferably ≤900 bp, ≤800 bp or ≤700 bp. When the replicon in the plasmid backbone of the antibiotic-free microplasmid is pUC57, ColE1 or pMB1, the length of the plasmid backbone is ≤1000 bp. When the replicon in the plasmid backbone of the antibiotic-free microplasmid is an R6K-γ replicon, the length of the plasmid backbone is ≤700 bp.

[0072] The plasmid contains a nucleotide sequence encoding an antitoxin protein, and the CpG motif in the nucleotide sequence encoding the antitoxin protein can be removed by codon optimization when used in mammals, such as humans, to reduce immunogenicity. However, the nucleotide sequence encoding the toxin protein is not on the same plasmid as the nucleotide sequence encoding the antitoxin protein, so the nucleotide sequence encoding the toxin protein can not have the CpG motif removed. In some embodiments, the nucleotide sequence encoding the antitoxin protein is as shown in SEQ ID NO: 5 or SEQ ID NO: 14.

[0073] The replicon and / or the nucleotide sequence encoding the antitoxin protein is optimized to not contain a CpG motif. In some embodiments, the nucleotide sequence encoding the antitoxin protein is as set forth in SEQ ID NO: 7, SEQ ID NO: 16, or SEQ ID NO: 23, preferably as set forth in SEQ ID NO: 23. The nucleotide sequence as set forth in SEQ ID NO: 23 corresponds to the amino acid sequence as set forth in SEQ ID NO: 1, which is a truncated sequence of the antitoxin protein 0637. In some embodiments, the R6K-gamma replicon does not contain a CpG motif, such as that as set forth in SEQ ID NO: 18 or SEQ ID NO: 22, preferably as set forth in SEQ ID NO: 22. The anti-free plasmid containing the replicon as set forth in SEQ ID NO: 22 has a higher proportion of supercoiling.

[0074] In the gene expression cassette of the anti-free mini plasmid expressing the antitoxin protein, a promoter, preferably a bacterial promoter, such as but not limited to EM2K, J23119, Tac, and the like, is contained to allow the antitoxin protein to be continuously expressed in the bacterial host cell. The anti-free mini plasmid of the present disclosure can not contain a gene of interest, and the empty plasmid can be directly obtained using the system for producing the anti-free mini plasmid of the present disclosure; the nucleotide sequence is as set forth in SEQ ID NO: 8 or 17. The anti-free mini plasmid of the present disclosure can contain a gene of interest. After the anti-free mini plasmid is loaded with the gene of interest, due to the smaller plasmid length, the anti-free mini plasmid has greater potential in non-viral vector delivery. The gene of interest can be an antibody gene, a chimeric antigen receptor gene, a gene editing enzyme gene, an antigen gene, a virus gene, and the like. In the examples of the present application, in order to verify the effect of the anti-free mini plasmid, the gene of interest can also be a reporter gene, such as an EGFP reporter gene.

[0075] In some embodiments, the anti-free mini plasmid comprises a structured DNA sequence selected from the group consisting of: a polyA repeat, an SV40 origin of replication, a viral LTR, a lentiviral LTR, a retroviral LTR, a transposon IR / DR repeat sequence, an AAV ITR, a transposon ITR, a CMV enhancer, and an SV40 enhancer. In some embodiments, the anti-free mini plasmid is selected from the group consisting of a Sleeping Beauty transposon vector, a PiggyBac transposon vector, a ZB transposon vector, a BZ transposon vector, a JL transposon vector, a PS transposon vector, a Tol2 transposon vector, and a polyA-containing mRNA vector. Among them, the ZB transposon vector can refer to CN105018523B, the PS transposon vector can refer to CN110257425B, the BZ transposon vector can refer to CN202211150935.9, and the JL transposon vector can refer to CN202310081106.8.

[0076] In some embodiments, the antibiotic-free miniplasmid comprises an exogenous gene, preferably, the exogenous gene encodes a chimeric antigen receptor and / or encodes an antibody. For example, the antibiotic-free miniplasmid can comprise an exogenous gene encoding a chimeric antigen receptor for preparing immune cells of chimeric antigen receptor, such as CAR-T; the antibiotic-free miniplasmid can comprise an exogenous gene encoding an antibody, for example, can comprise a PD-1 antibody gene; the antibiotic-free miniplasmid can also comprise an exogenous gene encoding a chimeric antigen receptor and an exogenous gene encoding an antibody, which can be on the same antibiotic-free miniplasmid, or can be used in combination on different antibiotic-free miniplasmids. The genetic expression frame of the antibiotic-free miniplasmid is used for preparing gene therapy drugs, preparing cell therapy drugs, preparing DNA vaccines, virus production, or antibody production.

[0077] In some embodiments, the antibiotic-free miniplasmid further comprises a genetic expression frame encoding a recombinant peptide, polypeptide, or protein of interest; at this time, during the culture of the production strain, during the amplification of the antibiotic-free miniplasmid, the recombinant peptide, polypeptide, or protein of interest can also be expressed, thereby being used for the production of the recombinant peptide, polypeptide, or protein. Examples of the recombinant peptide, polypeptide, or protein are, but not limited to, enzymes, regulatory proteins, receptors, peptides (such as peptide hormones), cytokines, antibodies, nanobodies, membrane proteins, or transport proteins.

[0078] The present disclosure also provides a method for preparing a production strain of the antibiotic-free plasmid of any of the embodiments herein, comprising: integrating a nucleotide sequence encoding a toxin protein into the genome of a PIR1 strain; and then transforming the antibiotic-free plasmid into the PIR1 strain. In some embodiments, the nucleotide sequence encoding the toxin protein is homologously recombined into the genome of the PIR1 strain by a RED recombination system. One specific implementation of homologous recombination is: introducing a tool plasmid carrying lambda-Red homologous recombination enzymes Exo, Gamma, Beta into the strain, screening positive strains successfully carrying the tool plasmid, then introducing a dsDNA fragment encoding the toxin protein into the strain, verifying the strain successfully integrating the gene, and then removing the tool plasmid and screening the gene, etc. In some embodiments, the nucleotide sequence encoding the toxin protein is integrated in the non-coding region between yjiP and yjiR (yjiP is a pseudogene, and yjiR is presumed to encode a DNA transcription binding regulator) or the non-coding region between thrW and ykfN.

[0079] The present disclosure also provides an anti-antibiotic-free plasmid with a high supercoiling ratio, comprising a nucleotide sequence encoding an anti-toxin protein and a replicon, wherein the amino acid sequence of the anti-toxin protein comprises: an amino acid sequence as set forth in SEQ ID NO: 1, or an amino acid sequence with one or more mutations of E24D, I36V, V43I compared with SEQ ID NO: 1, and the nucleotide sequence of the replicon is as set forth in SEQ ID NO: 22. The anti-antibiotic-free plasmid with the replicon as set forth in SEQ ID NO: 22 has a higher supercoiling ratio compared with the replicon as set forth in SEQ ID NO: 18.

[0080] In some embodiments, the amino acid sequence of the anti-toxin protein is as set forth in any one of SEQ ID NOs: 1-3, more preferably, the amino acid sequence of the anti-toxin protein is as set forth in SEQ ID NO: 1, and 50 amino acids are the shortest sequence maintaining the anti-toxin function. In some embodiments, the nucleotide sequence of the anti-toxin protein is as set forth in SEQ ID NO: 23.

[0081] In some embodiments, the plasmid backbone of the anti-antibiotic-free plasmid comprises a nucleotide sequence encoding an anti-toxin protein, a replicon, and a promoter of the anti-toxin protein; preferably, the plasmid backbone comprises, in order, a promoter of the anti-toxin protein, a nucleotide sequence encoding an anti-toxin protein, and a replicon, as shown in FIG. 16; more preferably, the nucleotide sequence of the plasmid backbone is as set forth in SEQ ID NO: 24. With the improvement of the anti-toxin protein sequence and the replicon sequence, the length of the plasmid backbone of the anti-antibiotic-free plasmid is slightly increased, still less than 700 bp, while the supercoiling ratio is significantly improved. The present disclosure also provides a method for producing an anti-antibiotic-free plasmid, comprising: culturing a production strain of the anti-antibiotic-free plasmid of any one of the embodiments herein, inducing expression of a toxin protein and expression of an anti-toxin protein, and obtaining the plasmid from the cultured production strain.

[0082] In a preferred embodiment, the expression of the gene encoding the toxin protein is induced by adding arabinose during culturing.

[0083] The present disclosure also provides use of the production strain of the anti-antibiotic-free plasmid of any one of the embodiments herein in the preparation of a gene therapy drug, the preparation of a cell therapy drug, the preparation of a DNA vaccine, virus production, or antibody production.

[0084] In some embodiments, the use thereof is selected from any one of (1)-(6) as follows:

[0085] (1) use in the preparation of a drug or reagent for integrating an expression frame of a gene of interest into a genome of a host cell;

[0086] (2) in the preparation of tools for integrating the gene expression frame of interest into the genome of a host cell;

[0087] (3) in the preparation of transgenic animals, transgenic cells;

[0088] (4) in the preparation of drugs or preparations for genomic research, gene therapy, cell therapy, or stem cell induction and post-induction differentiation;

[0089] (5) in the preparation of tools for genomic research, gene therapy, cell therapy, or stem cell induction and post-induction differentiation;

[0090] (6) in the preparation of kits, engineered immune cells, or pharmaceutical compositions.

[0091] The present disclosure also provides a method of producing a recombinant peptide, polypeptide, or protein of interest from a production strain of any of the embodiments herein that does not contain an anti-plasmid that also contains a gene expression frame encoding the recombinant peptide, polypeptide, or protein of interest, the method comprising culturing the system and recovering the recombinant peptide, polypeptide, or protein of interest. In some embodiments, the production strain cell also contains a nucleic acid sequence encoding a protein that induces expression of the peptide, polypeptide, or protein of interest, which is preferably placed in the gene expression frame encoding the recombinant peptide, polypeptide, or protein of interest.

[0092] The present application is further illustrated by the following examples, but the present application is not limited to the scope of the examples. The experimental methods in the following examples are not specified, and are selected according to conventional methods and conditions, or according to the instructions of the product.

[0093] Example 1. Insertion of a gene expression frame of a target toxin protein into the PIR1 genome

[0094] (1) Selection of integration site

[0095] The toxin protein integration site should have the characteristics of stable expression after insertion, and the destruction of the original site has no effect on the growth of the bacteria. E. coli One Shot TM PIR1 (Thermo Fisher Scientific) as the parent for integration experiments, the following 2 groups of insertion gene sites were selected, namely yjiP_yjiR (hereinafter referred to as yyPR), thrW_ykfN (hereinafter referred to as tyWN). The above-mentioned sites are non-coding regions between two genes in the genome of E. coli, and the two genes are either pseudogenes or have not been identified with accurate functions, and there is no obvious damage to the endogenous genes of the strain.

[0096] According to the E. coli MG1655 genome sequence, a primer was designed for amplifying and sequencing verification of yyPR, tyWN sites in PIR1 genome. The verified sequence was completely consistent with the E. coli MG1655 genome sequence.

[0097] (2) Construction of integration fragment

[0098] Based on the screening principle of positive clones of the RED integration system, a resistance gene needs to be added to the integration fragment containing the toxin operon as a screening marker, and FRT sequences are arranged on both sides of the resistance screening marker as the binding sites of site-specific recombinase FLP. In the previous test experiment of E. coli PIR1 strain self-resistance, it was tested that the strain cannot survive in the culture medium with a final concentration of 30 mg / L gentamicin, confirming that the strain itself does not carry the gentamicin resistance gene. Therefore, it is selected as a screening resistance and fused with the 0636 operon fragment by PCR connection.

[0099] Construction of Donor-FRT-Gam-FRT-0636(yyPR): FRT-Gm-FRT-0636 was synthesized by Genewiz, and the upper and lower arm sequences for homologous recombination with the yyPR site of PIR1 genome were introduced on both sides, respectively, to obtain the integration fragment Donor-FRT-Gam-FRT-0636(yyPR), as shown in the schematic diagram of FIG. 1, and the nucleotide sequence is shown in SEQ ID NO: 19. The similar method was used to construct Donor-FRT-Gam-FRT-0636(tyWN), as shown in the schematic diagram of FIG. 2, and the nucleotide sequence is shown in SEQ ID NO: 20.

[0100] (3) Preparation of E. coli PIR1 electrotransformation competent cells containing plasmid pKD46

[0101] Plasmid pKD46 carries λ-Red homologous recombinase Exo, Gamma, Beta. The pKD46 plasmid was introduced into E. coli PIR1 by transformation method, and the single colony was picked into ampicillin-resistant medium and cultured at 30°C for 18 h. The plasmid contained in the bacterial liquid in each test tube was extracted, and the extracted plasmid was identified by restriction enzyme SalI+BamHI+NotI digestion. The electrophoresis result is shown in FIG. 3, which shows that the extracted plasmid of the above-mentioned clone meets the expectation, and the recombinant bacteria have successfully carried the plasmid pKD46.

[0102] The successfully transformed PIR1 / pKD46 glycerol bacteria were transferred to ampicillin-resistant plates, and the single colony was picked into ampicillin-resistant medium and cultured at 30°C for 18 h to prepare PIR1 / pKD46 electrotransformation competent cells, and the steps are as follows:

[0103] 1) Transfer the bacterial culture from the test tube to 50 mL of ampicillin-resistant medium at an inoculation rate of 1%, and incubate at 30°C and 220 rpm until the bacterial culture reaches OD500. 600 The concentration was approximately 0.5-0.6. 0.6% (w / v) L-arabinose (final concentration 3 mg / mL) was added to induce the expression of homologous recombinant proteins (Exo, Beta, Gam). Induction was continued at 30°C for 2 hours, followed by an ice bath for 10 minutes. After the ice bath, the bacterial culture was transferred to pre-cooled 50 mL sterile centrifuge tubes under sterile conditions and collected by centrifugation at 4°C and 4500 rpm.

[0104] 2) Add 15 mL of pre-cooled 10% glycerol to the centrifuge tube to resuspend the cells, centrifuge at 4500 rpm for 5 min to collect the cells. Perform this step twice to wash away salt ions from the culture medium.

[0105] 3) Add 2 mL of pre-cooled 10% glycerol solution to centrifuge tubes and resuspend the bacterial cells by blowing and aspiration. Aliquot 100 μL per tube and store at -80°C.

[0106] (4) Electroporation transforms the corresponding integration fragment (Donor dsDNA fragment)

[0107] Mix 1 μg of the Donor dsDNA fragment with 100 μL of E. coli PIR1 / pKD46 electroporated competent cells and incubate on ice for 5 min. Immediately after electroporation at 1.78 kV for 5 ms, add 900 μL of pre-chilled fresh LB broth and mix well. Transfer to a 30°C shaker at 60 rpm for 8 h to allow homologous recombination between the Donor fragment and the PIR1 genome. Spread 300 μL of the recovery solution onto a Gm30 (gentamicin final concentration 30 μg / mL) + Glu20 (glucose final concentration 2 mg / mL) plate. The addition of glucose is to minimize toxin leakage due to basal expression of the arabinose operon. Incubate overnight at 37°C with the plate inverted position. Under these conditions, the temperature-sensitive pKD46 plasmid will be automatically lost. Single clones were picked and placed in liquid culture medium supplemented with Gm30 (final concentration 30 μg / mL) + Glu20 (final concentration 2 mg / mL). After incubation at 37°C for about 4-6 hours, the bacterial culture became turbid. Colony PCR was performed using the bacterial cultures of each group as templates to identify integrated positive clones.

[0108] The negative control bands of PIR1 genome yyPR site and tyWN site are 662 bp and 593 bp, respectively, the positive clone P band of yyPR site successfully inserted with toxin operon is 3001 bp, and the positive clone P band of tyWN site successfully inserted with toxin operon is 2975 bp. The integration of yyPR and tyWN sites of PIR1 genome is verified by several clones, wherein PR-1#, 5# and WN-1#, 2#, 3# clones have no negative control band and should be relatively pure integrated clones. The P product is sent for sequencing, and all the sequencing results are correct. The sequencing diagram is shown in FIG. 4.

[0109] (5) Correctly clone the tool plasmid

[0110] The PIR1-0636 pure clone with completely correct integrated toxin operon is streaked on a solid LB+Gm30+Glu20 (gentamicin final concentration 30 μg / mL, glucose final concentration 2 mg / mL) plate, cultured at 37°C to obtain single colonies, and then single colonies are picked and streaked on Gm30+Glu20 and Amp100+Glu20 plates, and the plates are cultured at 37°C overnight. Single colonies that grow normally on Gm30+Glu20 but do not grow on Amp100+Glu20 are PIR1-PR::0636 / WN correct clones with pKD46 plasmid removed. The results are shown in FIG. 5. The strain with correct sequencing and removed tool plasmid is named PIR1-PR::0636 and PIR1-WN::0636, respectively.

[0111] Example 2. Virulence verification of toxin protein

[0112] The method steps are as follows:

[0113] (1) Glycerol bacteria GT115::0636, self-made PIR1-PR::0636 1# and PIR1-WN::0636 2# are transformed into competent cells (20230914 batch), and each is inoculated into 2 test tubes (LB+0.2%Glu+Gm10, 3mL). The test tubes are cultured at 37°C overnight to obtain corresponding test tube seed liquids. PIR1-PR::0636 and PIR1-WN::0636 test tube seed liquids are inoculated into 2×2 30mL culture medium / 125mL shake flasks at the same time, the culture medium is LB+0.5%Glu, LB+0.2%Glu (positive control), LB+0.3%Ara, LB+0.6%Ara (virulence verification group), respectively, and induced at 37°C for 3.5h.

[0114] (2) After induction, the shake flask bacterial liquid is gradient diluted, a total of 6 gradients (10-10 6The 5ul dilution liquid was spotted on an antibiotic-free LB plate, and incubated at 37°C overnight. The number of colonies in the virulence verification group and the control group was checked, and the results are shown in Table 1 and Figure 6. The virulence verification group differed from the positive control group by 4 dilution gradients; the 3.5h killing effect induced by 0.3% Ara and 0.6% Ara was consistent, and the killing effect was less than 10 2 In the case of dilution, the colonies were basically 100% lethal.

[0115] (3) PCR was performed on the positive control bacterial solution of PIR1-PR::0636 and PIR1-WN::0636 in step (2) to verify whether the toxin gene was mutated (the verification primer is shown in Table 2), and sterile water and GT115::0636 bacterial solution (GT115::0636 is the strain of CN202310072956.1 Example 1) were set as controls, and the results are shown in Figure 7. The PIR1-PR::0636 1# and PIR1-WN::0636 2# strains were induced by 0.2% Glu or 0.5% Glu for 3.5h, and the toxin gene was not mutated, which was consistent with the control group with GT115::0636 as the template.

[0116] Table 1: OD of PIR1-PR::0636 and PIR1-WN::0636 bacterial solution in shake flask after 3.5h induction 600

[0117] Table 2: Verification primer of integrated positive clone

[0118] Example 3. Positive rate of transformation by pUC19 plasmid

[0119] The experimental steps are as follows:

[0120] 1) The above PIR1-PR::0636 and PIR1-WN::0636 test tube seed liquid was transferred to 50mL medium / 250mL shake flask, and the medium was LB+0.2% Glu+Gm10 to prepare a small amount of transformation competent. After the seed liquid was transferred to 50mL medium, it was cultured at 37°C for 2h, and then the bacteria were collected. A small amount of transformation competent was prepared by calcium chloride method. 80ul / tube, 14 tubes each.

[0121] 2) To PIR1-PR::0636, PIR1-WN::0636 and GT115::0636, 100 ng of pTini-JL(CIT)-1194NLA was introduced into the competent cells, ice bath for 30 min, 42°C heat shock for 90 s, add 800 uL of LB + 0.3% Ara, incubate at 37°C for 1.5 h, take 80 uL of recovery liquid to coat LB + 0.3% Ara + Gm10 plate, incubate at 37°C overnight to obtain single colonies. Take a single colony on the plate into 10 uL of sterile water for dilution, take 1 uL of bacterial liquid for PCR test of the target gene of the plasmid, and take 1 uL of bacterial liquid for PCR test of whether the toxin gene is mutated. The pTini-JL(CIT)-1194NLA is a too plasmid, the nucleotide sequence is shown as SEQ ID NO: 21, the nucleotide sequence of the replicon contained is shown as SEQ ID NO: 18, and the nucleotide sequence of the antitoxin protein gene contained is shown as SEQ ID NO: 7. The plasmid map is shown in FIG. 8.

[0122] 3) 5 uL of bacterial liquid corresponding to the positive clone was transferred to a test tube, the culture medium was LB + 0.3% Ara + Gm10, and the culture was carried out at 37°C for about 16 h, and the bacteria were preserved and extracted for too plasmid test.

[0123] Experimental results:

[0124] After introducing the too plasmid, the plate colony situation of PIR1-PR::0636, PIR1-WN::0636 and GT115::0636 (left, middle and right) is shown in FIG. 9; the positive rate of PIR1-PR::0636, PIR1-WN::0636 and GT115::0636 transformed too plasmid is shown in FIG. 10; and the test results of toxin gene of PIR1-PR::0636, PIR1-WN::0636 and GT115::0636 positive clones are shown in FIG. 11. According to the bacterial P results of the target gene of the too plasmid, the proportion of PIR1-PR::0636 1#, PIR1-WN::0636 2# and GT115::0636 positive clones transformed too plasmid pTini-JL(CIT)-1194NLA is 15 / 16, 16 / 16 and 14 / 16 respectively, and the positive rate of too plasmid transformation of PIR1-PR::0636, PIR1-WN::0636 and GT115::0636 is more than 14 / 16 (87.5%), and the positive rate of PIR1-PR::0636 and PIR1-WN::0636 transformation is higher, which can reach 93.7%-100%, meeting the expected transformation positive rate higher than 80%. At the same time, it can be observed from FIGS. 10-11 that the mutation of the toxin gene is closely related to the positive rate of too plasmid transformation: the corresponding clone with obvious insertion mutation of the toxin gene will also have abnormal PCR band of the target gene of the too plasmid.

[0125] The plasmid yield of PIR1-PR::0636, PIR1-WN::0636 is shown in Table 3, which is about 3.4 ug / 2mL bacterial solution and 2.9 ug / 2mL bacterial solution in the test tube.

[0126] Table 3: Plasmid yield of PIR1-PR::0636, PIR1-WN::0636

[0127] Example 4. Verification of strain passage virulence stability

[0128] PIR1-PR::0636, PIR1-WN::0636 were passaged in the medium with different concentrations of glucose (0.2% Glu, 0.5% Glu, 1% Glu, 5% Glu) for multiple times, the transfer ratio was 0.001%, and the culture was carried out at 37°C for 24h as one generation. The virulence stability was verified by the method of Example 2.

[0129] OD of PIR1-PR::0636, PIR1-WN::0636 passage bacterial solution 600 As shown in Figure 12, the four columns of each group from left to right are P1, P2, P3, P4 generations. The virulence verification of PIR1-PR::0636, PIR1-WN::0636 in the medium with different concentrations of glucose for P2 generation and P4 generation is shown in Figure 13. The comparison between the 0.3% Ara induction virulence group and the gradient Glu negative control group found that when the P2 generation was cultured in the four gradient Glu culture, the virulence of the strain was consistent with the expected result, that is, in the case of less than 10 -2 times dilution, the 0.3% Ara induction virulence group was 100% lethal, and there was more than 4 gradient difference with the Glu negative control group. Further comparison found that the concentration in the range of 0.2% Glu to 5% Glu was increased, which made the virulence of the strain show a trend of first increasing and then decreasing: when the passage Glu concentration was 0.2%, the virulence group had no colony growth after 100 times dilution; when the passage Glu concentration was higher than 0.2% (0.5%-1% was significantly effective), the virulence was enhanced, and it was observed that the number of colonies of the virulence group was significantly reduced after 10 times dilution; with the continuous increase of the passage Glu to 5%, the virulence did not continue to increase.

[0130] PIR1-PR::0636, PIR1-WN::0636 in the medium added with different concentrations of glucose P4 generation, P8 generation of virulence verification as shown in Figure 14. Four gradient Glu culture P4 generation, in the case of less than 10-2 times dilution, 0.3% Ara induced toxic killing group of bacteria 100% death, with more than 4 gradient difference with Glu negative control group. 0.2% Glu to 5% Glu concentration range shows the trend of first increase and then decrease of the killing efficiency of the strain, which is more obvious than P2: when the generation Glu concentration is 0.2%, the toxic killing group has basically no colony growth after dilution by 1000 times; when the generation Glu concentration is higher than 0.2% and is 0.5%-1%, the toxic killing effect is enhanced, and it is observed that the number of colonies of the toxic killing group is significantly reduced after dilution by 100 times.

[0131] The above results show that the LB medium with different concentrations of Glu as the library building medium can maintain the stability of the virulence of the competent cells for a long time.

[0132] Example 5. Optimization of the plasmid backbone of the T7 phage

[0133] The original plasmid backbone sequence spectrum of the T7 phage pTini-JL(CIT)-1194NLA used is shown in Figure 15, and the nucleotide sequence is shown in SEQ ID NO: 25, wherein the nucleotide sequence of the R6K-γ replicon is shown in SEQ ID NO: 18, and the nucleotide sequence of the antitoxin protein gene is shown in SEQ ID NO: 7. However, the T7 phage of the original plasmid backbone has the problems of low supercoiling and high proportion of multimers, and the backbone sequence needs to be optimized.

[0134] Experimental scheme: according to 1) the direction of the antagonistic element and the replicon, 2) the length of the R6K replicon sequence, and at the same time, the truncated antitoxin protein gene is cloned into the same vector of the target gene, wherein the nucleotide sequence of the R6K-γ replicon (R6K-γV3) is shown in SEQ ID NO: 22, and the nucleotide sequence of the antitoxin protein gene is shown in SEQ ID NO: 23. Extract monoclonal, gel electrophoresis / capillary electrophoresis synchronous comparison of the supercoiling state of the clones. The sequence spectrum of the improved plasmid backbone is shown in Figure 16, and the nucleotide sequence is shown in SEQ ID NO: 24.

[0135] The plasmids pTini-PB(CES)-dCGEGFP, pTini-JL(CES)-dCGEGFP, pTini-PB(DTS)-1444-8E, and JL-DE-wt-CACC were respectively introduced into the PIR1-WN::0636 strain using the improved plasmid backbone, cultured for production, and the supercoiling proportion before and after improvement of the plasmid backbone was detected. Taking pTini-JL(CES)-dCGEGFP as an example, the plasmid map of the plasmid with the improved plasmid backbone is shown in FIG. 25.

[0136] The electrophoresis results of the supercoiling of the three Tai plasmids before and after improvement of the plasmid backbone are shown in FIGS. 17-19, and the capillary electrophoresis results of the supercoiling of the Tai plasmid JL-DE-wt-CACC before and after improvement of the plasmid backbone are shown in FIG. 20. After optimization of the Tai plasmid backbone sequence, the supercoiling of the Tai plasmid with different target genes is improved from about 70% to about 80%, solving the problem of low supercoiling caused by a high proportion of Tai plasmid concatemers. Thus, the proportion of supercoiling can be significantly improved by using the improved plasmid backbone.

[0137] Example 6. Transformation positive rate of PIR1-WN::0636 competent strain

[0138] The pTini-JL(DTS)-E1 V2 Tai plasmid (using the improved plasmid backbone) was used to test the transformation positive rate (toxicity) of the PIR1-WN::0636 competent strain. Two experiments were performed, and 3 competent strains were randomly selected for each experiment for parallel transformation and plating. 200 ng of plasmid was transformed and heat-shocked with 100 ul of competent bacterial solution at 42°C for 90 s, and then ice-bathed for 3-5 min. 100 ul of YHN medium was added, and the mixture was incubated at 37°C with shaking at 220 rpm for 2-3 h, and then plated and cultured. Five clones were randomly selected from each plate for test tube expansion and then small-scale identification. The results of the two experiments are shown in FIGS. 21-22, and the small-scale yields are shown in Table 4. Gray indicates good supercoiling.

[0139] Table 4: Small-scale yield

[0140] Transformation positive rate: According to FIGS. 21-22, the positive rate of the first experiment was about 73.3% (11 / 15), and the positive rate of the second experiment was about 93.3% (14 / 15), both reaching a high level. The proportion of supercoiled monomers after transformation and preliminary screening can reach 76%-88%.

[0141] As can be seen from Table 4, at a culture temperature of 30°C, the OD 600 The yield per unit volume can reach about 0.5-1.0 ug / (ml*OD 600), comparable to conventional resistance plasmids, with little variation between clones.

[0142] Example 7. Production strain plasmid system fermentation study

[0143] Glycerol strains were generated by transforming PIR1-WN::0636 competent strains with plasmids pTini-JL(DTS)-2396(R73)v3, pTini-JL(DTS)-d1EGFP v3, and JL-DE-wt-CACC-Qa6 v3 (all using the improved plasmid backbone). Fermentation studies were performed on pTini-JL(DTS)-2396(R73)v3-PIR1 glycerol strain, pTini-JL(DTS)-d1EGFP v3-PIR1 glycerol strain, and JL-DE-wt-CACC-Qa6 v3-PIR1 glycerol strain using the platform fermentation process to evaluate scale up stability, reactor growth state, supercoiling, and yield. Fermentation data results are shown in Table 5, and JL-DE-WT-CACC Qa6 V3 fermentation mid-sample detection chart is shown in Figure 23, and d1EGFP V3, 2396(R73)V3 fermentation mid-sample detection chart is shown in Figure 24.

[0144] Table 5. Fermentation data summary

[0145] As can be seen from Table 5, at 5L reactor scale, PIR1 too plasmid systems were tested using the unoptimized platform process, with an OD600 of about 110 achieved at about 24h fermentation period, and a glycerol strain that was not screened for yield could achieve a 200-500mg / L of tank Titer, and 1-3mg / g of unit yield, all at a high level for normal resistance gene systems.

[0146] As can be seen from Figures 23 and 24, the PIR1-too plasmid system had a relatively stable supercoiling ratio during fermentation, with a tank ratio of about 80%, and the reactor tank supercoiling ratio was not less than that of the shake flask, indicating that the fermentation tank conditions were superior to the shake flask, which would promote the increase of the supercoiling monomer ratio.

Claims

1. An antibiotic plasmid-free production strain, characterized in that, The production strain is a genome-reconstructed strain using PIR1 as the original strain, and the production strain contains a nucleotide sequence encoding a toxin protein and an anti-plasmid containing a nucleotide sequence encoding an anti-toxin protein; the amino acid sequence of the toxin protein is as shown in SEQ ID NO: 4, and the amino acid sequence of the anti-toxin protein contains: an amino acid sequence as shown in SEQ ID NO: 1, or an amino acid sequence having one or more mutations of E24D, I36V, V43I compared with SEQ ID NO: 1; or the amino acid sequence of the toxin protein is as shown in SEQ ID NO: 13, and the amino acid sequence of the anti-toxin protein contains: an amino acid sequence as shown in SEQ ID NO: 9, or an amino acid sequence having one or more mutations of T6I, T43A, K47E, A50S, E51D, G52A, N54K compared with SEQ ID NO:

9.

2. A plasmid-free production strain according to claim 1, c h a r a c t e r i z e d in that the amino acid sequence of the toxin protein is as shown in SEQ ID NO: 4, and the amino acid sequence of the anti-toxin protein is as shown in SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3; or the amino acid sequence of the toxin protein is as shown in SEQ ID NO: 13, and the amino acid sequence of the anti-toxin protein is as shown in SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, or SEQ ID NO:

12.

3. A plasmid-free production strain according to claim 1 or 2, c h a r a c t e r i z e d in that The nucleotide sequence encoding the toxin protein is within the genome of the PIR1 strain; Preferably, the nucleotide sequence encoding the toxin protein is integrated in the non-coding region between yjiP and yjiR or the non-coding region between thrW and ykfN.

4. A plasmid-free production strain according to any one of claims 1 to 3, characterized in that, The PIR1 strain contains a gene expression cassette for inducible expression of a toxin protein, and the gene expression cassette for inducible expression of a toxin protein contains an operon or a promoter for inducible expression of a toxin protein, Preferably, the operon or the promoter is selected from a Lac lactose operon, an L-arabinose-induced pBAD promoter, an L-rhamnose-induced rhapBAD promoter, or a lambda PL / PR-clts857 temperature-sensitive promoter; More preferably, the nucleotide sequence encoding the toxin protein is as shown in SEQ ID NO: 6 or 15.

5. A plasmid-free production strain according to any one of claims 1 to 4, characterized in that, The anti-plasmid is an anti-microplasmid, which contains a nucleotide sequence encoding an anti-toxin protein and a replicon; the length of the replicon is ≤800bp, ≤600bp, or ≤400bp. Preferably, the replicon is selected from ColE1, ColE2, pMB1, pSC101, RSF, R6K, pUC57, RK2, and p15A; more preferably, R6K-γ or pUC57.

6. A plasmid-free production strain according to any one of claims 1 to 5, wherein the strain is a strain of Escherichia coli. the nucleotide sequence encoding the anti-toxin protein is as shown in SEQ ID NO: 5 or SEQ ID NO: 14; or, the nucleotide sequence encoding the antitoxin protein does not contain CpG motifs, preferably, the nucleotide sequence encoding the antitoxin protein is as set forth in SEQ ID NO: 7 or SEQ ID NO:

16.

7. A plasmid-free production strain according to any one of claims 1 to 6, characterized in that, the nucleotide sequence of the replicon is as set forth in SEQ ID NO: 18 or 22, preferably as set forth in SEQ ID NO:

22.

8. A plasmid-free production strain according to any one of claims 1 to 7, characterized in that, the antivector plasmid comprises a structured DNA sequence selected from the group consisting of: polyA repeats, SV40 origin of replication, viral LTRs, lentiviral LTRs, retroviral LTRs, transposon IR / DR repeats, AAV ITRs, transposon ITRs, CMV enhancer, and SV40 enhancer.

9. A plasmid-free production strain according to any one of claims 1 to 8, characterized in that, the antivector plasmid is selected from the group consisting of Sleeping Beauty transposon vector, PiggyBac transposon vector, ZB transposon vector, PS transposon vector, Tol2 transposon vector, and polyA-containing mRNA vector.

10. A plasmid-free production strain according to any one of claims 1 to 9, characterized in that, the antivector plasmid comprises an exogenous gene, preferably, the exogenous gene encodes a chimeric antigen receptor and / or encodes an antibody.

11. A method for producing an antibiotic plasmid-free production strain according to any one of claims 1 to 10, characterized in that, comprising: integrating a nucleotide sequence encoding a toxin protein into the genome of the PIR1 strain; and then transforming an antivector plasmid into the PIR1 strain; preferably, the nucleotide sequence encoding the toxin protein is homologously recombined into the genome of the PIR1 strain by a RED recombination system. more preferably, the nucleotide sequence encoding the toxin protein is integrated in the non-coding region between yjiP and yjiR or the non-coding region between thrW and ykfN.

12. A method for producing an antibiotic-free plasmid, characterized by, comprising: culturing a production strain of any one of the antivector plasmids of claims 1-10, inducing expression of the toxin protein and expression of the antitoxin protein, and obtaining the plasmid from the cultured production strain.

13. A supercoiled plasmid with a high supercoil to relaxed ratio, characterized in that, comprising a nucleotide sequence encoding an antitoxin protein comprising an amino acid sequence as set forth in SEQ ID NO: 1 or an amino acid sequence having one or more mutations of E24D, I36V, V43I compared to SEQ ID NO: 1, and a replicon having a nucleotide sequence as set forth in SEQ ID NO: 22; preferably, the amino acid sequence encoding the antitoxin protein is as set forth in any one of SEQ ID NOs: 1-3, more preferably, the amino acid sequence encoding the antitoxin protein is as set forth in SEQ ID NO:

1.

14. A miniplasmid of high superhelix ratio according to claim 13, wherein, the plasmid backbone of the antivector plasmid comprises a nucleotide sequence encoding an antitoxin protein, a replicon, and a promoter of the antitoxin protein gene; preferably, the nucleotide sequence encoding the antitoxin protein is as set forth in SEQ ID NO: 23; more preferably, the nucleotide sequence of the plasmid backbone is as set forth in SEQ ID NO:

24. ​ 15. Use of a production strain of any one of the antivector plasmids of claims 1-10 in the preparation of a gene therapy drug, a cell therapy drug, a DNA vaccine, virus production, or antibody production.

16. A method for producing a recombinant peptide, polypeptide or protein of interest by a plasmid-free production strain according to any one of claims 1 to 10, characterized in that, the plasmid further comprises a gene expression cassette encoding a recombinant peptide, polypeptide, or protein of interest, the method comprising culturing the system and recovering the recombinant peptide, polypeptide, or protein of interest.

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