Plasmid system without selectable markers and production method thereof

Abstract

To provide a precursor plasmid for producing a plasmid without selectable markers.SOLUTION: A precursor plasmid comprises: 1) a replication origin site; 2) a selectable marker gene; 3) a target gene or a cloning site for inserting the target gene; and 4) paired recombination sites. The paired recombination sites allow the precursor plasmid to perform self-recombination in the presence of recombinase to form a daughter plasmid molecule and a circular double-stranded DNA molecule. The daughter plasmid comprises the replication origin site and the cloning site or the target gene. The circular double-stranded DNA comprises the selectable marker gene. The present invention further provides a method for producing a plasmid without selectable markers using the precursor plasmid. The plasmid without selectable markers produced can be used as a DNA delivery vector or a virus packaging plasmid vector in the field of gene and cell therapy to improve the safety of the plasmid.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to a plasmid and a precursor plasmid for producing a markerless plasmid. The present invention further relates to a method for producing the markerless plasmid and a set (kit) for producing the markerless plasmid. [Background technology]

[0002] In recent years, gene and cell therapy technologies have become an important new method for treating human diseases. Safe and efficient DNA delivery vectors will enable gene and cell therapy to play a greater role in the prevention and treatment of human diseases in the future. Over the past two decades, plasmid vector-based non-viral delivery systems and plasmid-based viral packaging / delivery systems have attracted widespread attention in the areas of repairing gene defects, treating and preventing diseases, etc. Improving the safety, stability, and yield of plasmids, as well as reducing cytotoxicity, have become the main direction of plasmid research in the field of gene and cell therapy.

[0003] Plasmid DNA molecules used in gene therapy typically have a modular structure containing a eukaryotic transcription unit and a prokaryotic replication unit. In addition to sequences necessary for DNA replication, conventional plasmids typically contain at least one antibiotic resistance gene to facilitate positive clone screening and genetic stabilization during the cloning process. Commonly used selectable markers include ampicillin, kanamycin, chloramphenicol, neomycin, and tetracycline. [1] However, when applied to gene therapy, plasmid DNA can be absorbed by bacteria present on the surface of the respiratory or digestive tract, and the antibiotic resistance gene carried by the plasmid can cause side effects such as antibiotic resistance in patients. Therefore, the harmful side effects of conventional plasmids have prompted the development of plasmids without antibiotic resistance genes.

[0004] Currently widely used antibiotic resistance gene-free plasmids mainly include: 1) minicircles (circular DNA molecules). Minicircles are derived from conventional plasmids containing recombination sites by self-recombination in E. coli to form a circular DNA molecule containing only the target gene sequence and a small, replicative plasmid containing the antibiotic resistance gene. Minicircles have the shortest plasmid backbone sequence, but lack replication capacity and are limited by recombination efficiency and purification methods, resulting in a cumbersome manufacturing process, low yields, and low purity, making mass production difficult [2]. 2) RNA-OUT selection technology-based plasmids. These plasmids encode an RNA-OUT antisense strand to suppress the expression of the negative selection gene SacB in the host strain, allowing for screening by growing transformed positive clones on sucrose-containing media [3]. This screening system is easy to scale up for production. The plasmid backbone contains a DNA replication element and an RNA-OUT element, and its sequence (450-500 bp) is more complex than that of minicircles. Summary of the Invention

[0005] According to one aspect, the present invention provides a precursor plasmid, the precursor plasmid comprising: 1) a replication initiation site; 2) a selectable marker gene; and 3) a target gene or a cloning site for inserting said target gene; 4) paired recombination sites, wherein: These paired recombination sites allow the precursor plasmid to self-recombine in the presence of a recombinase to form daughter plasmid molecules lacking a selectable marker gene and circular double-stranded DNA molecules; The daughter plasmid contains the replication origin and the target gene, or the replication origin and the cloning site, and the circular double-stranded DNA contains a selectable marker gene.

[0006] In some embodiments, the sequences of the paired recombination sites are in the same orientation.

[0007] In some embodiments, the replication initiation site is adjacent to the target gene or the cloning site, and the paired recombination sites are adjacent to the replication initiation site and the target gene upstream and downstream, respectively, or these paired recombination sites are adjacent to the replication initiation site and the cloning site upstream and downstream, respectively. In some preferred embodiments, the replication initiation site is adjacent to the target gene or the cloning site, and the paired recombination sites are located at both ends of the "replication initiation site-target gene," respectively, or these paired recombination sites are located at both ends of the "replication initiation site-cloning site," respectively. In some other preferred embodiments, the replication initiation site is located at one end of the target gene or the cloning site, and the paired recombination sites are located at both ends of the "replication initiation site-target gene," respectively, or these paired recombination sites are located at both ends of the "replication initiation site-cloning site," respectively.

[0008] In some embodiments, the paired recombination sites are loxP sequences in the same orientation, FRT sequences in the same orientation, or attB / attP sequences in the same orientation.

[0009] In some embodiments, the paired recombination sites are lox71 and lox66 sequences in the same orientation. In some embodiments, the paired recombination sites are attB and attP sequences in the same orientation.

[0010] In some embodiments, the precursor plasmid may comprise one or more pairs of paired recombination sites. In some embodiments, the precursor plasmid comprises one pair of paired recombination sites. In other embodiments, the precursor plasmid comprises at least two pairs of paired recombination sites.

[0011] In some embodiments, the replication origin may be from a bacterial or phage replication origin. In some specific embodiments, the replication origin is a bacterial replication origin. In some preferred embodiments, the replication origin is selected from the replication origin of pUC, the replication origin of pMB1 and its derivatives, the replication origin of ColE1, and the replication origin of R6Kγ. In some embodiments, the replication origin comprises a nucleotide sequence set forth in SEQ ID NOs: 43-46, or a nucleotide sequence that has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the nucleotide sequence set forth in SEQ ID NOs: 43-46 and that can serve as an origin of replication. In some other embodiments, the origin of replication comprises a nucleotide sequence that has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to, and is capable of serving as, an origin of replication with, the nucleotide sequence set forth in SEQ ID NO: 43, 44, 45, or 46. In some embodiments, the origin of replication comprises the nucleotide sequence set forth in SEQ ID NO: 43, 44, 45, or 46. In particular embodiments, the nucleotide sequence of the origin of replication is as set forth in SEQ ID NO: 43, 44, 45, or 46.

[0012] In some embodiments, the precursor plasmid further comprises a rop (primer repressor of primer) gene sequence, a coding sequence for an endonuclease, and a coding sequence for a plasmid replication helper protein. The endonuclease may be I-SceI. The plasmid replication helper protein may be a π protein. In some other embodiments, the circular double-stranded DNA comprises one or more of a rop gene sequence, a coding sequence for an endonuclease, and a coding sequence for a plasmid replication helper protein.

[0013] In some embodiments, the precursor plasmid comprises other plasmid backbone sequences. In some other embodiments, the circular double-stranded DNA comprises other plasmid backbone sequences.

[0014] In some embodiments, the selectable marker gene is an antibiotic resistance gene, hi some other embodiments, the selectable marker gene is selected from genes conferring resistance to ampicillin, kanamycin, chloramphenicol, neomycin, or tetracycline.

[0015] In some embodiments, the precursor plasmid further comprises a gene encoding a recombinase. In some other embodiments, the circular double-stranded DNA may comprise a gene encoding a recombinase. In some embodiments, the precursor plasmid is capable of expressing the recombinase under appropriate conditions. The appropriate conditions may be any conditions that allow the precursor plasmid to express the recombinase, and may include, for example, an appropriate host cell, growth conditions, induction conditions, etc. In addition to the gene encoding the recombinase, the precursor plasmid may further comprise elements necessary for expressing the recombinase, such as a promoter, an enhancer, a terminator, etc.

[0016] In some embodiments, the precursor plasmid comprises a nucleotide sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the nucleotide sequence set forth in SEQ ID NO: 8, 34, 39-42 or 47. In some embodiments, the precursor plasmid comprises the nucleotide sequence set forth in SEQ ID NO: 8, 34, 39-42 or 47. In embodiments, the nucleotide sequence of the precursor plasmid is as set forth in SEQ ID NO: 8, 34, 39-42 or 47.

[0017] In another aspect, the present invention provides the use of the precursor plasmid described above in producing a daughter plasmid lacking a selectable marker gene.

[0018] In another aspect, the present invention provides a method for producing a daughter plasmid without a selectable marker gene, the method comprising: 1) introducing the precursor plasmid into a host cell capable of expressing or supporting the expression of a recombinase, and screening for host cells that express the selectable marker gene; 2) culturing the host cells screened in step 1) to allow expression of the recombinase in the host cells, and culturing the host cells to screen for host cells that do not express the selectable marker gene; and 3) culturing the host cells screened in step 2) to obtain a daughter plasmid, and extracting the plasmid.

[0019] In some embodiments, the production method further comprises, prior to step 1), a step of obtaining or producing the precursor plasmid described above.

[0020] In some embodiments, the precursor plasmid comprises an origin of replication, a selectable marker gene, a target gene, and paired recombination sites. These paired recombination sites allow the precursor plasmid to self-recombine in the presence of a recombinase to form a daughter plasmid molecule and a circular double-stranded DNA molecule. The daughter plasmid comprises an origin of replication and the target gene. The circular double-stranded DNA comprises a selectable marker gene. In some embodiments, the daughter plasmid does not comprise an antibiotic resistance gene.

[0021] In some embodiments, the sequences of the paired recombination sites are in the same orientation.

[0022] In some embodiments, the paired recombination sites are loxP sequences oriented in the same direction and the recombinase is Cre recombinase, the paired recombination sites are FRT sequences oriented in the same direction and the recombinase is Flp recombinase, or the paired recombination sites are attB / attP sequences oriented in the same direction and the recombinase is PhiC31 recombinase.

[0023] In some embodiments, the paired recombination sites are lox71 and lox66 sequences in the same orientation, and the recombinase is Cre recombinase. In some other embodiments, the paired recombination sites are FRT sequences in the same orientation, and the recombinase is Flp recombinase. In some embodiments, the paired recombination sites are attB / attP sequences in the same orientation, and the recombinase is PhiC31 recombinase.

[0024] In some embodiments, the origin of replication is from a bacterial or phage origin of replication. In some specific embodiments, the origin of replication is selected from the group consisting of the origin of replication of pUC, the origin of replication of pMB1 and its derivatives, the origin of replication of ColE1, and the origin of replication of R6Kγ. In some other embodiments, the origin of replication comprises a nucleotide sequence set forth in SEQ ID NOs: 43-46, or a nucleotide sequence that has at least 80%, 85%, 90%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the nucleotide sequence set forth in SEQ ID NOs: 43-46 and that can serve as an origin of replication.

[0025] In some embodiments, the selectable marker gene is an antibiotic resistance gene, hi some other embodiments, the selectable marker gene is selected from genes conferring resistance to ampicillin, kanamycin, chloramphenicol, neomycin, or tetracycline.

[0026] In some embodiments, the precursor plasmid comprises a gene encoding a recombinase, and the precursor plasmid is capable of expressing the recombinase in a host cell.

[0027] In some embodiments, the precursor plasmid comprises a nucleotide sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the nucleotide sequence set forth in SEQ ID NO: 8, 34, 39-42, or 47. In some other embodiments, the precursor plasmid comprises the nucleotide sequence set forth in SEQ ID NO: 8, 34, 39-42, or 47. In embodiments, the nucleotide sequence of the precursor plasmid is as set forth in SEQ ID NO: 8, 34, 39-42, or 47.

[0028] In some embodiments, the host cell comprises a gene encoding a recombinase in its genome, or the host cell comprises an expression vector containing a gene encoding the recombinase.

[0029] In some embodiments, the recombinase is inducibly expressed in the host cell. In some other specific embodiments, the host cell comprises an inducible promoter, and the inducible promoter allows the recombinase to be inducibly expressed in the presence of an inducer. For example, the inducible promoter may be a lactose promoter (lactose operon), an arabinose promoter (arabinose operon), a temperature-inducible promoter, a metal ion-inducible promoter, or the like. In some other embodiments, the recombinase gene is integrated into the host cell genome via a plasmid containing the inducible promoter and the recombinase-encoding gene. Integration methods include, but are not limited to, CRISPR / Cas9 methods.

[0030] In some embodiments, the expression vector comprises a conditionally inducible deletion plasmid replication element. In some specific embodiments, the expression vector comprises a conditionally inducible deletion replicon, such as a temperature-sensitive replicon or a metal ion replicon. Under inducing conditions, the expression vector loses replication function and is gradually lost within the host cell.

[0031] In some embodiments, the host cell is E. coli. For example, the host cell may be E. coli JM108, TOP10, DH5α, GT115, pir1, pir2, etc., and other modified strains derived from related strains.

[0032] All the definitions and technical features of the precursor plasmid mentioned above are applicable to the precursor plasmid in the method for producing the daughter plasmid, and are all incorporated herein, and no further details will be provided.

[0033] The present invention also includes daughter plasmids obtained by the above-mentioned production methods.

[0034] In another aspect, the present invention provides a set for producing a daughter plasmid lacking a selectable marker gene, the set comprising the precursor plasmid described above.

[0035] In some embodiments, the set further includes a host cell capable of expressing or supporting the expression of the recombinase. The host cell is Escherichia coli. For example, the host cell may be E. coli JM108, TOP10, DH5α, GT115, pir1, pir2, etc., and other modified strains derived from related strains.

[0036] In some embodiments, in the precursor plasmid, the paired recombination sites are loxP sequences oriented in the same direction and the recombinase is Cre recombinase; the paired recombination sites are FRT sequences oriented in the same direction and the recombinase is Flp recombinase; or the paired recombination sites are attB / attP sequences oriented in the same direction and the recombinase is phiC31 recombinase. In some embodiments, the paired recombination sites are lox71 and lox66 sequences oriented in the same direction and the recombinase is Cre recombinase. In other embodiments, the paired recombination sites are FRT sequences oriented in the same direction and the recombinase is Flp recombinase.

[0037] In some embodiments, the host cell comprises a gene encoding a recombinase in its genome, or the host cell comprises an expression vector containing a gene encoding the recombinase.

[0038] In some embodiments, the recombinase is inducibly expressed in the host cell. In some other specific embodiments, the host cell comprises an inducible promoter, and the inducible promoter allows the recombinase to be inducibly expressed in the presence of an inducer. For example, the inducible promoter may be a lactose promoter (lactose operon), an arabinose promoter (arabinose operon), a temperature-inducible promoter, a metal ion-inducible promoter, or the like. The recombinase gene is integrated into the host cell genome via a plasmid containing the inducible promoter and the recombinase-encoding gene. Integration methods include, but are not limited to, CRISPR / Cas9 methods.

[0039] In some embodiments, the expression vector comprises a conditionally inducible deletion plasmid replication element. In some specific embodiments, the expression vector comprises a conditionally inducible deletion replicon, such as a temperature sensitive replicon and a metal ion replicon.

[0040] In some embodiments, a target gene is inserted into the cloning site so that the daughter plasmid formed after recombination contains the target gene.

[0041] All the definitions and technical features of the precursor plasmids mentioned above are applicable to the precursor plasmids in the above set, and are all incorporated herein, and the details will not be further described.

[0042] In yet another aspect, the present invention provides a daughter plasmid comprising an origin of replication and a target gene, wherein the daughter plasmid does not comprise an antibiotic resistance gene. In some embodiments, the daughter plasmid does not comprise a selectable marker gene. In some embodiments, the daughter plasmid consists essentially of the origin of replication and the target gene.

[0043] In some embodiments, the origin of replication is from a bacterial or phage origin of replication. In some other embodiments, the origin of replication is selected from the group consisting of the origin of replication of pUC, the origin of replication of pMB1 and its derivatives, the origin of replication of ColE1, and the origin of replication of R6Kγ. In some other embodiments, the origin of replication comprises a nucleotide sequence set forth in SEQ ID NOs: 43-46, or a nucleotide sequence that has at least 80%, 85%, 90%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the nucleotide sequence set forth in SEQ ID NOs: 43-46 and that can serve as an origin of replication.

[0044] In some embodiments, the target gene includes a promoter, a gene encoding an expressed protein, and a terminator sequence. In some other embodiments, the target gene includes a gene encoding an expressed protein and may further include a promoter, a terminator, an enhancer, etc.

[0045] In some embodiments, the daughter plasmid can replicate within the host cell. The host cell is E. coli. For example, the host cell may be E. coli JM108, TOP10, DH5α, GT115, pir1, pir2, etc., or other modified strains derived from related strains. The daughter plasmid is cultured by fermentation within the host cell and then obtained by plasmid extraction. This method can be scaled up to meet production demands.

[0046] According to yet another aspect, the present invention further provides a host cell comprising the aforementioned daughter plasmid, wherein the daughter plasmid is capable of replicating within the host cell. In some embodiments, the daughter plasmid is obtained from the host cell by culturing, harvesting, and extraction.

[0047] All the definitions and technical characteristics of the daughter plasmids mentioned above are applicable to the daughter plasmids in the host cell, and are all incorporated herein, and no further details will be provided.

[0048] According to yet another aspect, the present invention further provides a composition comprising the aforementioned daughter plasmid, wherein the daughter plasmid content is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%.

[0049] In some embodiments, the method for measuring the daughter plasmid content is an NGS analysis method or a gel electrophoresis imaging analysis method. In some embodiments, the daughter plasmid content is determined by performing an NGS analysis on the composition to determine the proportion of daughter plasmid sequences in the composition. In some embodiments, the daughter plasmid content in the composition measured by the NGS analysis method is at least 80%, 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%. In some embodiments, the daughter plasmid content in the composition measured by the NGS analysis method is at least 98%. In other embodiments, the daughter plasmid content in the composition measured by the NGS analysis method is at least 99%. In some embodiments, the composition is analyzed using an NGS analysis method, wherein the content of resistance gene sequences is less than 0.1%, 0.08%, 0.06%, 0.04%, 0.02%, 0.01%, or 0.008%. In some other embodiments, the composition is analyzed using an NGS method, wherein the content of the host cell genome is less than 10%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1%. In some other embodiments, the composition is analyzed by an NGS method, wherein the target daughter plasmid accounts for more than 90% of the composition, the residual resistance gene sequence is less than 0.02%, and the content of the host cell genome is less than 5%.

[0050] In another embodiment, the daughter plasmid content is determined by performing gel electrophoresis imaging analysis on the composition and measuring the band intensity at the position corresponding to the supercoiled monomer form of the daughter plasmid in the gel image. In some embodiments, the daughter plasmid content in the composition measured by gel electrophoresis imaging analysis is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%. In some embodiments, the daughter plasmid content in the composition measured by gel electrophoresis imaging analysis is at least 90%.

[0051] Plasmids without a selectable marker produced using precursor plasmids can be used in gene and cell therapy as DNA delivery vectors or virus packaging plasmid vectors to improve plasmid safety. Compared to minicircles, the production of plasmids without antibiotic resistance genes according to the present invention has the following advantages: 1) high plasmid yields. The plasmids without a selectable marker according to the present invention contain replication elements that can replicate and amplify autonomously, resulting in yields comparable to those of conventional plasmids; 2) high purity of the plasmid product. Precursor plasmids are recombined into host bacteria, and screening for antibiotic-susceptible strains yields strains free of the precursor plasmid. The recombined circular DNA containing the antibiotic resistance gene is replication-incompetent and naturally metabolized during bacterial culture, thereby enabling the production of strains carrying only the plasmid without a resistance selection marker, and these strains can produce highly pure plasmid DNA; and 3) the production process can be easily scaled up. Strains carrying the antibiotic resistance gene-free plasmid produced by a single recombination process can be used for the fermentation production of different lots of the same plasmid without repeated recombination production. [Brief explanation of the drawings]

[0052] [Figure 1] 1 is a schematic diagram of the structure of a precursor plasmid, where ori: origin of replication, RS: recombination site, Redundant backbone include ABR gene: backbone sequence including antibiotic resistance gene, and Target Gene: target gene. [Figure 2] Electrophoresis gel of cre fragment amplification, where lane 1 is the marker, and lanes 2 and 3 are two parallel cre gene amplification products. [Figure 3]Electrophoresis gel of pSC101-araBAD linear vector preparation, where lane 1 is the marker and lanes 2-5 are the four parallel digestion products. [Figure 4] Electrophoresis gel diagram of PCR verification results of pSC101-araBAD-cre(AmpR) plasmid, where lanes 1 to 12 are PCR products from 12 colonies, and lane 13 is the marker. [Figure 5] FIG. 1 shows the Sanger sequencing results of the pSC101-araBAD-cre(AmpR) plasmid, where each arrow above represents the actual sequence obtained by one sequencing reaction, and two sequencing reactions completely cover the cre gene fragment to be sequenced. [Figure 6] Electrophoresis gel of the pSC101(ts)-araBAD-cre plasmid fragment preparation, where lane 1 is the marker, lanes 2 and 3 are the amplified pSC101 ori (ts) fragment products, and lanes 4 and 5 are the pSC101-araBAD-cre(AmpR) plasmid digestion products. [Figure 7] FIG. 1 shows the results of Sanger sequencing of the pSC101-araBAD-cre(ts) plasmid. [Figure 8] Electrophoresis gel diagram of PCR verification of JM108 genetically modified bacteria, where lane 1 is the marker, lane 2 is the PCR product of the wild-type JM108 strain, and lane 3 is the PCR product of the colony into which the araBAD-cre fragment was knocked in. [Figure 9] FIG. 1 shows the results of Sanger sequencing of the JM108 genetically modified bacteria. [Figure 10] 1 is a gel diagram of the results of FLP fragment amplification, in which lane 1 is the marker, and lanes 2 and 3 are the amplification products of the FLP gene. [Figure 11]Electrophoresis gel of the prepared pSC101(ts)-araBAD linear vector, where lane 1 is the marker and lanes 2 to 5 are the pSC101(ts)-araBAD-cre digestion products (parallel). [Figure 12] FIG. 1 shows the Sanger sequencing results of pSC101(ts)-araBAD-flp, where each arrow above represents the actual sequence obtained by one sequencing reaction, and two sequencing reactions completely cover the FLP fragment to be sequenced. [Figure 13] Schematic diagram of precursor plasmid vectors containing lox71 / lox66 recombination sites, where the backbone includes pMB1 ori: backbone sequence containing the pMB1 replication origin site, MCS: multiple cloning site, and Redundant backbone includes KanR Gene: backbone sequence containing the kanamycin resistance gene. [Figure 14] Electrophoresis gel diagram of the pMF9-loxP plasmid digestion verification results, where lane 1 is the marker, and lanes 2 to 5 are the pMF9-loxP digestion products (parallel). [Figure 15] Electrophoresis gel of the prepared pMF9-loxP linear vector, where lane 1 is the marker, and lanes 2 and 3 are the pMF9-loxP digestion products (parallel). [Figure 16] Figure 1 shows the Sanger sequencing results of pMF9-loxP-5.5 kb, where each arrow above represents the actual sequence obtained by one sequencing reaction, and nine sequencing reactions completely cover the 5.5 kb fragment to be sequenced. [Figure 17] 1 is a schematic diagram of the structure of the pMF65-loxP precursor empty vector plasmid containing the loxP-specific recombination site in the same direction, where pMB1 ori is the pMB1 replication origin, MCS is the multiple cloning site, and Redundant backbone includes KanR Gene is a backbone sequence containing the kanamycin resistance gene. [Figure 18]Electrophoresis gel of the prepared pUC57(KanR) linear vector template, where lane 1 is the marker and lanes 2 and 3 are the pUC57(KanR) plasmid digestion products (parallel). [Figure 19] Electrophoresis gel of the pMF65-loxp precursor empty vector plasmid fragment preparation, where lane 1 is the marker, lanes 2 and 3 are the amplification products (parallel) of pUC57 (KanR) containing the pMB1 replicon fragment, and lanes 4 and 5 are the amplification products (parallel) of the fragment containing the KanR selectable marker gene and loxp71 / 66 sites. [Figure 20] Figure 1 shows the sequencing results of the pMF65-loxp precursor empty vector plasmid, where each arrow above represents the actual sequence obtained from one sequencing reaction, and six sequencing reactions completely cover the entire sequenced plasmid. [Figure 21] 1 is a schematic diagram of the structure of the pMF65-loxP-RFP precursor plasmid containing the loxP-specific recombination site in the same direction, where pMB1 ori is the pMB1 replication origin, RFP cassette is the inserted target gene, and Redundant backbone includes KanR Gene is the backbone sequence containing the kanamycin resistance gene. [Figure 22] Electrophoresis gel of the fragment preparation of pMF65-loxp-RFP precursor plasmid, where lane 1 is the marker, lanes 2 and 3 are RFP amplified fragments, and lanes 4 and 5 are pMF65-loxp vector fragments. [Figure 23] Figure 1 shows the sequencing results of the pMF65-loxp-RFP precursor plasmid, where each arrow above represents the actual sequence obtained by one sequencing reaction, and five sequencing reactions completely cover the entire sequenced plasmid. [Figure 24]1 is a schematic diagram of the structure of the pMF7-loxP-RFP precursor plasmid containing the loxp-specific recombination site in the same direction, where pUC ori: pUC replication origin, RFP cassette: inserted target gene, Redundant backbone includes KanR Gene: backbone sequence containing the kanamycin resistance gene, and rop: rop gene. [Figure 25] 1 is a schematic diagram of the structure of the pMF5-loxP-RFP precursor plasmid containing the loxp-specific recombination site in the same direction, where R6Kγ ori: the R6Kγ replication origin, RFP cassette: the inserted target gene, and redundant backbone includes KanR Gene: a backbone sequence containing the kanamycin resistance gene. [Figure 26] 1 is a schematic diagram of the structure of a precursor plasmid containing the specific recombination site FRT in the same direction, where the backbone includes pMB1 ori: a backbone sequence containing the pMB1 replication origin site, RFP cassette: an expression element containing the target gene, and redundant backbone includes KanR Gene: a backbone sequence containing the kanamycin resistance gene. [Figure 27] Electrophoresis gel of FRT-RFP fragment and vector amplification, where lane 1 is the marker, lanes 2 and 3 are the amplification products of the FRT-RFP fragment (parallel), and lanes 4 and 5 are the amplification products of the pMF9-loxP-RFP vector fragment (parallel). [Figure 28] FIG. 1 shows the Sanger sequencing results of pMF9-FRT-RFP, where each arrow above represents the actual sequence obtained by one sequencing reaction, and four sequencing reactions completely cover the fragment to be sequenced. [Figure 29]Electrophoresis gel showing the verification of induced Cre-loxP 5.5 kb plasmid recombination, where lane 1 is the unrecombined precursor plasmid and lane 2 is the product 1 h after recombination: the main band is the resulting resistance gene-less plasmid, and the band corresponding to the precursor plasmid is clearly weaker; lanes 4 and 5 are the linear DNA bands of the digested plasmids in lanes 1 and 2; and lane 3 is the marker. [Figure 30] This figure shows the results of antibiotic plate screening. Here, the left panel shows the screening results for antibiotic-susceptible strains, and none of the 10 selected clonings grew colonies on the antibiotic plate. Test tubes #1 to #5 in the right panel show the growth status of colonies #1 to #5 in the left panel in non-antibiotic medium, where the colonies grew normally (the white dots on the plate in the left panel are holes created in the medium by the monocloning dotting procedure). [Figure 31] Electrophoresis gel of plasmid verification after antibiotic plate screening, where lanes #1-5 on the left are the plasmid products extracted from test tubes #1-5 in Figure 30 (five parallel clonings), lane 6 in the middle is the marker, and lanes #1-5 on the right are the digestion products of the plasmids in lanes #1-5 on the left. [Figure 32] Electrophoresis gel showing the results of induced pMF65-loxp-RFP plasmid recombination. Lane 1 is the DL3000 marker, lane 2 is the unrecombined precursor plasmid, and lane 3 is the product 1 h after recombination. The main band is the resulting resistance gene-free plasmid, and the band corresponding to the precursor plasmid is clearly weaker. [Figure 33]Electrophoresis gel of plasmid verification after antibiotic plate screening, where lane 1 is the marker, lane 2 is the precursor plasmid control without recombination induction, and lane 3 is the daughter plasmid obtained 1 h after recombination induction. [Figure 34] Figure 1 shows the sequencing results of the plasmid after antibiotic plate screening, where each arrow above represents the actual sequence obtained by one sequencing reaction, and three sequencing reactions completely cover the entire plasmid to be sequenced, and the hollow arrow represents the map sequence where the actual sequence was sequenced, the gene being deleted. [Figure 35] Electrophoresis gel showing the results of induced pMF7-loxp-RFP plasmid recombination. Lane 1 is the Kb Ladder Marker, lane 2 is the unrecombined precursor plasmid, and lane 3 is the product 1 h after recombination. The main band is the resulting resistance gene-free plasmid, and the band corresponding to the precursor plasmid is clearly faint. [Figure 36] Electrophoresis gel diagram of plasmid validation after antibiotic plate screening, where lane 1 is the marker and lanes 1-7 are purified plasmid products (seven parallel plasmid products). [Figure 37] Figure 1 shows the sequencing results of the plasmid after antibiotic plate screening, where each arrow above represents the actual sequence obtained by one sequencing reaction, and three sequencing reactions completely cover the entire plasmid to be sequenced, and the hollow arrow represents the map sequence where the actual sequence was sequenced, the gene being deleted. [Figure 38]Electrophoresis gel of the digested plasmid after FLP-FRT recombination, where lanes 1 to 7 on the left and right respectively represent no recombination induction, 2-hour recombination induction, 5-hour recombination induction, 8-hour recombination induction, 2-hour induction followed by overnight incubation at 37°C, 5-hour induction followed by overnight incubation at 37°C, and 8-hour induction followed by overnight incubation at 37°C. Lanes 1 to 7 on the left are the plasmid products, lanes 1 to 7 on the right are the plasmid digestion products, and lane 8 in the middle is the marker. [Figure 39] FIG. 1 shows the screening of non-resistant strains, where the left panel shows the growth of 10 colonies on kanamycin and ampicillin plates, respectively, after inducing recombination for 2 h, 5 h, and 8 h, and the right panel shows the growth of 5 colonies cultured in a test tube without antibiotic medium after inducing recombination for 5 h. [Figure 40] Electrophoresis gel of the results of validation of plasmids from antibiotic-susceptible strains obtained by resistance screening, where different lanes are parallel clonings of the same strain selected under conditions inducing recombination for 2 h, 5 h, and 8 h, and lane 1 is the marker. [Figure 41] Electrophoresis gel showing the results of induced pMF5-loxp-RFP plasmid recombination. Lane 1 is a Kb ladder marker, lane 2 is the unrecombined precursor plasmids (1): pSC101(ts)-araBAD-cre and pMF5-loxp-RFP precursor plasmid, and lane 3 is the product (2) 1 h after recombination: pSC101(ts)-araBAD-cre, pMF5-loxp-RFP precursor plasmid, circular double-stranded DNA carrying the KanR gene, and pMF5-loxp-RFP. The band corresponding to the pMF5-loxp-RFP precursor plasmid is clearly weakened. [Figure 42]Electrophoresis gel of the results of validation of plasmids from antibiotic-susceptible strains obtained by resistance screening, where lanes 1 to 5 are five parallel clonings of the same strain, and lane 6 is the marker. [Figure 43] Figure 1 shows the sequencing results of the plasmid extracted from the antibiotic-susceptible strain obtained by resistance screening, where the precursor plasmid sequence is used as reference (plasmid elements are labeled with a black line below), and the upper region covered by the solid arrow represents the actual sequence sequenced. The results suggest that only the plasmid replication element and target gene sequence were detected in the plasmid product, but the antibiotic resistance gene KanR and other redundant vector sequences were not detected, which is consistent with the desired sequence of the marker-free plasmid product. [Figure 44] Electrophoresis gel of the verification results of the digestion product of pMF9-5.5 kb plasmid, where lane 1 is the pMF9-5.5 kb plasmid product, lane 2 is the digestion product of the pMF9-5.5 kb plasmid, and lane 3 is the marker. [Figure 45] FIG. 1 shows the results of Sanger sequencing of the bulk extracted pMF9-5.5 kb plasmid. [Figure 46] 1 is an electrophoresis gel diagram of the verification results of the digestion product of pMF65-RFP plasmid, in which lane 1 is the pMF65-RFP plasmid product, lane 2 is the digestion product of the pMF65-RFP plasmid, and lane 3 is a marker. [Figure 47] FIG. 1 shows the results of Sanger sequencing of bulk extracted pMF65-RFP plasmid. [Figure 48] 1 is an electrophoresis gel diagram of the verification results of the digestion product of pMF7-RFP plasmid, in which lane 1 is the pMF7-RFP plasmid product, lane 2 is the digestion product of the pMF7-RFP plasmid, and lane 3 is a marker. [Figure 49]FIG. 1 shows the results of Sanger sequencing of bulk extracted pMF7-RFP plasmid. DETAILED DESCRIPTION OF THE INVENTION

[0053] Unless otherwise defined, all scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. In order to facilitate understanding of the technical solutions according to this specification, some technical terms will be briefly explained below.

[0054] Unless the context permits or unless otherwise defined, the terms "comprise" or "comprises" herein include "consisting of" and / or "consisting essentially of," and the meaning of such terms is governed by the provisions of the Patent Act.

[0055] The terms "plasmid" and "plasmid vector" are used interchangeably herein and refer to a circular DNA molecule that has a replication origin (also called a DNA replication element) and is capable of autonomous replication in a host cell. A plasmid may be a natural plasmid or a modified plasmid. A plasmid may contain a selectable marker gene, such as an antibiotic resistance gene, so that host cells containing the plasmid can grow under specific culture conditions, while host cells not containing the plasmid cannot grow normally under those conditions. For example, host cells (e.g., E. coli) containing a plasmid with a tetracycline resistance gene can grow in a medium containing tetracycline, while host cells that do not contain or have lost the plasmid cannot grow or their growth is suppressed in a medium containing tetracycline. Therefore, by using a selectable marker gene, those skilled in the art can determine which host cells contain the desired plasmid and complete the screening process. A plasmid or a modified plasmid may further contain a cloning site to facilitate the insertion of a target gene. After insertion into a plasmid via a cloning site, the target gene can be replicated along with the plasmid to achieve target gene amplification, or, when constructed as an expression plasmid, the plasmid can be used to express the target gene in a host cell. The cloning site may be a single cloning site or a multiple cloning site. For ease of experimental manipulation, a multiple cloning site is usually preferred. A multiple cloning site, as used herein, refers to a DNA segment containing multiple sites recognized by a restriction endonuclease or other endonuclease (e.g., a homing endonuclease). For example, the restriction endonuclease may be AhdI, AclI, HindIII, SspI, MluCI, Tsp509I, PciI, AgeI, BspMI, BfuAI, SexAI, MluI, BceAI, NdeI, or EcoR I.

[0056] As used herein, a "precursor plasmid" refers to a parent plasmid for producing a target / daughter plasmid (e.g., a plasmid lacking a selectable marker gene). A "precursor plasmid" contains the following elements: a replication origin that allows the plasmid to replicate in a host cell to increase or maintain the number of the precursor plasmid or the produced target plasmid in the host cell; a selectable marker gene for screening the host cell; a target gene or a cloning site for inserting the target gene; and paired recombination sites for recombination in the presence of a corresponding recombinase to remove the sequence between the paired recombination sites. To produce a plasmid lacking a selectable marker gene, a selectable marker gene is typically inserted between the paired recombination sites (see Figure 1). Thus, during the recombination process, the precursor plasmid molecule forms two circular DNA molecules. One circular DNA molecule contains the replication origin and the target gene or cloning site, but does not contain the selectable marker gene. Correspondingly, the other circular DNA molecule contains the selectable marker gene but does not contain the replication origin and cloning site. The former is also referred to herein as a daughter or target plasmid, i.e., a plasmid lacking a selectable marker gene. For purposes of the present invention, a cloning site includes the cloning site itself, or is a cloning site into which a target gene has already been inserted. Alternatively, a precursor plasmid that does not contain a target gene in the cloning site (or may be referred to as a "platform plasmid" or "precursor empty vector plasmid") is considered to be a parent plasmid of a precursor plasmid that does contain a target gene in the cloning site, and both are included within the meaning of "precursor plasmid" herein.

[0057] A "daughter plasmid" (also referred to herein as a "mini-plasmid," "target plasmid," and "plasmid without a selection marker") refers to a sequence containing an origin of replication and a cloning site and / or a target gene, produced by recombination of precursor plasmids containing recombination sites (e.g., loxP sequences in the same orientation) in the presence of a recombinase (e.g., Cre recombinase). In some examples herein, the daughter plasmid contains an origin of replication and a cloning site and / or a target gene sequence, but does not contain a selection marker gene. In some other examples herein, the backbone sequence of the daughter plasmid refers to a sequence that does not contain a target gene or a cloning site, and the sequence length of the sequence is less than 1000 bp, less than 900 bp, less than 800 bp, less than 700 bp, less than 600 bp, less than 500 bp, less than 400 bp, less than 300 bp, or less than 200 bp. In some specific embodiments, the length of the backbone sequence of the daughter plasmid may be 878 bp, 864 bp, 708 bp, 623 bp, or 429 bp.

[0058] A "daughter plasmid" is a plasmid having the specific structure and characteristics described and claimed in the specification of this application. That is, it is a special plasmid, and therefore, the definition may exist independently of a "precursor plasmid" or a "parent plasmid." The inventors do not exclude that such a special plasmid (i.e., daughter plasmid) may be obtained by a method other than the present invention, for example, that such a special plasmid may be produced by another method without using a precursor plasmid, but as long as the resulting plasmid conforms to the structure and / or definition of a "daughter plasmid" herein, it will still fall within the scope of protection of this application.

[0059] As used herein, the term "recombination site" refers to a nucleotide sequence that can be specifically recognized by a corresponding recombinase. When a precursor plasmid contains paired recombination sites in the same orientation, recombination occurs between the recombination sites in the precursor plasmid through the action of the corresponding recombinase, generating a daughter plasmid molecule and a circular DNA molecule. The sequence orientation of the paired recombination sites in the precursor plasmid is the same. Through the action of the corresponding recombinase, the DNA fragment between the two recombination sites is deleted, generating two DNA molecules. In some embodiments, the recombination site is a loxP sequence, and the corresponding recombinase is Cre recombinase. In other embodiments, the recombination site is an FRT sequence, and the corresponding recombinase is Flp recombinase. In still other embodiments, the recombination site is an attB / attP sequence, and the recombinase is PhiC31 recombinase. Those skilled in the art will understand that the recombination sites used in the precursor plasmid may be such that, upon recombination, the sequence between them (including the selectable marker gene) is removed from the precursor plasmid and the remaining sequence is formed in the daughter plasmid. Therefore, these recombination sites are not limited to the specific sequences mentioned herein, but may be variants thereof or other recombination sites. For example, the loxP recombination site mutant may be lox75, lox44, lox76, lox43, lox72, lox78, lox65, lox511, lox5171, or lox2272. The FRT recombination site may be wild-type or a mutant such as FRT3 or FRT5. In a specific embodiment herein, the loxP recombination site is a lox71 or lox66 sequence, and the nucleotide sequence is as set forth in SEQ ID NOs: 25 and 26. In another specific embodiment, the nucleotide sequence of the FRT recombination site is as set forth in SEQ ID NO: 27. In a specific embodiment, the attB and attP recombination site sequences are as set forth in SEQ ID NOs: 48 and 49, respectively.

[0060] The term "recombinase" refers to an enzyme involved in the gene positioning and recombination process. It recognizes and cuts specific recombination sites, linking two molecules involved in recombination. As used herein, a recombinase may be Cre recombinase, Flp recombinase, or phiC31 recombinase. In some embodiments, a gene encoding a recombinase comprises a sequence having at least 80%, 85%, 90%, 91%, 93%, 95%, 97%, or 99% identity to the nucleotide sequence set forth in SEQ ID NO: 1, 24, or 50. For example, a gene encoding Cre recombinase comprises the nucleotide sequence set forth in SEQ ID NO: 1, Flp recombinase comprises the nucleotide sequence set forth in SEQ ID NO: 24, and phiC31 recombinase comprises the nucleotide sequence set forth in SEQ ID NO: 50.

[0061] As used herein, the term "selection marker" or "selection marker gene" refers to a selectable marker gene segment in a plasmid, vector, or cell. Introducing a gene into cells, particularly cultured bacteria or cells, can result in the expression of a suitable trait for artificial selection. It is a reporter gene, used in the fields of microbiology, molecular biology, and genetic engineering to indicate whether exogenous DNA has been successfully transfected or transformed into cells by other methods. A selectable marker gene may typically be an antibiotic resistance gene, or some auxotrophic selectable markers, such as glucosamine synthase selectable markers and mannose phosphate isomerase selectable markers, or a negative selectable marker, such as an antivirulence gene or an anti-SacB selectable marker. As used herein, a selectable marker gene may be an expression cassette and may contain other sequences for expressing the selectable marker gene, such as a promoter, enhancer, and terminator. The SacB gene is a structural gene encoding levosucrose in Bacillus subtilis. Expression of SacB in Gram-negative bacteria is toxic in the presence of sucrose.

[0062] The terms "screening stress" and "selection stress" are used interchangeably. As used herein, they refer to the application of a specific substance or condition to an organism, such as a host cell, in a culture condition (e.g., a medium), allowing organisms that are adapted to these specific substances or conditions to survive and selecting out organisms that are not adapted. For example, as used herein, an antibiotic such as kanamycin or ampicillin can be added to a culture medium for host cells, and the medium can contain an antibiotic selection stress to screen for host cells that have the corresponding antibiotic resistance.

[0063] As used herein, "adjacent" refers to two DNA sequence fragments located upstream and downstream in the direction of gene replication, and being very close to each other. For example, the distance between the two gene fragments is less than 100 bp, less than 90 bp, less than 80 bp, less than 70 bp, less than 60 bp, less than 50 bp, less than 40 bp, less than 30 bp, less than 20 bp, less than 10 bp, less than 5 bp, less than 3 bp, or less than 1 bp. In some embodiments, the two gene fragments are directly linked. As used herein, in a precursor plasmid, the distance between the replication origin and the target gene or cloning site in the replication direction may be less than 50 bp, less than 45 bp, less than 40 bp, less than 35 bp, less than 30 bp, less than 25 bp, less than 20 bp, less than 15 bp, less than 10 bp, less than 5 bp, less than 3 bp, or less than 1 bp. In some embodiments, the replication origin is directly linked to the target gene or cloning site. In some other embodiments, the distance in the replication direction between the replication initiation site and the target gene or cloning site is 30 bp, 25 bp, 20 bp, 15 bp, 14 bp, 13 bp, 12 bp, 11 bp, 10 bp, 9 bp, 8 bp, 7 bp, 6 bp, 5 bp, 4 bp, 3 bp, 2 bp, or 1 bp.

[0064] As used herein, "paired recombination sites adjacent to the replication initiation site and the target gene, respectively" means that the distance between any one of the paired recombination sites and one of the replication initiation site and target gene closer to the replication site can be less than 100 bp, less than 90 bp, less than 80 bp, less than 70 bp, less than 60 bp, less than 50 bp, less than 40 bp, less than 30 bp, less than 20 bp, less than 10 bp, less than 5 bp, 3 bp, or less than 1 bp. In some embodiments, one or two of the paired recombination sites are directly linked to the replication initiation site and the target gene, i.e., the distance is 0 bp. In some other embodiments, the distance between any one of the paired recombination sites and the location of one of the replication initiation sites and target genes closer to the replication site may be 70 bp, 65 bp, 60 bp, 55 bp, 50 bp, 45 bp, 40 bp, 35 bp, 30 bp, 25 bp, 20 bp, 15 bp, 14 bp, 13 bp, 12 bp, 11 bp, 10 bp, 9 bp, 8 bp, 7 bp, 6 bp, 5 bp, 4 bp, 3 bp, 2 bp, or 1 bp. In some embodiments, one of the paired recombination sites is directly linked to the replication origin and the target gene, and the distance between the other of the paired recombination sites and the replication origin or the target gene may be 70 bp, 65 bp, 60 bp, 55 bp, 50 bp, 45 bp, 40 bp, 35 bp, 30 bp, 25 bp, 20 bp, 15 bp, 14 bp, 13 bp, 12 bp, 11 bp, 10 bp, 9 bp, 8 bp, 7 bp, 6 bp, 5 bp, 4 bp, 3 bp, 2 bp, or 1 bp. In some other embodiments, both of the paired recombination sites are directly linked to the replication origin and the target gene.Similarly, "paired recombination sites are adjacent to the replication initiation site and the cloning site, respectively" means that the replication initiation site is adjacent to the cloning site, and the distance between any one of the paired recombination sites and the position of the replication initiation site and the cloning site closer to the replication site can be less than 100 bp, less than 90 bp, less than 80 bp, less than 70 bp, less than 60 bp, less than 50 bp, less than 40 bp, less than 30 bp, less than 20 bp, less than 10 bp, less than 5 bp, 3 bp, or less than 1 bp. In some embodiments, one or two of the paired recombination sites are directly linked to the replication initiation site and the cloning site, i.e., the distance is 0 bp. In some other embodiments, the distance between any one of the paired recombination sites and the location of one of the replication initiation sites and cloning sites closer to the replication site may be 70 bp, 65 bp, 60 bp, 55 bp, 50 bp, 45 bp, 40 bp, 35 bp, 30 bp, 25 bp, 20 bp, 15 bp, 14 bp, 13 bp, 12 bp, 11 bp, 10 bp, 9 bp, 8 bp, 7 bp, 6 bp, 5 bp, 4 bp, 3 bp, 2 bp, or 1 bp. In some embodiments, one of the paired recombination sites is directly linked to the origin of replication and the cloning site, and the distance between the location of the other of the paired recombination sites and the origin of replication or the cloning site may be 70 bp, 65 bp, 60 bp, 55 bp, 50 bp, 45 bp, 40 bp, 35 bp, 30 bp, 25 bp, 20 bp, 15 bp, 14 bp, 13 bp, 12 bp, 11 bp, 10 bp, 9 bp, 8 bp, 7 bp, 6 bp, 5 bp, 4 bp, 3 bp, 2 bp, or 1 bp. In some other embodiments, both of the paired recombination sites are directly linked to the origin of replication and the cloning site. Preferably, in the related plasmids described, "the paired recombination sites are adjacent to the origin of replication and the target gene upstream and downstream, respectively," and the origin of replication is also adjacent to the target gene.

[0065] As used herein, the term "target gene" refers to a gene in which different nucleotide sequences can be designed according to different needs, and may include different element sequences such as a promoter, a gene encoding an expressed protein, and a terminator, and may further include regulatory elements such as an enhancer and poly A. The target gene may further include mRNA genes encoding protein or peptide antigens and protein or peptide therapeutic agents, mRNA, shRNA, RNA, or microRNA encoding RNA therapeutic agents, mRNA, shRNA, RNA, or microRNA encoding RNA vaccines, etc.

[0066] As used herein, the term "host cell" refers to a cell capable of maintaining and / or replicating a plasmid therein, including prokaryotic and eukaryotic cells such as bacteria (Escherichia coli), fungi (yeast), insect cells, and mammalian cells. In the method for producing a selectable marker gene-free plasmid according to the present invention, the host cell provides the components necessary for replication of the plasmid therein (e.g., various enzymes and nucleotide monomer molecules) and further provides the recombinase required for recombination. Expression of the recombinase in the host cell is preferably controllable or inducible. In some embodiments, the recombinase-encoding gene is integrated into the genome of the host cell. In some other embodiments, the recombinase-encoding gene is inserted into a separate expression vector, which can be introduced into the host cell together with the precursor plasmid, either before or after the precursor plasmid. In some embodiments, the recombinase-encoding gene is contained in the precursor plasmid, and can express the recombinase after introduction into the host cell. In either embodiment, it is preferable to control the recombinase gene with an inducible promoter, allowing the operator to control the onset time of recombination.

[0067] As used herein, the term "conditionally inducible deletional plasmid replication element" refers to a replication element that loses its replication ability under certain conditions (e.g., high temperature and inducer induction), thereby causing the plasmid to be gradually lost during propagation in host cells. The replication element may be a temperature-sensitive replication element or a metal ion-inducible replication element, such as pSC101 ori (ts).

[0068] In the case of sequences, the term "identity" refers to the degree of identity between two sequences (e.g., a query sequence and a reference sequence), and is usually expressed as a percentage. Usually, before calculating the percent identity between two sequences, sequence alignment is first performed and gaps, if any, are introduced. If the base or amino acid in the two sequences is the same at a certain alignment position, the two sequences are considered to be identical or matched at that position; if the base or amino acid in the two sequences is different, the two sequences are considered to be unequal or mismatched at that position. In some algorithms, the sequence identity is obtained by dividing the number of matching positions by the total number of positions within the alignment window. In some other algorithms, the number and / or length of gaps are also taken into account. For the purposes of the present invention, the disclosed alignment software BLAST (which can be found at ncbi.nlm.nih.gov) may be used to obtain optimal sequence alignment by using default settings and calculate the sequence identity between two nucleotide or amino acid sequences.

[0069] As used herein, "plasmid copy number" refers to the number of copies of a plasmid in each cell. Single copy refers to a cell containing only one plasmid, while multicopy refers to a cell containing multiple plasmids. Increasing the plasmid copy number increases the production yield of the plasmid. As used herein, the copy number of a daughter plasmid is typically 10-20, and a high copy number can reach 500-800 or even higher.

[0070] "Plasmid backbone sequence" refers to a sequence in a genetic engineering plasmid that loads a target gene as a template so that the target gene sequence can be selected and amplified in a host cell. The plasmid backbone sequence may contain functional elements for plasmid replication and other functional elements related to plasmid productivity. The plasmid backbone sequence may be of eukaryotic, prokaryotic, or viral origin and does not contain a target gene.

[0071] The term "cloning site" refers to any nucleotide or sequence of nucleotides that serves to link one polynucleotide (e.g., a polynucleotide of a target gene) to another polynucleotide (e.g., a cloning vector). Typically, a cloning site contains one or more sites recognized by a restriction endonuclease, e.g., a multiple cloning site. In some examples, a "cloning site" may be a multiple cloning site (also called an "MCS" or "polylinker"). A multiple cloning site refers to a DNA segment that contains multiple sites recognized by a restriction endonuclease or other endonucleases (e.g., a homing endonuclease).

[0072] PolyA refers to a polyadenylation signal or site. Polyadenylation refers to the addition of a poly(A) tail to an RNA molecule. Polyadenylation signals contain sequence motifs recognized by RNA cleavage complexes. Most human polyadenylation signals contain the AAUAAA sequence and its conserved sequences 5' and 3'. Commonly used polyA signals are from rabbit beta-globin, bovine growth hormone, and SV40 early or SV40 late polyA signals.

[0073] The pMB1 origin of replication (pMB1 ori) refers to the origin of replication derived from the pMB1 plasmid or may be a derivative of the origin of replication from the pMB1 plasmid. The sequence of the pMB1 origin of replication may be as set forth in SEQ ID NO: 44, and the sequence of the pMB1 origin of replication derivative may be as set forth in SEQ ID NO: 43.

[0074] pUC origin of replication (pUC ori) refers to a replication origin derived from pBR322 with a G to A substitution. It lacks the rop negative regulator and can increase copy number at elevated temperatures. Its sequence may be as set forth in SE ID NO: 45.

[0075] The R6K replication initiation site (R6K ori) refers to the region specifically recognized by the R6K Rep protein to initiate DNA replication. It includes, but is not limited to, the R6Kγ replication initiation site sequence shown in SEQ ID NO: 46, and further includes the CpG-free version described by Drocourt et al. in U.S. Patent No. 7,244,609, which is incorporated herein by reference.

[0076] The rop gene sequence is a repressor of the primer. As used herein, the precursor plasmid may contain the rop gene sequence. Removal of the rop sequence from the daughter plasmid by a recombinase results in a significant increase in the plasmid copy number.

[0077] The term "transfection" refers to methods for delivering nucleic acids into cells, such as poly(lactic-co-glycolic acid) (PLGA), ISCOMs, liposomes, nonionic surfactant vesicles (niosomes), virosomes, Pluronic block copolymers, chitosan, and other biodegradable polymers, particles, microspheres, calcium phosphate nanoparticles, nanoparticles, nanocapsules, nanospheres, poloxamine nanospheres, electroporation, nucleofection, piezoelectric penetration, sonoporation, iontophoresis, ultrasound, SQZ high-speed cell deformation-mediated membrane disruption, corona plasma, plasma-enhanced delivery, tissue-resistant plasma, laser microporation, shock wave energy, magnetic fields, non-contact magnetic field penetration, gene guns, microneedles, microabrasion, hydrodynamic delivery, high-pressure tail vein injection, and the like, are known in the art and are incorporated herein by reference.

[0078] As used herein, "terminator" or "transcription terminator" refers to a DNA sequence at the end of a marker gene or operon for transcription in bacteria. This may be an endogenous transcription terminator or a Rho-dependent transcription terminator. In the case of an internal terminator, such as the trpA terminator, a hairpin structure is formed in the transcript, disrupting the mRNA-DNA-RNA polymerase ternary complex. Alternatively, Rho-dependent transcription terminators require Rho factors (RNA helicase protein complexes) to disrupt the nascent mRNA-DNA-RNA polymerase ternary complex. In eukaryotes, the poly(A) signal is not a "terminator"; it is cleaved within the poly(A) site, leaving an unblocked 5' end of the 3' UTR RNA for nuclease digestion. The nuclease then catches up with RNA Pol II, resulting in termination. Transformation of an RNA Pol II pause site (a eukaryotic transcription terminator) can promote termination within a short region of the poly(A) site. The pause of RNA Pol II allows the nuclease that transforms the 3'UTR mRNA after polyA cleavage to catch up with RNA Pol II at the pause site. A non-limiting list of eukaryotic transcription terminators known in the art includes C2x4 and gastrin terminators. Eukaryotic transcription terminators can improve mRNA levels by enhancing the proper 3' end processing of mRNA.

[0079] The present invention further provides a method for establishing a recombinase-inducible expression system, including a method for constructing an expression vector containing a recombinase-encoding gene and transforming a host bacterium with the vector, and a method for constructing a host cell genome containing the recombinase gene. Taking a Cre recombinase-inducible expression vector as an example, the method for constructing an expression vector containing a recombinase-encoding gene includes constructing a cre gene fragment and a pSC101-araBAD linear vector, assembling the cre gene fragment and the pSC101-araBAD linear vector into a pSC101-araBAD-cre(AmpR) plasmid, transforming the plasmid into competent cells, culturing and verifying, selecting clones verified as correct, and extracting the plasmid. For example, the method for constructing a Cre recombinase temperature-sensitive inducible expression vector includes constructing a temperature-sensitive replicon pSC101 ori (ts) fragment in a pCP20 plasmid, and assembling the pSC101-araBAD-cre(AmpR) plasmid into a pSC101-araBAD-cre(AmpR) plasmid. R ) plasmid to generate araBAD-cre(Amp R ) fragment and the pSC101 ori (ts) fragment and araBAD-cre (Amp R ) Assembling the fragments, transforming the assembled fragments into competent cells, culturing and verifying them, selecting clones that are verified as successful, extracting the plasmids, and obtaining expression vectors.

[0080] The Cre recombinase can be integrated into the host cell genome by preparing a cre gene fragment and a pSC101-araBAD linear vector, or by integrating the cre gene fragment and the pSC101-araBAD linear vector into pSC101-araBAD-cre(Amp R) Assembling the plasmid, transforming the plasmid into competent cells, culturing and verifying; selecting clones that are verified to be correct, extracting the plasmid; using λRed recombination technology and CRISPR / Cas9 technology to perform intact knock-in of a Cre-specific recombinase inducible expression system into host cells, and culturing the host cells to obtain a strain that can express Cre recombinase inducibly. The method for incorporating Flp recombinase into host cells includes preparing the flp gene fragment and pSC101(ts)-araBAD linear vector; assembling the flp gene fragment and pSC101(ts)-araBAD into the pSC101(ts)-araBAD-flp plasmid; transforming the plasmid into competent cells, culturing and verifying; selecting clones that are verified to be correct; extracting the plasmid; using λRed recombination technology and CRISPR / Cas9 technology to perform intact knock-in of the Cre-specific recombinase-inducible expression system into host cells; and culturing the host cells (e.g., E. coli) to obtain an inducible expression strain of Flp recombinase.

[0081] For example, in some embodiments, a host cell (e.g., E. coli) into which a recombinase-encoding gene has been integrated may first be transformed with a precursor plasmid. E. coli containing the precursor plasmid are screened for a selectable marker and cultured. A portion of the culture is exposed to a recombinase-expressing inducer (the other portion of the culture may be saved for later use). Upon expression of the recombinase, the precursor plasmid recombines between the recombination sites to form daughter plasmid molecules lacking the selectable marker gene and circular DNA molecules containing the selectable marker gene. The host cells are continued to be cultured, and the host cells are screened for the selectable marker. Host cells containing the precursor plasmid (unrecombined) or circular DNA will have the selectable marker characteristic (e.g., antibiotic resistance), while host cells containing only the daughter plasmid will not have the selectable marker characteristic (i.e., no antibiotic resistance). Host cells that cannot grow on the appropriate antibiotic are selected. Host cells lacking the selectable marker characteristic are cultured to obtain a plasmid without the selectable marker gene.

[0082] In embodiments where the recombinase is introduced into the host cell via an expression vector, to prevent contamination of the final product (i.e., the selectable marker gene plasmid) with the expression vector, the expression vector preferably has an inducible deletion expression, for example, contains an inducible deletion replication element, or uses the temperature sensitive replication element from the pSC101(ts) plasmid.

[0083] In some particular embodiments, the method for producing a selectable marker gene-free plasmid according to the present invention comprises: (1) constructing precursor plasmids that can be recombined into circular DNA; (2) constructing a site-specific recombinase-inducible expression system in E. coli; (3) transforming the precursor plasmid into a site-specific recombinase-expressing strain and screening for positive clones; (4) culturing the cells, inducing expression of the recombinase, and allowing the precursor plasmids to self-recombine, forming a circular double-stranded DNA molecule containing a selectable marker-free mini-plasmid molecule containing the target gene and a replication origin, and a plasmid backbone sequence (e.g., a selectable marker gene); (5) isolating, purifying, and screening the monoclonal strains without a selectable marker gene; (6) culturing the cells and extracting the plasmid.

[0084] Those skilled in the art will understand that in some cases, one or more of the above-mentioned steps may be omitted or the order thereof may be changed, so long as the daughter plasmid of the present invention, i.e., the unmarked plasmid, is ultimately obtained. Therefore, in some other specific embodiments, the method for producing the unmarked plasmid according to the present invention comprises: 1) introducing the precursor plasmid of the present invention into a host cell capable of expressing or supporting the expression of a recombinase, and screening for host cells that express a selectable marker gene; 2) culturing the host cells screened in step 1) to allow expression of the recombinase in the host cells, and culturing the host cells to screen for host cells that do not express the selection marker gene; 3) culturing the host cells screened in step 2) to obtain a plasmid without a selection marker gene, and extracting the plasmid.

[0085] In some embodiments, the production method further comprises, prior to step 1), a step of obtaining or producing a precursor plasmid of the present invention. In some embodiments, the screening in steps 1) and 2) both uses a screening stress corresponding to the selectable marker. In some embodiments, the cell culture in step 3) is free of a screening stress.

[0086] The recombinase system, whether present on the precursor particle, in the host cell, or both, may be an inducible or constitutive expression system.

[0087] As used herein, "constitutive expression" refers to gene expression (e.g., a recombinase system) that is not affected by time, location, or environment and has no temporal or spatial specificity. In contrast to inducible expression, constitutive expression allows stable expression without the need for induction of other factors, whereas inducible expression requires the induction of other factors for expression.

[0088] More specifically, the method for producing a markerless plasmid according to the present invention comprises the following steps:

[0089] (1) Construct a precursor plasmid that can be recombined into a circular DNA.

[0090] The precursor plasmid contains a replication initiation site (e.g., a DNA replication element), a selectable marker gene, and a pair of unidirectional specific recombination sites and a target gene. The target gene does not contain a specific recombination site of the same type as the plasmid backbone. In addition to the DNA replication element, the plasmid backbone sequence (e.g., a selectable marker gene) is located within the pair of specific recombination sites. The precursor plasmid containing the target gene sequence and DNA replication element can be reconstituted by the action of a site-specific recombinase and split into two circular double-stranded DNA fragments. Here, one contains the plasmid backbone sequence (e.g., a selectable marker gene), has no replication ability, and is gradually lost during cell amplification. The other is a mini-plasmid containing only the DNA replication element and target gene sequence, which can be continuously amplified along with the cell amplification process.

[0091] (2) Construct a site-specific recombinase-inducible expression system in E. coli.

[0092] A site-specific recombinase-inducible expression system can be constructed in a plasmid vector and transformed into an E. coli host cell for expression, or integrated into the E. coli host cell genome for expression. The expression system comprises an inducible prokaryotic transcription promoter (e.g., the araBAD promoter), a site-specific recombinase gene, and a transcription terminator. The site-specific recombinase gene may comprise Cre recombinase derived from the P1 phage Cre-loxP recombination system, which corresponds to a pair of unidirectional specific recombination sites, the loxP sequences (e.g., lox71 / lox66), in a. (1), or Flp recombinase derived from the brewer's yeast Flp-FRT recombination system, which corresponds to a pair of unidirectional specific recombination sites, the FRT sequences, in b. (1). When the recombinase expression system is constructed on a plasmid vector, the vector plasmid may be a conditionally inducible deletion type plasmid replication element, such as the temperature-sensitive replication element pSC101(ts) plasmid, to ensure that the selectable marker-free plasmid product is free of recombinase expression plasmid contamination.

[0093] (3) Transform the precursor plasmid into a site-specific recombinase-expressing strain and screen for positive clones.

[0094] Competent cells are prepared from an E. coli strain carrying a site-specific recombinase-inducible expression system, and the precursor plasmid is transformed into the cells. Positive transformed clones are screened using a selective medium corresponding to the selectable marker gene in the precursor plasmid. If the recombinase system is constructed on a plasmid, the recombinase expression plasmid and the precursor plasmid may be co-transformed into conventional E. coli competent cells and cultured using a double selective medium to screen for positive transformed clones.

[0095] (4) The cells are cultured to induce expression of the recombinase and allow the precursor plasmid to self-recombine.

[0096] (3) Select positive clones transformed with the method (3), and culture and enrich the strain overnight in a selective medium corresponding to the selection marker. Wash the cells with a liquid medium without selection stress, and resuspend the strain in a medium without selection stress containing an inducer to induce expression of the recombinase, which causes recombination between the two recombination sites carried by the plasmid, and deletes the plasmid backbone sequence (e.g., the selection marker gene) between the recombination sites to form a mini-plasmid without the selection marker.

[0097] (5) Isolate, purify, and screen for monoclonal strains without selectable marker genes.

[0098] (4) The fully recombinant bacterial solution is lined on a plate without selective stress to isolate a single colony. The single colony is then replicated on a selective culture plate containing the selection marker gene and a non-selective culture plate (e.g., no antibiotic) and cultured overnight. Colonies on the antibiotic-free plate that cannot grow on the corresponding antibiotic plate are selected and cultured, thereby obtaining a strain containing only the selection marker plasmid.

[0099] (6) The strain obtained in (5) is cultured in a non-selective (e.g., antibiotic-free) medium, and the plasmid is extracted to obtain the final product, a mini-plasmid without a selection marker.

[0100] To avoid the safety risks of antibiotic resistance gene-based gene therapy and achieve mass production of highly pure plasmids, the mini-plasmid and production method of the present invention, which lacks a selectable marker gene (e.g., an antibiotic resistance selectable marker) and whose product plasmid backbone contains only a replication origin (DNA replication element), significantly reduces the proportion of bacterial-derived sequences in the product plasmid, thereby lowering potential safety risks. Furthermore, the isolation, purification, culture, and amplification of positive bacterial strains containing the plasmid product are possible, resolving the problem of low purity and difficulty in mass production of selectable marker-free plasmids.

[0101] The beneficial technical effects of the present invention include, but are not limited to: the plasmid obtained by the method of the present invention does not have an antibiotic selection marker and does not have any extra prokaryotic DNA elements other than the replication origin, and no antibiotic drug is added during the production process of the antibiotic selection marker-free plasmid, making it easy to scale up production and realizing mass production.

[0102] The selectable marker-free plasmid according to the present invention can be used in the field of gene and cell therapy as a DNA delivery vector or a virus packaging plasmid vector to improve the safety and stability of the plasmid and reduce cytotoxicity.

[0103] The technical solutions of the present invention will be further described in detail below through examples and with reference to the drawings. Unless otherwise specified, the methods and materials in the examples described below are all common products that are commercially available. Those skilled in the art will understand that the methods and materials described below are merely illustrative and should not be construed as limiting the scope of the present invention.

[0104] Example 1: Establishment of a Cre recombinase expression system

[0105] In this example, a site-specific recombinase inducible expression system was constructed in E. coli JM108. This system includes the inducible prokaryotic transcription promoter araBAD promoter, the site-specific recombinase gene cre, and a transcription terminator. The system was constructed and expressed in the host bacterium using the following two methods.

[0106] 1.1 A Cre-specific recombinase-inducible expression system was constructed on the pSC101(ts) replication system vector plasmid and transformed into JM108 for expression. The specific steps are as follows:

[0107] (1) Preparation of the cre gene fragment and the pSC101-araBAD linear vector Preparation of cre gene fragment: The cre fragment was amplified with primers cre-F and cre-R (primer sequences are shown in SEQ ID NO: 2 and SEQ ID NO: 3, respectively). The cre fragment was synthesized by Nanjing GenScript Biotechnology Co., Ltd. (the cre gene sequence is shown in SEQ ID NO: 1). The amplification system and PCR system are shown in Tables 1 and 2. Phusion® HF DNA polymerase is commercially available from New England Biotechnology Co., Ltd., product number 10058481. The theoretical size of the cre gene fragment is 1116 bp. The amplified product was subjected to agarose gel electrophoresis, and the fragment size of the electrophoresed amplified product was confirmed to be accurate (as shown in Figure 2). The gel was recovered to obtain the cre gene fragment.

[0108] [Table 1]

[0109] [Table 2]

[0110] Preparation of pSC101-araBAD linear vector: The pSC101-araBAD linear vector was derived from the pKD46 plasmid, which has the NCBI sequence ID: AY048746.1. The pKD46 plasmid was digested with EcoRI and the linear vector was recovered. The digestion system was as shown in Table 3. EcoRI restriction endonuclease is commercially available from New England Biotechnology, Inc., product number 10079659. The digestion system was reacted at 37°C for 45 minutes. The digestion product was loaded onto an agarose gel and electrophoresed. As shown in Figure 3, two bands were observed after digestion, with sizes of 4820 bp + 1509 bp (4820 bp and 1509 bp, respectively), which was consistent with the theoretical values ​​(4820 bp and 1509 bp). The 4820 bp vector fragment was recovered by cutting the gel, thereby obtaining the pSC101-araBAD linear vector.

[0111] [Table 3]

[0112] (2) pSC101-araBAD-cre(Amp R ) Plasmid organization The assembly system is shown in Table 4. The Gene Builder™ Cloning Kit is available from Nanjing Jinsirui Biotechnology Co., Ltd., product number C20012009. The reaction mixture was mixed and incubated at 50°C for 15 minutes. The product was transformed into DH10b competent cells and cultured in a culture chamber at 37°C.

[0113] [Table 4]

[0114] (3) pSC101-araBAD-cre(Amp R ) Plasmid verification Single colonies were selected from the transformation plates from the previous step and transferred to 48-well plates and grown overnight at 37°C and 220 rpm. Colony PCR was performed using primers cre-seq1 / cre-seq2 (the sequences of the two primers are shown in SEQ ID NO: 4 and SEQ ID NO: 5, respectively, and the amplified fragment size was 1069 bp). The products were verified by agarose gel electrophoresis. As shown in Figure 4, colonies 1-12 show bands in the agarose gel electrophoresis of the colony PCR products. All clones except clone #7 showed bands, indicating potential successful assembly. Positive clones with the correct fragment size were selected, and the plasmids were extracted. These plasmids were sent to Nanjing Jinsirui Biotechnology Co., Ltd. for Sanger sequencing. The sequencing results were accurate (as shown in Figure 5, the tested sequence was completely consistent with the mapped sequence (black line in the figure), indicating no gene deletions or mutations and successful plasmid assembly). pSC101-araBAD-cre(Amp R ) plasmid was successfully organized.

[0115] (4) Construction of temperature-sensitive plasmid pSC101(ts)-araBAD-cre Preparation of the temperature-sensitive replicon pSC101 ori (ts) fragment: Using the pCP20 plasmid (NCBI sequence ID: MK178598.1) as a template, PCR amplification was performed on the pSC101 ori (ts) fragment (the sequence of which is shown in SEQ ID NO: 23). The amplification primers, pSC101(ts)-F and pSC101(ts)-R, are shown in SEQ ID NO: 6 and SEQ ID NO: 7, respectively. The PCR system and procedure are shown in Tables 5 and 6. The pSC101 ori (ts) fragment product obtained by amplification was loaded onto an agarose gel and electrophoresed. As shown in lanes 2 and 3 in Figure 6, the exact size of the fragment is 1434 bp. Phusion® HF DNA polymerase is commercially available from New England Biotechnology, Inc., product number 10058481.

[0116] araBAD-cre(Amp R ) fragment: Digest the pSC101-araBAD-cre(Amp) fragment prepared in step (3) with NcoI and NotI. R The digestion system is shown in Table 7. As shown in lanes 4 and 5 in Figure 6, the exact fragment size of the digested product is 4431 bp. The large fragment was recovered by cutting the gel, thereby araBAD-cre(Amp R ) was obtained. Both Nco I and Not I restriction endonucleases are commercially available from New England Biotechnology, Inc., product numbers 10080424 and 10046577, respectively.

[0117] [Table 5]

[0118] [Table 6]

[0119] [Table 7]

[0120] pSC101(ts)-araBAD-cre(Amp R ) plasmid was assembled. As shown in Table 8, the reaction system was reacted at 50°C for 15 minutes. The assembled product was transformed into DH10b competent cells and cultured in a culture chamber at 30°C. The Gene Builder™ Cloning Kit is available from Nanjing Jinsirui Biotechnology Co., Ltd., product number C20012009.

[0121] [Table 8]

[0122] PCR verification was performed on the pSC101(ts)-araBAD-cre plasmid. Single colonies from the transformation plate in the previous step were selected and transferred to a 48-well plate and cultured at 30°C and 220 rpm. Eight clones that were correctly verified by PCR were randomly selected, and the plasmids were extracted and subjected to Sanger sequencing (provided by Nanjing Jinsirui Biotechnology Co., Ltd.). Referring to Figure 7, the sequencing results showed that the tested sequence was completely consistent with the map sequence (black line in the figure), indicating no gene deletions or mutations and successful plasmid organization. The pSC101(ts)-araBAD-cre plasmid was successfully organized.

[0123] 1.2 An inducible Cre recombinase expression system was integrated into the JM108 genome via λRed recombination and CRISPR / Cas9 editing. Strain editing and validation procedures 3)-5) were completed by Nanjing Jinsirui Biotechnology Co., Ltd. (CRISPR Products and Services (genscript.com.cn)). The specific steps are as follows:

[0124] 1) A CRISPR / Cas9 specific targeting point was designed between the cyaA and cyaY genes, where the length of the gRNA was 20 bp and the specific gRNA sequence was as shown in SEQ ID NO: 9.

[0125] 2) JM108-araBAD-cre(Amp) L-homology arm and JM108-araBAD-cre(Amp) R-homology arm were designed on both sides of the insertion site between the cyaA and cyaY genes. The specific homology arm sequences are SEQ ID NO: 10 and SEQ ID NO: 11, respectively, as shown in the sequence listing.

[0126] 3) Using λRed recombination and CRISPR / Cas9 technology, intact knock-in of a Cre-specific recombinase-inducible expression system (the sequence of which is shown in SEQ ID NO: 22) was performed into the E. coli JM108 strain.

[0127] 4) Colony PCR and agarose gel electrophoresis screening verification was performed on the edited strain (primers: C4818FK060-6JJF1 and C4818FK060-6JJR1, sequences are shown in SEQ ID NO: 14 and SEQ ID NO: 15, respectively). The theoretical size of the gel electrophoresis band obtained by colony PCR for successful knock-in should be greater than 3000 bp, while the theoretical size of the band for unsuccessful knock-in was 1297 bp. As shown in Figure 8, the results suggest that the band sizes of the colony PCR gel electrophoresis bands for successful knock-in are accurate.

[0128] 5) Sanger sequencing was performed on the PCR-verified clones. As shown in Figure 9, the results suggest that the sequencing results are accurate and there are no mutations.

[0129] Example 2: Establishment of Flp recombinase expression system

[0130] The Cre recombinase expression system described in Example 1 can be used to establish an Flp recombinase-inducible expression system. The Flp recombinase-inducible expression system includes an inducible prokaryotic transcription promoter (araBAD promoter), a site-specific recombinase gene (flp), and a transcription terminator. The system can be constructed and expressed in host bacteria using the following two methods:

[0131] 2.1 The Flp-specific recombinase-inducible expression system was constructed on the pSC101(ts) temperature-sensitive replication system vector plasmid, and JM108 was transformed with it for expression. The specific procedures are as follows:

[0132] (1) Construction of the flp gene fragment and the pSC101(ts)-araBAD linear vector

[0133] Preparation of flp gene fragment: The flp gene (the sequence of which is shown in SEQ ID NO: 24) was obtained from the pCP20 plasmid, and the flp gene was amplified using primers flp-F and flp-R (the sequences of the two primers are shown in SEQ ID NO: 16 and SEQ ID NO: 17, respectively). The amplification system and PCR system are shown in Tables 9 and 10. As shown in Figure 10, the agarose gel electrophoresis result shows that the correct size of the fragment is 1333 bp. Gel recovery was performed. Phusion® HF DNA polymerase is commercially available from New England Biotechnology, Inc., product number 10058481.

[0134] [Table 9]

[0135] [Table 10]

[0136] Preparation of pSC101(ts)-araBAD linear vector: The pSC101(ts)-araBAD-cre plasmid was digested with NdeI and EcoR I-HF (available from New England Biotechnology, product numbers 10064897 and 10079659, respectively) to prepare a linear vector. The size of the pSC101(ts)-araBAD-cre plasmid is 5876 bp. The digestion product was analyzed by agarose gel electrophoresis. As shown in the gel image in Figure 11, two bands were observed after digestion, with sizes of 4841 bp + 1034 bp, which were consistent with the theoretical values. The digestion system is shown in Table 11. The digestion system was treated at 37°C for 60 minutes. The large fragment was recovered by cutting the gel.

[0137] [Table 11]

[0138] (2) Organization of the pSC101(ts)-araBAD-flp plasmid

[0139] The assembly system is shown in Table 12. The assembly system was reacted at 50°C for 15 minutes. After assembly was completed, the assembly product was transformed into DH10b competent cells and cultured in a culture chamber at 30°C. The Gene Builder™ cloning kit is available from Nanjing Jinsirui Biotechnology Co., Ltd., product number C20012009.

[0140] [Table 12]

[0141] (3) Verification of pSC101(ts)-araBAD-flp plasmid

[0142] Eight single colonies were randomly selected from the plate and inoculated into 4 mL of LB liquid medium, followed by cultivation at 30°C and 220 rpm. The plasmid was extracted and subjected to Sanger sequencing, which was completed by Nanjing Jinsirui Biotechnology Co., Ltd. Referring to Figure 12, the sequencing results showed that the tested sequence was completely consistent with the map sequence (black line in the figure), indicating no gene deletions or mutations and successful plasmid organization.

[0143] 2.2 The inducible expression system of Flp recombinase was integrated into the JM108 genome and expressed by λRed recombination and CRISPR / Cas9 editing. The specific steps were the same as step 1.2 in Example 1.

[0144] Example 3: Construction of precursor plasmids containing lox71 / lox66 recombination sites

[0145] 3.1 Construction of pMF9-loxp precursor empty vector plasmid

[0146] The pMF9-loxp precursor empty vector plasmid contains a DNA replication element (pMB1 derivative, 784 bp, SEQ ID NO: 43), a resistance selection marker gene KanR, a pair of lox71 / lox66 specific recombination sites in the same direction (as shown in the sequence listing, the lox71 sequence is as shown in SEQ ID NO: 25, and the lox66 sequence is as shown in SEQ ID NO: 26), and a multiple cloning site (MCS). The multiple cloning site and replication element are located between lox71 and lox66 (as shown in Figure 13), in the following order: (1) The plasmid sequence is as shown in SEQ ID NO: 8 and was synthesized by Nanjing Jinsirui Biotechnology Co., Ltd. The synthesized pMF9-loxp plasmid was verified by digestion. The restriction endonuclease used is Hind III, available from New England Biotechnology, Inc., product number 10055811. The digestion system is shown in Table 13. The digestion system was incubated at 37°C for 45 minutes. The theoretical size of the target band should be 1176 bp + 2015 bp. As shown in Figure 14, agarose gel electrophoresis of the digestion products revealed that lanes 2 to 5 were four parallel digestion products with accurate band sizes, indicating that the resulting plasmid was accurate.

[0147] [Table 13]

[0148] 3.2. Preparation of pMF9-loxp linear vector

[0149] The pMF9-loxp plasmid was digested with BamHI-HF (New England Biotechnology, 10081013). The digestion system is shown in Table 14. The digestion system was incubated at 37°C for 45 minutes. The size of the pMF9-loxp plasmid is 3191 bp. As shown in Figure 15, agarose gel electrophoresis of the digestion products showed that lanes 2 and 3 were two parallel digestion products, and the band sizes after digestion were accurate. Gel recovery was performed.

[0150] [Table 14]

[0151] 3.3 Assembly of pMF9-loxp-5.5 kb precursor plasmid

[0152] A 5.5 kb fragment (target gene) without loxP sites was inserted between the loxP sites. It was amplified and recovered using a primer pair with homologous arms. The primer sequences are listed in the Sequence Listing (the sequences are shown as SEQ ID NO: 12 and SEQ ID NO: 13, respectively). The recovered fragment was assembled with the pMF9-loxp linear vector prepared in Example 3.2. The assembly system is shown in Table 15. Assembly was performed at 50°C for 15 minutes. The assembly system was transformed into JM108. 50 μL / 100 μL of the transformation product was added and cultured overnight at 37°C. The Gene Builder™ Cloning Kit is available from Nanjing Jinsirui Biotechnology Co., Ltd., product number C20012009.

[0153] [Table 15]

[0154] 3.4 Verification of pMF9-loxp-5.5 kb plasmid

[0155] The assembled pMF9-loxp-5.5 kb plasmid grew as single colonies on plates after transformation. Eight monoclonal clones were randomly selected and inoculated into 4 mL of LB liquid medium and cultured at 37°C and 220 rpm. The plasmid was extracted and subjected to Sanger sequencing. As shown in Figure 16, the Sanger sequencing results indicated successful assembly. Sanger sequencing was completed by Nanjing Jinsirui Biotechnology Co., Ltd.

[0156] Example 4. Construction of precursor plasmids containing lox71 / lox66 recombination sites and the pMB1 minimal replication element

[0157] 4.1 Construction of pMF65-loxp precursor empty vector plasmid

[0158] Plasmid safety is crucial for gene and cell therapy. Sequences on the plasmid backbone have potential immunogenicity and can cause adverse reactions. Shortening the plasmid backbone has become a trend in optimizing plasmids for gene and cell therapy. The precursor plasmid vector designed in this example was able to recombine to yield daughter plasmids containing only approximately 0.65 KB of the pMB1 replication element sequence, reducing the introduction of exogenous gene sequences, potentially lowering cytotoxicity and immunogenicity, and improving safety and stability.

[0159] The plasmid was constructed using the DNA replication element pMB1 (589 bp, SEQ ID NO: 44) and the resistance selection marker gene Kan R pUC57 (Kan R ) plasmid (the sequence of which is shown in SEQ ID NO: 29 and which was synthesized by Nanjing Jinsirui Biotechnology Co., Ltd.) The replication element and cloning site are located between lox71 and lox66 (the plasmid is shown in Figure 17), and the order includes, but is not limited to, this example.

[0160] The restriction endonucleases sal / BamHI at the multiple cloning insertion site of pUC57(Kan R ) plasmid and pUC57(Kan R ) plasmid was digested to form a linear vector. The digestion system is shown in Table 16. Referring to Figure 18, the results show that pUC57 (Kan R The plasmid is 2579 bp, and the band size is accurate. The gel was collected according to the gel kit. pUC57 (Kan R ) fragments, as well as Kan R pUC57 (Kan R The fragments were amplified with two primer pairs, respectively. The primer sequences for amplifying the fragment containing the pMB1 replicon are shown in SEQ ID NO: 30 and SEQ ID NO: 31. R The sequences of the primers for amplifying the fragment containing the selection marker and the loxp71 / 66 site are shown in SEQ ID NO: 32 and SEQ ID NO: 33. The PCR system and PCR procedure are shown in Table 17 and Table 18. As shown in Figure 19, the size of the amplified product is 668 bp in lanes 1 and 2, and the size of the fragment 1 containing the pMB1 replicon is 668 bp in lanes 3 and 4. R The size of fragment 2, which contains the resistance selection marker and the loxp71 / 66 site, is 1574 bp. All band sizes are accurate. The gel was collected according to the instructions in the gel kit. Primer Star GXL DNA polymerase is available from Takara Biomedical Technology (Beijing) Co., Ltd., product number R050A.

[0161] [Table 16]

[0162] [Table 17]

[0163] [Table 18]

[0164] The pMF65-loxp plasmid was assembled using the system shown in Table 19. The system was reacted at 50°C for 15 minutes. After assembly was completed, the assembled product was transformed into JM108 competent cells and cultured at 37°C. The Gene Builder™ Cloning Kit is available from Nanjing Jinsirui Biotechnology Co., Ltd., product number C20012009.

[0165] [Table 19]

[0166] Verification of pMF65-loxp plasmid

[0167] After transformation, the assembled pMF65-loxp plasmid grew as a single colony on the plate. A single clone was selected and inoculated into 4 mL of LB liquid medium and grown at 37°C and 220 rpm. The plasmid was extracted and subjected to Sanger sequencing. As shown in Figure 20, the Sanger sequencing results showed that the primer sequences covered the entire plasmid sequence, with no mutations, indicating successful assembly. Sanger sequencing was completed by Nanjing Jinsirui Biotechnology Co., Ltd.

[0168] 4.2 Construction of pMF65-loxp-RFP precursor plasmid

[0169] For the construction of the pMF65-loxp-RFP precursor plasmid, see the construction of pMF9-loxp-5.5 kb in Example 3. That is, the rfp red pigment synthesis gene (the sequence of which is shown in SEQ ID NO: 28) was inserted as an exogenous fragment (see Figure 21 for a structural diagram) into the MCS site of the pMF65-loxp plasmid.

[0170] The pMF65-loxp vector was amplified by PCR and the RFP fragment was inserted. The vector fragment size was 2182 bp, and the RFP fragment size was 1150 bp (the sequences of the two primer pairs are shown in SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, and SEQ ID NO: 38, respectively). The amplification system is shown in Table 20. The amplification procedure is shown in Table 21. Referring to Figure 22, the results show that lanes 2 and 3 are RFP amplified fragments, and lanes 4 and 5 are pMF65-loxp vector fragments, and their sizes are accurate. The gel was collected according to the gel kit and assembled using a Gene Builder™ cloning kit.

[0171] [Table 20]

[0172] [Table 21]

[0173] The pMF65-loxp-RFP precursor plasmid was assembled using the system shown in Table 22. The system was incubated at 50°C for 15 minutes. After assembly was complete, the assembled product was transformed into JM108 competent cells and cultured at 37°C. The Gene Builder™ Cloning Kit is available from Nanjing Jinsirui Biotechnology Co., Ltd., product number C20012009.

[0174] [Table 22]

[0175] Validation of pMF65-loxp-RFP precursor plasmid

[0176] After transformation, the assembled pMF65-loxp-RFP precursor plasmid grew as a single colony on the plate. A single clone was selected and inoculated into 4 mL of LB liquid medium and cultured at 37°C and 220 rpm. The plasmid was extracted and subjected to Sanger sequencing. As shown in Figure 23, the Sanger sequencing results showed that the primers used covered the entire plasmid sequence, with no mutations, indicating successful assembly. Sanger sequencing was completed by Nanjing Jinsirui Biotechnology Co., Ltd.

[0177] Example 5. Construction of high copy precursor plasmids containing lox71 / lox66 recombination sites

[0178] 5.1 Construction of pMF7-loxp-RFP plasmid

[0179] The plasmid in this example contains a high-copy pUC replicon, which improves the yield and purity of the selection marker-free plasmid, simplifies the manufacturing flow, reduces production costs, and is advantageous for mass production.

[0180] The plasmid was constructed using the DNA replication element pUC ori (674 bp, SEQ ID NO: 45) and the resistance selection marker gene Kan R pUC57 (Kan) containing a gene sequence encoding the Rop protein, a pair of specific recombination sites lox71 / lox66 in the same direction, and a target gene RFP sequence. R) plasmid (the sequence is shown in SEQ ID NO: 29). The replication element and target gene are located between lox71 and lox66 (the structure is shown in Figure 24) (the entire sequence of the plasmid is shown in SEQ ID NO: 39 in the Sequence Listing). The plasmid was synthesized by the Genetics Department of Nanjing Jinsirui Biotechnology Co., Ltd. The rop gene sequence is from the pBR322 replication element and is a replication control protein that reduces the copy number of the pUC replication element plasmid. Before recombination, the precursor plasmid expressed the Rop protein, and the copy number of the precursor plasmid was suppressed. After recombination, the plasmid without the resistance selection marker lost the rop gene sequence and was converted into a high-copy plasmid, which increased the proportion of plasmid without the resistance selection marker in the recombinant strain, promoted the loss of the precursor plasmid, and improved the screening efficiency of strains containing plasmids without the resistance selection marker.

[0181] Example 6. Construction of a precursor plasmid containing lox71 / lox66 recombination sites and an R6Kγ replicon

[0182] 6.1 Construction of pMF5-loxp-RFP precursor plasmid

[0183] This example demonstrates that the method of the present invention is suitable for producing selectable marker-free plasmids of different types of replication elements, demonstrating its versatility. The R6Kγ ori (389 bp, SEQ ID NO: 46) is a different replicon from pMB1 or pUC in Examples 3, 4, and 5. The R6Kγ sequence is shorter in length, which can further shorten the backbone length of the daughter plasmid and reduce cytotoxicity.

[0184] The pMF5-loxP-RFP precursor plasmid contains the DNA replication element R6Kγ ori and the resistance selection marker gene Kan RThe plasmid contains a pair of lox71 / lox66 specific recombination sites in the same direction, and a target gene to be inserted into the RFP sequence. The replication element and inserted gene are located between lox71 and lox66 (the structure is shown in Figure 25) (the complete sequence of the plasmid is shown in SEQ ID NO: 40 in the sequence listing). The plasmid was synthesized by Nanjing Jinsirui Biotechnology Co., Ltd.

[0185] Example 7. Construction of precursor plasmids containing the specific recombination site FRT in the same orientation

[0186] 7.1 Preparation of FRT-RFP gene fragment and vector

[0187] The pMF9-FRT-RFP precursor plasmid (the structure of which is shown in Figure 26) was constructed based on the pMF9-loxP-RFP plasmid. For the construction of the pMF9-loxP-RFP plasmid, see the construction method of pMF9-loxP-5.5 kb in Example 3. Specifically, the rfp red pigment synthesis gene (the sequence of which is shown in SEQ ID NO: 28) was inserted as the target gene between the loxP sites of pMF9-loxP-RFP. A fragment between the loxP sites of the pMF9-loxP-RFP plasmid was amplified using primers carrying the FRT sequence (the FRT sequence is shown in SEQ ID NO: 27 in the Sequence Listing). The specific primer sequences are shown in the Sequence Listing (the FRT-F1 sequence is shown in SEQ ID NO: 18, and the FRT-R1 sequence is shown in SEQ ID NO: 19). The PCR system and PCR procedure are shown in Tables 23 and 24. The amplified product was subjected to gel electrophoresis. As shown in Figure 27, the sizes of lanes 2 and 3 were both 2123 bp, which is correct. The gel was collected according to the instructions in the gel kit.

[0188] Preparation of pMF9 vector fragment: PCR amplification was performed on the vector using the pMF9-loxP-RFP plasmid as a template. The primers FRT-F2 and FRT-R2 are shown in the primer sequence table (SEQ ID NO: 20 and SEQ ID NO: 21, respectively). The PCR system and PCR procedure are shown in Table 16 and Table 17. The amplified product was subjected to agarose gel electrophoresis. As shown in lanes 4 and 5 in Figure 27, the size of the amplified product is 2236 bp. The band of the correct size was excised and the gel was recovered according to the gel kit. The Phusion® HF DNA polymerase used in PCR is commercially available from New England Biotechnology, Inc., product number 10058481.

[0189] [Table 23]

[0190] [Table 24]

[0191] 7.2 Assembling of pMF9-FRT-RFP Plasmid. The assembly system is shown in Table 25. The assembly system was reacted at 50°C for 15 minutes. After assembly was completed, the assembly product was transformed into DH10b competent cells and cultured at 37°C. The Gene Builder™ Cloning Kit is available from Nanjing Jinsirui Biotechnology Co., Ltd., product number C20012009.

[0192] [Table 25]

[0193] 7.3 Verification of pMF9-FRT-RFP plasmid

[0194] Eight single colonies were randomly selected from the transformation plate and sent to Nanjing Jinsirui Biotechnology Co., Ltd. for Sanger sequencing. Referring to Figure 28, the sequencing results showed that the tested sequence was consistent with the map sequence (black line in the figure), indicating no gene deletion or mutation and successful plasmid organization.

[0195] Example 8: Recombination induction, resistance screening, and plasmid purification based on single-plasmid transformation of pMF9-loxp vector

[0196] This example uses JM108 araBAD-cre + pMF9-loxp-5.5kb as an example to demonstrate the recombination induction, resistance screening, and plasmid purification methods based on single-plasmid transformation, but is not limited to the Cre-loxP recombination system. The specific procedures are as follows:

[0197] 8.1 Single-plasmid transformation

[0198] The genetically modified strain JM108 araBAD-cre in Example 1 was used to prepare competent cells, which were then transformed with the precursor plasmid pMF9-loxp-5.5kb and plated onto kanamycin plates. The specific transformation steps were as follows:

[0199] JM108 araBAD-cre competent cells were placed on ice and allowed to thaw. 1 μL of the plasmid was added to the competent cells on ice. The competent cells and the plasmid were mixed uniformly. The mixture was then placed in an ice bath at 42°C for 30 min, heat-shocked for 90 s, and then placed in an ice bath for 3 min. The mixture was added to 800 μL of fresh LB liquid medium and incubated at 37°C and 220 rpm for 45 min in a temperature-controlled shaking culture chamber. An appropriate amount of the bacterial solution was poured onto a solid plate of LB + Kan + 1% glucose and cultured overnight in a culture chamber at 37°C. Single colonies that had grown to a normal size were selected for subsequent experiments.

[0200] 8.2 Induction of Cre-loxP recombination by addition of arabinose

[0201] A certain amount of arabinose was added to the JM108 araBAD-cre pMF9-loxp-5.5kb culture system to induce the expression of Cre recombinase and achieve Cre-loxP recombination. The specific procedure is as follows:

[0202] Three to four JM108 araBAD-cre pMF9-loxp-5.5 kb colonies were selected and incubated in 4 mL of LB + Kan (kanamycin, 30 μg / mL) + 1% glucose liquid medium. The strains were concentrated by overnight cultivation at 37°C and 220 rpm. The strains were collected, washed twice with LB without antibiotics, and resuspended in induction solution (induction solution: LB + 2% arabinose). Recombinant induction was carried out for 1 h at 37°C and 220 rpm. 5 μL of the recombinant bacterial solution was streaked onto an LB solid plate without antibiotics and cultivated overnight in an incubator at 37°C. Plasmids were extracted from the remaining bacterial solution and verified. The digestion system is shown in Table 26. Ahd I restriction endonuclease is available from New England Biotechnology, Inc., product number 10079968. Agarose gel electrophoresis was performed. The original plasmid was used as a control, and the electrophoresis results are shown in FIG.

[0203] Results: The size of the precursor plasmid pMF9-loxp-5.5 kb is 8720 bp, the size of the resistant circular DNA generated after recombination is 2270 bp, and the size of the generated pMF9-5.5 kb plasmid is 6450 bp. As can be seen from Figure 29, Cre-loxP recombination was successfully achieved by adding 2% arabinose and inducing for 1 hour, resulting in a markerless plasmid of approximately 6.5 kb, but a small amount of precursor plasmid still remained.

[0204] [Table 26]

[0205] 8.3 Kan resistance screening and purification to obtain unmarked plasmids

[0206] 1) After streaking an antibiotic-free plate with the recombinant bacterial solution, 10 single colonies were selected. As shown in the left panel of Figure 30, each colony was spotted on an LB + Kan plate and cultured overnight in an incubator at 37°C. As shown in the right panel of Figure 30, each colony was also inoculated into an LB liquid medium without antibiotics and cultured at 37°C and 220 rpm for resistance screening.

[0207] 2) Five colonies that did not grow on the LB + Kan plate but grew normally in LB liquid medium without antibiotics were selected (as shown in Figure 30), and after storage, plasmids were extracted and subjected to plasmid verification.

[0208] 3) The extracted plasmid was subjected to digestion verification. The digestion system is shown in Table 26. Agarose gel electrophoresis was performed. The pMF9-5.5 kb plasmid is 6450 bp. As the obtained unmarked plasmid product, a plasmid with the correct plasmid size and correct digestion product was selected (as shown in lanes #1 and #4 on the left side of Figure 31).

[0209] Resistance screening plate and plasmid validation results showed that Kan resistance reverse screening eliminated the remaining precursor plasmid and resistance gene, and marker-free plasmid was obtained with high efficiency.

[0210] Example 9: Recombination induction, resistance screening and plasmid purification based on single-plasmid transformation of the shortest backbone precursor plasmid containing only the pMB1 replication element

[0211] This example tested the feasibility of producing a selectable marker-free plasmid of the present invention by minimizing the pMB1 replication element in the precursor plasmid based on the pMF65-loxp-RFP precursor plasmid in Example 4. The specific embodiment is the same as in Example 8. Recombination of the precursor plasmid was achieved by induction. As shown in Figure 32, gel electrophoresis results show that lane 2 is the precursor plasmid control without recombination induction, measuring 3272 bp in size, and lane 3 is the MF plasmid obtained after 1 h of recombination induction, measuring 1743 bp in size, and both band sizes are correct. Subsequent resistance screening and purification yielded the pMF65-RFP plasmid without a resistance selectable marker, with the correct size. As shown in Figure 33 (three parallel plasmid products), gel electrophoresis results show that the bands are correct in size, simple, and haploid. The pMF65-loxp-RFP plasmid was sequenced for verification. As shown in Figure 34, the verification results indicate successful recombination.

[0212] Example 10: Recombination induction, resistance screening and plasmid purification based on single-plasmid transformation of high-copy precursor plasmid backbone

[0213] This example tested the feasibility of producing a selectable marker-free plasmid of the present invention using a high-copy precursor plasmid backbone based on the pMF7-loxp-RFP precursor plasmid in Example 5. The specific embodiment is the same as in Example 8. Recombination of the precursor plasmid was achieved by induction. As shown in Figure 35, gel electrophoresis showed that lane 2 was the precursor plasmid without induced recombination, measuring 3879 bp in size, and lane 3 was the resistance gene-free plasmid produced after 1 h of induced recombination. The band corresponding to the precursor plasmid was clearly weakened and the size was correct. Subsequent resistance screening and purification yielded the pMF7-RFP plasmid without a resistance selection marker, measuring 1828 bp in size. As shown in Figure 36 (seven parallel plasmid products), the gel electrophoresis results indicated the correct size. The purified pMF7-RFP plasmid was subjected to sequencing verification. As shown in Figure 37, the verification results indicated successful recombination.

[0214] Example 11: Recombination induction, resistance screening and plasmid purification based on a dual-plasmid system

[0215] In the dual-plasmid recombination system, the recombinase expression system is loaded onto a plasmid vector and transformed into a host strain along with the precursor plasmid. The multi-copy plasmid can increase the expression level of the recombinase and improve recombination efficiency. The dual-plasmid recombination system does not require strain modification and is generally suitable for producing selectable marker-free plasmids in commercially available strains.

[0216] This example demonstrates the methods for recombination induction, resistance screening, and plasmid purification based on dual-plasmid transformation using the pSC101(ts)-araBAD-flp temperature-sensitive plasmid prepared in Example 2 and the pMF9-FRT-RFP prepared in Example 7. However, this example is not limited to the FLP-FRT recombination system. Specific procedures are as follows:

[0217] 11.1 Co-transformation of pSC101(ts)-araBAD-flp and pMF9-FRT-RFP plasmids

[0218] JM108 competent host bacteria was transformed with 1 μL of pSC101(ts)-araBAD-flp plasmid and 1 μL of pMF9-FRT-RFP plasmid. The transformation steps were the same as in Example 8.1.

[0219] 11.2 Induction of Flp-FRT recombination by addition of arabinose

[0220] In this example, arabinose was added to the JM108 pSC101(ts)-araBAD-flp pMF9-FRT-RFP culture system to induce Flp enzyme expression and achieve Flp-FRT recombination. The control group was treated in the same way as the experimental group. The specific procedures were as follows:

[0221] 1) Three to four JM108 pSC101(ts)-araBAD-flp pMF9-FRT-RFP colonies were selected and incubated in 4 mL of LB + Amp (100 μg / mL) + Kan (30 μg / mL) + 1% glucose liquid medium. The culture was grown overnight at 30°C and 220 rpm to concentrate the strain.

[0222] 2) The strain was harvested, washed twice with LB without antibiotics, and resuspended in induction solution (induction solution: LB + 2% arabinose). Recombination induction was carried out at 30°C and 220 rpm for 2-8 hours.

[0223] 3) After the recombination time was reached, the recombinant strain was transferred to a 4 mL LB tube without antibiotics at 1‰, and the loss of pSC101(ts)-araBAD-flp was induced at 37°C, and the strain was cultured overnight at 220 rpm.

[0224] 4) 5 μL of bacterial solution was taken from each of the three overnight culture tubes, streaked onto an LB solid plate without antibiotics, and cultured in an incubator at 37°C. Plasmids were extracted from the remaining bacterial solution and verified. The digestion system is shown in Table 27. AhdI is commercially available from New England Biotechnology, Inc., product number 10079968. Agarose gel electrophoresis was performed (shown in Figure 38). The precursor plasmid pMF9-FRT-RFP is 4309 bp in size. The unmarked recombinant plasmid is 2025 bp. The electrophoresis results show that after 2 to 8 hours of induction, a clear unmarked plasmid band appears (plasmid lanes 2 to 4 and plasmid digestion lanes 2 to 4), and after overnight induction at 37°C, the pSC101(ts)-araBAD-flp plasmid is clearly lost (plasmid lanes 5 to 7 and plasmid digestion lanes 5 to 7).

[0225] [Table 27]

[0226] 11.3 Kan and Amp Resistance Reverse Screening and Purification to Obtain Marker-Unmarked Plasmids

[0227] 1) Ten single colonies were selected from each of the three antibiotic-free plates. LB + Amp and LB + Kan plates were dotted with colonies and cultured in an incubator at 37°C. Each colony was then inoculated into antibiotic-free LB liquid medium and cultured at 37°C and 220 rpm for resistance screening. Screening results showed that 10% to 50% of the recombined single colonies had lost the precursor plasmid and Kan resistance gene. After induction at 37°C, pSC101-araBAD-flp(ts) was completely lost, resulting in non-resistant strains (Figure 39, using the bacterial solution sample from the 5-hour group as an example).

[0228] 2) Colonies that did not grow on LB + Amp plates or LB + Kan plates but grew in LB liquid medium without antibiotics were selected and preserved, after which plasmids were extracted and digested for verification. The digestion system is shown in Table 20. Agarose gel electrophoresis was performed. Verification revealed that the plasmids and digestion products of 2 h#2, 5 h#6, and 8 h#7 were the correct size and were unmarked plasmid products (as shown in Figure 40).

[0229] Example 12: Recombination induction, resistance screening and plasmid purification based on dual-plasmid transformation of R6Kγ replicon

[0230] This example illustrates the methods for recombination induction, resistance screening, and plasmid purification based on dual-plasmid transformation using the pSC101(ts)-araBAD-cre plasmid prepared in Example 1(4) and the pMF5-loxp-RFP precursor plasmid prepared in Example 6.

[0231] 12.1 Co-transformation of pSC101(ts)-araBAD-cre plasmid and pMF5-loxp-RFP precursor plasmid

[0232] The host bacteria used were pir2 competent strains provided by Nanjing Jinsirui Biotechnology Co., Ltd. Transformation was performed using 1 μL of pSC101(ts)-araBAD-cre and 1 μL of pMF5-loxp-RFP precursor plasmid. The transformation steps were the same as in Example 8.1.

[0233] 12.2 The specific embodiment of recombination, screening, and purification is the same as in Examples 8.2 and 8.3. Recombination of the precursor plasmids was achieved by induction. As shown in Figure 41, the results show that lane 2 is the unintroduced plasmid control, containing two bands, pSC101(ts)-araBAD-cre and pMF5-loxp-RFP precursor plasmids, with sizes of 5867 bp and 3369 bp, respectively. Lane 3 is the band of the mixed plasmid obtained by recombination induction, which, from top to bottom, are pSC101(ts)-araBAD-cre, pMF5-loxp-RFP precursor plasmid, and Kan. R The resulting plasmids are the circular double-stranded DNA carrying the gene and pMF5-loxp-RFP. Both band sizes are correct. Subsequent resistance screening and purification yielded the pMF5-loxp-RFP plasmid without a resistance selection marker, with the correct size of 1533 bp. As shown in Figure 42, the results demonstrate that the size is correct. Sequencing verification was performed on the purified pMF5-loxp-RFP plasmid. As shown in Figure 43, the verification results demonstrate successful recombination.

[0234] Example 13: Large-scale extraction of unmarked plasmids

[0235] In this example, pMF9-5.5 kb, pMF65-RFP, and pMF7-RFP plasmids in Examples 8, 9, and 10 are used as examples, which show accurate digestion and good supercoiled bands. The host E. coli cells carrying the above plasmids are sent to Nanjing Genscript Biotechnology Co., Ltd. for mass production of the plasmids. See https: / / www.genscript.com.cn / industrial-grade-plasmid.html.

[0236] QC detection results: pMF9-5.5 kb plasmid: 0.5-1.2 mg / L. Digestion verification was performed on 300 ng of the plasmid. The size of the plasmid is 6450 bp. Gel electrophoresis detection results show that the band size is correct (as shown in Figure 44). Analysis by Tanon GIS shows that the proportion of monomer supercoiled plasmid bands is over 90%. Sanger sequencing of the plasmid was performed. The sequencing was completed by Nanjing Jinsirui Biotechnology Co., Ltd. The sequencing results show that the sequence is correct and there is no resistance gene sequence (as shown in Figure 45).

[0237] pMF65-RFP plasmid: 0.3-1 mg / L. Digestion verification was performed on 300 ng of the plasmid. Gel electrophoresis detection results showed that the plasmid size was 1743 bp and the band size was accurate (as shown in Figure 46). Analysis by Tanon GIS showed that the monomer supercoiled plasmid band rate was over 90%. Sanger sequencing was performed on the plasmid, and the sequencing was completed by Nanjing Jinsirui Biotechnology Co., Ltd. The sequencing results showed that the sequence was accurate and there was no resistance gene sequence (as shown in Figure 47). NGS analysis of the plasmid showed that the content of the target daughter plasmid was over 98% and the content of the precursor plasmid was less than 0.02%.

[0238] pMF7-RFP plasmid: 3.2-4.1 mg / L. Digestion verification was performed on 300 ng of the plasmid. Gel electrophoresis detection results showed that the plasmid size was 1828 bp and the band size was accurate (as shown in Figure 48). Analysis by Tanon GIS showed that the monomer supercoiled plasmid band rate was over 90%. Sanger sequencing was performed on the plasmid, and the sequencing was completed by Nanjing Jinsirui Biotechnology Co., Ltd. The sequencing results showed that the sequence was accurate and there was no resistance gene sequence (as shown in Figure 49). NGS analysis was performed on the plasmid, and the content of the target daughter plasmid was over 98% and the content of the precursor plasmid was less than 0.02%.

[0239] [Table 28]

[0240] The embodiments of the present invention are not limited to those described in the above examples. Those skilled in the art can make various changes and modifications to the present invention in form and detail without departing from the spirit or scope of the present invention, and all of them are considered to fall within the protection scope of the present invention.

[0241] Some nucleic acid sequence information referred to herein is as follows: TIFF2025128158000029.tif234153TIFF2025128158000030.tif231152TIFF2025128158000031.tif231152TIFF20251281580 00032.tif229152TIFF2025128158000033.tif233152TIFF2025128158000034.tif232152TIFF2025128158000035.tif234152 TIFF2025128158000036.tif234152TIFF2025128158000037.tif232152TIFF2025128158000038.tif230152TIFF20251281580 00039.tif230152TIFF2025128158000040.tif232152TIFF2025128158000041.tif232152TIFF2025128158000042.tif231152

[0242] Some primer sequence information referred to herein is as follows: TIFF2025128158000043.tif238170TIFF2025128158000044.tif122170Citations: [1] Davies, J; Smith, DI (1978). Plasmid-Determined Resistance to Antimicrobial Agents. Annual Review of Microbiology, 32(1), 469-508 [2] Schleef, M. (2013) Minicircle and Miniplasmid DNA Vectors: The Future of Non-Viral and Viral Gene Transfer. Wiley-VCH [3] Luke, J. et al. (2009) Improved antibiotic-free DNA vaccine vectors utilizing a novel RNA based plasmid selection system. Vaccine 27, 6454-6459

Claims

1. A precursor plasmid comprising: 1) a replication initiation site; 2) a selectable marker gene; and 3) a target gene or a cloning site for inserting said target gene; 4) paired recombination sites, wherein: the paired recombination sites enable the precursor plasmid to self-recombine in the presence of a recombinase to form a daughter plasmid molecule lacking a selectable marker gene and a circular double-stranded DNA molecule; the daughter plasmid comprises the replication origin and the target gene, or the replication origin and the cloning site; and A precursor plasmid, wherein the circular double-stranded DNA comprises the selectable marker gene.

2. 2. The precursor plasmid of claim 1, wherein the sequences of the paired recombination sites are in the same orientation.

3. 3. The precursor plasmid of claim 1, wherein the replication initiation site is adjacent to the target gene or the cloning site, and the paired recombination sites are adjacent to the replication initiation site and the target gene upstream and downstream, respectively, or the paired recombination sites are adjacent to the replication initiation site and the cloning site upstream and downstream, respectively.

4. 4. The precursor plasmid of claim 1, wherein the paired recombination sites are selected from the group consisting of unidirectional loxP sequences, unidirectional FRT sequences, and unidirectional attB / attP sequences.

5. 5. The precursor plasmid of claim 4, wherein the paired recombination sites are lox71 and lox66 sequences in the same orientation.

6. 6. The precursor plasmid according to claim 1, wherein the replication origin is selected from the group consisting of the replication origin of pUC, the replication origin of pMB1 and its derivatives, the replication origin of ColE1, and the replication origin of R6Kγ.

7. The precursor plasmid of claim 6, wherein the replication origin comprises a nucleotide sequence set forth in SEQ ID NOs: 43 to 46 and a nucleotide sequence that has at least 80% identity with the nucleotide sequence set forth in SEQ ID NOs: 43 to 46 and can serve as an origin of replication.

8. 8. The precursor plasmid of claim 1, further comprising one or more of a rop gene sequence, an endonuclease coding sequence, and a plasmid replication helper protein coding sequence.

9. The precursor plasmid of any one of claims 1 to 8, wherein the selectable marker gene is an antibiotic resistance gene.

10. 10. The precursor plasmid of any one of claims 1 to 9, further comprising other plasmid backbone sequences.

11. The precursor plasmid according to any one of claims 1 to 10, further comprising a gene encoding the recombinase.

12. 12. The precursor plasmid of claim 11, wherein the precursor plasmid is capable of expressing the recombinase under appropriate conditions.

13. The precursor plasmid of any one of claims 8 to 11, wherein the circular double-stranded DNA further comprises one or more of a rop gene sequence, other plasmid backbone sequences, and a gene encoding the recombinase.

14. 14. The precursor plasmid of any one of claims 1 to 13, comprising the nucleotide sequence set forth in SEQ ID NO: 8, 34, 39-42 or 47, or a nucleotide sequence having at least 80% identity to the nucleotide sequence set forth in SEQ ID NO: 8, 34, 39-42 or 47.

15. Use of the precursor plasmid according to any one of claims 1 to 14 in producing a daughter plasmid lacking a selectable marker gene.

16. 1. A method for producing a daughter plasmid lacking a selectable marker gene, comprising: 1) introducing the precursor plasmid of any one of claims 1 to 14 into a host cell capable of expressing or supporting the expression of the recombinase, and screening for host cells that express the selectable marker gene; 2) culturing the host cells screened in step 1) to allow expression of the recombinase in the host cells, culturing the host cells, and screening for host cells that do not express the selection marker gene; 3) culturing the host cells screened in step 2) to obtain the daughter plasmids, and extracting the plasmids.

17. 17. The method of claim 16, wherein the paired recombination sites are loxP sequences in the same orientation and the recombinase is Cre recombinase, the paired recombination sites are FRT sequences in the same orientation and the recombinase is Flp recombinase, or the paired recombination sites are attB / attP sequences in the same orientation and the recombinase is PhiC31 recombinase.

18. 18. The method of claim 16 or 17, wherein the paired recombination sites are lox71 and lox66 sequences in the same orientation, and the recombinase is Cre recombinase.

19. The method according to any one of claims 16 to 18, wherein the host cell comprises a gene encoding the recombinase in its genome, or the host cell comprises an expression vector containing a gene encoding the recombinase.

20. The method according to any one of claims 16 to 18, wherein the precursor plasmid comprises a gene encoding the recombinase, and the precursor plasmid is capable of expressing the recombinase in the host cell.

21. The method of any one of claims 16 to 20, wherein the recombinase is inducibly expressed in the host cell.

22. 22. The method of claim 19 or 21, wherein the expression vector comprises a conditionally inducible deletional plasmid replication element.

23. The method of any one of claims 16 to 22, wherein the host cell is Escherichia coli.

24. A set for producing a daughter plasmid lacking a selectable marker gene, the set comprising the precursor plasmid according to any one of claims 1 to 14.

25. 25. The set of claim 24, further comprising a host cell capable of expressing or supporting the expression of said recombinase.

26. 26. The set of claim 24 or 25, wherein the paired recombination sites are loxP sequences in the same orientation and the recombinase is Cre recombinase, the paired recombination sites are FRT sequences in the same orientation and the recombinase is Flp recombinase, or the paired recombination sites are attB / attP sequences in the same orientation and the recombinase is phiC31 recombinase.

27. The set according to any one of claims 24 to 26, wherein the paired recombination sites are lox71 and lox66 sequences in the same orientation, and the recombinase is Cre recombinase.

28. The set according to any one of claims 25 to 27, wherein the host cell comprises a gene encoding the recombinase in its genome, or the host cell comprises an expression vector containing a gene encoding the recombinase.

29. The set according to any one of claims 25 to 28, wherein the recombinase is inducibly expressed in the host cell.

30. 30. The set of claim 28 or 29, wherein the expression vector comprises a conditionally inducible deletion-type plasmid replication element.

31. The set according to any one of claims 25 to 30, wherein the host cell is Escherichia coli.

32. The set according to any one of claims 24 to 31, wherein the daughter plasmid formed after recombination contains the target gene by inserting the target gene into the cloning site.

33. A daughter plasmid containing an origin of replication and a target gene, but not an antibiotic resistance gene.

34. 34. The daughter plasmid of claim 33, wherein the replication origin is selected from the replication origin of pUC, the replication origin of pMB1 and its derivatives, the replication origin of ColE1, and the replication origin of R6Kγ.

35. The daughter plasmid of claim 33 or 34, wherein the replication origin comprises a nucleotide sequence shown in SEQ ID NO: 43 to 46 and a nucleotide sequence that has at least 80% identity with the nucleotide sequence shown in SEQ ID NO: 43 to 46 and can serve as an origin of replication.

36. The daughter plasmid of any one of claims 33 to 35, wherein the target gene comprises one or more of a promoter, a coding gene for expressing a protein, and a terminator sequence.

37. A daughter plasmid according to any one of claims 33 to 36, capable of replicating in a host cell.

38. 38. The daughter plasmid of claim 37, which is capable of replicating in the host cell under cell culture conditions without screening stress.

39. A daughter plasmid obtained by the method of any one of claims 16 to 23.

40. A host cell comprising a daughter plasmid according to any one of claims 33 to 39, wherein said daughter plasmid is capable of replicating within said host cell.

41. 41. The host cell of claim 40, wherein the daughter plasmid is obtained from the host cell by culturing, harvesting, and extraction.

42. 42. The host cell of claim 40 or 41, which is capable of amplifying the daughter plasmid under cell culture conditions without screening stress.

43. 40. A composition comprising the daughter plasmid of any one of claims 33 to 39, wherein the daughter plasmid content is at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%.

44. 44. The composition of claim 43, wherein the method for measuring the content of daughter plasmids in the composition is an NGS analysis method or a gel electrophoresis imaging analysis method.

45. 45. The composition of claim 43 or 44, wherein the daughter plasmid content in the composition as measured by the NGS analytical method is at least 90%, 95%, 96%, 97%, 98% or 99%.

46. 46. ​​The composition of any one of claims 43 to 45, wherein the daughter plasmid content in the composition is at least 90%, 95%, 96%, 97%, 98% or 99% as measured by the gel electrophoresis imaging analysis method.

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