Non-resistant plasmid production system and application thereof

Abstract

A non-resistance plasmid production system constructs a plasmid containing a nucleic acid fragment 3 for coding small RNA, a host cell expression cassette containing a toxic gene expression cassette and an anti-toxic gene expression cassette through combined design of a toxic transcript / anti-toxic transcript, an operon related element and the small RNA. The toxic gene expression cassette comprises a promoter and a nucleic acid fragment 1 for transcribing a toxic transcript, and the anti-toxic gene expression cassette comprises a promoter, an operon and a nucleic acid fragment 2 for transcribing an anti-toxic transcript. Through reverse complementation of the nucleic acid fragment 3 and the nucleic acid fragment 1, a steric effect is generated, and a toxic transcript is inhibited, so that the screening production of plasmids can be realized without adding antibiotics or other inhibitors in the whole plasmid production process, and the harm of the antibiotics and the inhibitors to the plasmids in cell gene therapy is solved.

Description

A non-resistance plasmid production system and its application Technical Field

[0001] The present invention relates to the field of biotechnology, in particular to a non-resistance plasmid production system and application thereof. Background Art

[0002] In recent years, gene therapy methods have attracted more and more attention, especially DNA or RNA vaccines, which have overcome the defects of traditional vaccines or viral vector vaccines in many aspects. Whether it is DNA or RNA vaccines or viral vector vaccines, the demand for plasmids is usually very large.

[0003] Traditional plasmid systems have numerous drawbacks in gene therapy applications. For one thing, traditional plasmids carry antibiotic marker genes, but overexpression of resistance genes can negatively impact E. coli metabolism and growth. Furthermore, the spread of antibiotics in the environment can lead to widespread antibiotic resistance in the population, and residual antibiotics in the plasmid may trigger allergic reactions in susceptible individuals. Furthermore, traditional plasmids carry prokaryotic bacterial backbones, which often contain unmethylated CpG sequences and can easily trigger the innate immune system. Whether carrying an antibiotic marker gene or a prokaryotic bacterial backbone, sequences outside the exogenous gene expression cassette occupy a disproportionate portion of the plasmid, increasing its size. This not only reduces plasmid yield but also decreases transfection efficiency, a negative impact in gene therapy. Smaller supercoiled plasmids are more efficient in reaching the cell nucleus and enabling sustained, long-lasting expression of the exogenous gene. Therefore, plasmids lacking resistance genes and possessing shorter prokaryotic backbones offer significant advantages.

[0004] To improve the safety of gene therapy, a new generation of plasmid backbone systems without antibiotic resistance markers has been developed. These systems primarily include the following: 1. Complementation of malnourished strains: This involves modifying a bacterial strain by introducing a deletion or mutation into a gene essential for bacterial growth, rendering it malnourished and unable to grow. Subsequently, introduction of a plasmid carrying the deleted gene restores growth. 2. Virulence-antidoxogenetic systems: A toxic gene is inserted into the bacterial genome, rendering it incapable of growth. An anti-virulence gene is cloned onto a plasmid and introduced into the strain to restore growth. 3. Operator / repressor systems: A repressor protein is introduced upstream of an essential growth gene in the bacterial genome to inhibit its expression. Plasmids containing one or more operator sequences are then introduced into the bacteria to competitively titrate the repressor, allowing gene expression. 4. Overexpression of essential growth genes: Overexpression of certain essential growth genes (fabl or murA) in Escherichia coli can reduce their susceptibility to antimicrobial compounds, but the inhibitors are present in the culture medium and must be removed from the purified plasmid DNA. Although the above-mentioned novel antibiotic-free plasmid systems have solved the use of antibiotic marker genes, there are still two major problems: 1. The sequences outside the target gene expression cassette account for too large a proportion in the plasmid, resulting in an increase in the plasmid volume. Therefore, under the same unit weight, part of the plasmid yield is contributed by the ineffective prokaryotic sequence skeleton, and the copy number of the target gene expression cassette is relatively reduced, affecting the yield of the target expression cassette. 2. Although some systems do not require the use of antibiotics, other auxiliary inhibitors still need to be added, and these inhibitors cannot be guaranteed to be 100% removed in the final plasmid product, which not only increases the cost of plasmid preparation, but also the impact of their residues in gene therapy is uncontrollable. Therefore, these resistance-free vector systems cannot be effectively popularized at present.

[0005] Therefore, there is still a need in the art to develop plasmid production systems that do not require the use of antibiotics and other auxiliary inhibitors.

[0006] Summary of the Invention

[0007] To address the shortcomings of the prior art, the present invention provides an antibiotic-free plasmid production system and its application. This production system eliminates the need for antibiotics and other co-inhibitors, and can achieve a copy number of some plasmids that is 4-5 times higher than conventional plasmid yields, significantly reducing production costs while improving application safety.

[0008] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0009] In one aspect, the present invention provides a plasmid production system, characterized in that it comprises a host cell expression cassette and a plasmid;

[0010] The host cell expression cassette includes a toxic gene expression cassette and an anti-toxic gene expression cassette;

[0011] The toxic gene expression cassette includes a promoter and a nucleic acid fragment 1 that transcribes a toxic transcript; the toxic transcript may include a gene and / or non-coding RNA that is toxic to the host;

[0012] The anti-toxic gene expression cassette includes a promoter, an operator, and a nucleic acid fragment 2 that transcribes an anti-toxic transcript; the anti-toxic transcript has a resistance effect on the toxicity produced by the toxic transcript; wherein the anti-toxic transcript may include an anti-toxic gene transcript and / or a non-coding RNA that has a resistance effect on the toxicity of the toxic transcript;

[0013] The plasmid includes a nucleic acid segment 3 encoding a small RNA and an Ori replication initiation site, wherein the nucleic acid segment 3 is reversely complementary to the nucleic acid segment 1, thereby inhibiting the function of the toxic transcript.

[0014] In the plasmid production system described herein, an operon regulates the on / off switching of a promoter, thereby manipulating the expression of an anti-toxic gene. When the toxic anti-toxic transcript is expressed alone, host cell growth is inhibited or prevented. The product of the anti-toxic transcript resists the toxicity produced by the toxic transcript. When both the toxic transcript and the anti-toxic transcript are expressed simultaneously, the toxic effects of the toxic transcript are suppressed, allowing normal host cell growth.

[0015] In some embodiments, the operon is any one of the arabinose operon, the Lac operon, the rhamnose catabolism operon, the tryptophan operon, the gab operon, and the Gal operon. The promoter regulated by the operon is any one of the araBAD promoter, the T7lac promoter, the Tac promoter, the Trc promoter, the LacUV5 promoter, the tetA promoter, the rhaBAD promoter, the gabP promoter, and the galP1 promoter; and the repressor protein gene of the operon is AraC, LacI, CAP, rhaS / R, tetracycline, csiR, or galR, including but not limited to these.

[0016] In some embodiments, the nucleic acid fragment 3 is completely complementary to the nucleic acid fragment 1 in reverse sequence, or has at least 20% reverse complementarity in sequence, or has at least 10 consecutive bp of reverse complementarity.

[0017] In some embodiments, the nucleic acid fragment 1 includes a UTR sequence and a toxic gene, and the UTR sequence is selected from any one of 1) to 4):

[0018] 1) the nucleic acid sequence shown in positions 1 to 252 of SEQ ID NO: 1;

[0019] 2) Sequences in which one or more nucleotides are substituted, deleted, or added to the sequence shown in 1) and which have the same or similar functions;

[0020] 3) A sequence complementary to 1), 2) or the sequence shown;

[0021] 4) A sequence that has at least 50% homology with the sequences shown in 1), 2) and 3) and whose function is not changed.

[0022] In some embodiments, the UTR sequence can also be selected from the nucleic acid sequence shown in positions 1 to 64 of SEQ ID NO: 2.

[0023] In some embodiments, the UTR sequence may also be selected from SEQ ID NO: 10, 12 and 14.

[0024] In the present invention, the toxic gene is selected from any one of ccdB, ParE, MazF, Kid, HicA, RelE, VapC, Doc, RatA, HipA, Zeta, ToxN, YeeV, CptA, GhoT, Hok, TisB, SymE, and PasA; the anti-toxic transcript is an anti-toxic gene; wherein, the anti-toxic gene is selected from any one of ccdA, ParD, MazE, Kis, HicB, RelB, VapB, Phd, RatB, HipB, Epsilon, ToxI, YeeU, CptB, GhoS, Sok, IstR-1, SymR, and PasB / C.

[0025] In some embodiments, the toxic gene is the ccdB gene, and the toxic gene expression cassette includes a promoter, a UTR sequence, and the ccdB gene; the nucleic acid fragment 2 that transcribes the anti-toxic transcript is the ccdA gene, and the anti-toxic gene expression cassette includes the araBAD promoter, the arabinose operator, and the ccdA gene.

[0026] The toxic gene and anti-toxic gene can also be Zeta gene or Doc gene, and Epsilon gene or Phd gene, respectively.

[0027] In some embodiments, the toxic gene expression cassette includes a promoter, UTR sequences, and a Zeta gene transcript; the anti-toxic gene expression cassette includes an araBAD promoter, an arabinose operator, and an Epsilon gene. In some embodiments, the toxic gene expression cassette includes a promoter, UTR sequences, and a Doc gene transcript; the anti-toxic gene expression cassette includes an araBAD promoter, an arabinose operator, and a Phd gene.

[0028] In some specific embodiments, the UTR sequence is a 5'UTR sequence, and the 5'UTR sequence is fused with the toxic gene for expression, and the fused sequence is shown in SEQ ID NO: 1 or SEQ ID NO: 2:

[0029] SEQ ID NO: 1

[0030] SEQ ID NO:2

[0031] In some specific embodiments, the nucleotide sequence of the anti-toxic gene expression cassette is shown in SEQ ID NO:9.

[0032] SEQ ID NO:9

[0033] In some specific embodiments, the nucleotide sequence of the anti-toxic gene expression cassette is shown in SEQ ID NO: 17 or 19.

[0034] In some specific embodiments, in the host cell expression cassette of the plasmid production system described in the present invention, the number of the anti-toxic gene expression cassettes and the number of the toxic gene expression cassettes are both selected from integers ≥1. Those skilled in the art can select an appropriate number based on the selected toxic genes and their toxicity as well as the inhibitory effect of the anti-toxic genes on toxicity, so as to ensure that when the toxic transcripts and the anti-toxic transcripts are expressed simultaneously, the toxic effect of the toxic transcripts is inhibited. In some specific embodiments, the number of the toxic gene expression cassette is 1, and the number of the anti-toxic gene expression cassettes is specifically 1, 2, 3, 4 or more. In a specific embodiment, the toxic gene in the toxic gene expression cassette is the ccdB gene, the anti-toxic gene in the anti-toxic gene expression cassette is the ccdA gene, the number of the toxic gene expression cassette is 1, and the number of the anti-toxic gene expression cassette is 2.

[0035] In the present invention, according to the type of replication origin Ori, the host cell expression cassette can also introduce a Rep protein required for plasmid replication, such as R6KRep, an essential protein for R6K plasmid replication, which is suitable for the preparation of plasmids such as R6K that require auxiliary proteins for conditional replication.

[0036] The plasmid of the present invention comprises a nucleic acid fragment 3 for transcribing small RNA; the sequence of the nucleic acid fragment 3 is selected from any one of the following a) to d):

[0037] a) a sequence as shown in any one of SEQ ID NOs: 3 to 7;

[0038] b) a sequence in which one or more nucleotides are substituted, deleted or added to the sequence shown in a) and which has the same or similar functions;

[0039] c) a sequence complementary to the sequence shown in a) or b);

[0040] d) A sequence that has at least 50% homology to the sequence shown in a), b) or c) and whose function is not altered.

[0041] Wherein, the sequence shown in SEQ ID NO: 3 is:

[0042] The sequence shown in SEQ ID NO:4 is:

[0043] The sequence shown in SEQ ID NO:5 is:

[0044] The sequence shown in SEQ ID NO:6 is:

[0045] The sequence shown in SEQ ID NO:7 is:

[0046] In some embodiments, the nucleic acid fragment 3 may also be selected from the sequence shown in SEQ ID NO: 8:

[0047] In some embodiments, the nucleic acid fragment 3 can also be selected from the sequence shown in any one of SEQ ID NOs: 11, 13 and 15.

[0048] In some embodiments, the sequence of the nucleic acid fragment 3 of the present invention may be 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, 100% homology to the sequence shown in any one of SEQ ID NOs: 3 to 8 or 11, 13, 15, or their complementary sequences.

[0049] In some embodiments, the Ori replication origin is selected from ColE1, pBR322, pMB1, R6K, pUC, F1, p15A, 2μori or oriV.

[0050] The plasmid provided by the present invention may also include a target gene. Specifically, the plasmid may include a promoter, a target gene, an Ori replication initiation site, and a nucleic acid fragment for transcribing small RNA 3.

[0051] In the present invention, the promoter in the plasmid and the promoter in the toxic gene expression cassette can be a viral, prokaryotic and eukaryotic promoter or a synthetic promoter commonly used in the art, such as CMV, EF1A, EFS, CAG, CBh, SFFV, MSCV, SV40, mPGK, hPGK, UBC, RSV, Nanog, Nes, Tubala, Camk2a, SYN1, Hb9, Th, NSE, GFAP, Ibal, ProA1, hRK, hRHO, hBEST1, Prnp, Cnp, K14, BK5, mTyr, cTnT, aMHC, Myog, ACTA1, MHCK7, SM22a, EnSM22a, Runx2OC, Collal, Col2al, aP2, Adipoq, Tiel, Cd144, CD68, CD1b, Afp, Alb, TBG, MMTV, Wap, HIP , Pdxl, Ins2, Hcn4, NPHS2, SPB, CD144, TERT, TRE, TRE3G, GAL1, MET17, CUP1, AOX1, sCMV, bactin2, Ubi, cmlc2, zK5, 503unc, HSP70, 5x UAS, CaMV35S, Nos, ZmUbi, TEF1, GPD, ADH1, GAP, actin5C, Polyubiquitin, α1-tubulin, Rh2, Mtn, U6, U3, H1, U6-26, TK, RSV, MC1, GAL1, PH, p5, p10, p40, p41, araBAD, cspA, Hsp68, pL, pR, EM7, T7, SP6, AmpR promoter, etc., including but not limited to.

[0052] In some embodiments, a polyA sequence may be added to the 3' end of the target gene in the plasmid of the present invention. The plasmid comprises a promoter, a target gene, a polyA sequence, an Ori replication initiation site, and a nucleic acid fragment encoding a small RNA inhibitory factor, which are sequentially connected.

[0053] In some embodiments, the resistance-free plasmid production system provided by the present invention further includes a host, that is, the production system includes a host cell expression cassette, a plasmid and a host, wherein the host is a host bacteria or a host cell, and the host bacteria and the host cell are derived from Escherichia coli, Agrobacterium, Bacillus, Bacillus or yeast. Those skilled in the art can select a suitable host type according to actual conditions, and the present invention is not particularly limited. In a specific embodiment of the present invention, the host is Escherichia coli. The host cell expression cassette of the present invention is integrated into the genome of the host bacteria or host cell to obtain a strain integrated with the host cell expression cassette of the present invention. Based on this, the resistance-free production plasmid provided by the present invention includes the above-mentioned strain and the above-mentioned plasmid.

[0054] The present invention also provides the use of the plasmid production system in producing plasmids or in preparing products for cell gene therapy.

[0055] The present invention also provides a transformed Escherichia coli strain, the host cell expression cassette is integrated into the genome of the strain.

[0056] The E. coli is a strain commonly used in the art, including DH5α, TOP10, NEB stable, BL21, EPI300, DH10B, JM109, SURE, Stbl3, XL10, and variants thereof. The integration site is a safe site in the E. coli genome, any commonly used site in the art.

[0057] Taking the arabinose operon as an example, the principle of plasmid screening in the resistance-free plasmid production system provided by the present invention is explained: when the strain is not supplemented with arabinose, the araC gene inhibits the activity of the araBAD promoter, preventing normal expression of the anti-toxic gene, while the toxic gene is expressed normally and the strain dies. With the addition of arabinose, the anti-toxic gene is expressed normally, thereby inhibiting the toxic gene and enabling normal growth of the strain. The survival rate of the strain is significantly increased when the number of anti-toxic gene expression cassettes is ≥ 2, and the specific number is preferably 2, 3, 4, 5, or more.

[0058] When a host strain is transformed with the plasmid of the present invention, the small RNA transcribed from the plasmid binds to the UTR of the toxic gene in the host cell expression cassette through base complementarity. The two can form a complex, and the resulting steric hindrance effect can effectively inhibit the translation of the toxic protein, thereby allowing the strain to survive. Specifically, experiments have shown that the steric hindrance effect generated by the binding of the small RNA transcribed from the plasmid to the UTR of the toxic gene can effectively control the expression of the toxic gene in the toxic transcript.

[0059] When the strain does not contain the plasmid of the present invention, the lack of arabinose in the culture medium cannot activate the expression of the anti-toxic protein, and thus cannot inhibit the toxicity of the toxic protein. Therefore, the strain that does not contain the plasmid of the present invention will die during the culture process and cannot continue to divide.

[0060] The present invention also provides a method for producing a plasmid, wherein the plasmid in the plasmid production system of the present invention is transferred into a host cell into which the host cell expression cassette is integrated. In some specific embodiments, the transfer is carried out by chemical transformation or electroporation. The specific steps are not limited and can be carried out according to conventional procedures in the art.

[0061] The present invention combines toxic transcripts / antitoxic transcripts, operon-related elements, and small RNAs to construct a novel resistance-free plasmid production system, including a host cell expression cassette and a plasmid. By binding the small RNA sequence in the plasmid to the toxic transcript in the host cell expression cassette, the expression of the toxic gene in the modified strain is controlled without the use of antibiotics or other auxiliary inhibitors. The resistance-free backbone can be combined with various types of plasmid replication origin sites (ORIs) to form a novel resistance-free plasmid production system. Compared with the existing technology, the present invention has the following advantages:

[0062] 1. The entire plasmid production process does not require the addition of antibiotics or other auxiliary inhibitors, which solves the harm of antibiotics and inhibitors to plasmids in gene therapy and reduces the cost of plasmid preparation and purification.

[0063] 2. According to different Ori lengths and replication characteristics, the prokaryotic backbone sequence can be greatly shortened. The copy number of some plasmids can reach 4-5 times the conventional plasmid yield, which improves application safety while greatly reducing production costs.

[0064] 3. The plasmid backbone provided by the present invention contains only the Ori required for self-replication and small RNA inhibitors, and the remaining sequences are for target gene expression. The prokaryotic backbone region of traditional plasmids is between 2000-2500bp, which is usually 4-5 times the prokaryotic backbone region of the plasmid of the present invention. The proportion of the prokaryotic backbone region of the plasmid of the present invention is greatly reduced, solving the problem that the sequence outside the exogenous gene expression cassette accounts for too large a proportion in the plasmid and the yield cannot be improved. BRIEF DESCRIPTION OF THE DRAWINGS

[0065] Figure 1 shows an example diagram of the functional elements of the miniVec stable strain;

[0066] Figure 2 shows the spectrum of miniVec Plasmid;

[0067] FIG3 shows a diagram of the synergistic effect of VecSeqA / VecSeqB and araBAD-ccdB / cddA;

[0068] Figure 4 shows the Intermediate Vector 1 map;

[0069] Figure 5 shows the Intermediate Vector2 map;

[0070] Figure 6 shows a pHelper Vector 1 map;

[0071] Figure 7 shows a pHelper Vector2 map;

[0072] Figure 8 shows the Intermediate Vector3 map;

[0073] Figure 9 shows the Intermediate Vector4 map;

[0074] Figure 10 shows the Intermediate Vector5 map;

[0075] Figure 11 shows the Intermediate Vector 6 map;

[0076] Figure 12 shows the Intermediate Vector7 map;

[0077] Figure 13 shows the Intermediate Vector8 map. DETAILED DESCRIPTION

[0078] The present invention provides an antibiotic-free plasmid production system and its applications. Those skilled in the art can refer to the contents herein and appropriately modify the process parameters to achieve the desired results. It should be noted that all similar substitutions and modifications will be apparent to those skilled in the art and are considered to be encompassed by the present invention. The methods and applications of the present invention have been described through preferred embodiments, and relevant personnel can obviously modify or appropriately alter and combine the methods and applications herein without departing from the content, spirit, and scope of the present invention to implement and apply the technology of the present invention.

[0079] The present invention constructs a novel resistance-free plasmid production system and its application. The system combines toxic transcripts / antitoxic transcripts, operon-related elements and small RNAs to construct a novel resistance-free plasmid production system. The system includes a host cell expression cassette and a plasmid. The expression of toxic genes in the strain integrated with the host expression cassette is controlled by the transcriptional small RNA sequence of the plasmid, without the need for antibiotics and other auxiliary inhibitors. The resistance-free skeleton can be combined with various types of plasmid replication origins (Ori) to form a set of novel resistance-free plasmid production systems. Furthermore, according to different Ori lengths and replication characteristics, the prokaryotic skeleton sequence can be greatly shortened, and the plasmid copy number can reach 4-5 times that of conventional plasmid production, thereby greatly reducing production costs while improving application safety.

[0080] The terms used in this application are defined as follows:

[0081] "Transcript" refers to a single-stranded ribonucleic acid (RNA) product synthesized by DNA transcription, which can be processed to produce various mature RNA products, such as non-coding RNA such as mRNA, tRNA or rRNA.

[0082] "Transcriptional toxic transcripts" and "toxic transcripts" are used interchangeably and refer to transcripts expressing toxic substances that are toxic to an organism, preventing the organism from growing normally. Toxic genes capable of producing the toxic transcripts of the present invention are known in the art, and include, but are not limited to, any one selected from ccdB, ParE, MazF, Kid, HicA, RelE, VapC, Doc, RatA, HipA, Zeta, ToxN, YeeV, CptA, GhoT, Hok, TisB, SymE, and PasA.

[0083] "Transcriptional anti-toxicity transcripts" and "anti-toxicity transcripts" are used interchangeably and refer to transcripts that express anti-toxicity substances capable of resisting toxicity. Anti-toxicity genes capable of producing the anti-toxicity transcripts of the present invention are known in the art. For example, the anti-toxicity gene is selected from any one of ccdA, ParD, MazE, Kis, HicB, RelB, VapB, Phd, RatB, HipB, Epsilon, ToxI, YeeU, CptB, GhoS, Sok, IstR-1, SymR, and PasB / C, including but not limited to these.

[0084] "Nucleic acid fragment that transcribes a small RNA" refers to the expression cassette for transcribing a small RNA, including the promoter, non-coding RNA, and terminator. "Small RNA inhibitor" and "small RNA" are used interchangeably to refer to non-coding RNA that reverse-complements the toxic transcripts of the present invention, generating a steric hindrance. Typically, expressed non-coding RNAs are less than 300 nucleotides in length.

[0085] "Steric effect" refers to the complementary binding of RNA sequences to DNA or RNA target sequences to form a double-stranded complex, which changes the spatial structure of the target sequence, making it impossible for the target sequence to effectively bind to enzymes related to replication, transcription or translation, thereby affecting the replication and expression of the target sequence itself.

[0086] "UTR" or untranslated region is located at the 5' end or 3' end of mature mRNA. It does not encode protein, but has translational regulatory function and participates in regulating the stability and intracellular localization of mRNA. UTR sequences that can form hairpin loops or can interact with stem-loop structures are all suitable for the present invention. UTR sequences can be selected from RNAII of ColEI, repA of IncF, hok of IncFII, repZ of IncI and ColIb, rep of CoIE2, rep1 of R1162, repR of pIP501, repC of pT181, repB of IncB and pLS1, Q and cII of lambda, ant of P22, P1 and P7, tnp of IS10, ompF, crp, fhlA, pks12, dinQ, tatC, bsrG, bsrE, RalR and rpoS of bacteria, including but not limited to this. Once the UTR sequence is determined, small RNA inhibitors that are reverse complementary to it and produce a steric hindrance effect can be designed according to conventional techniques.

[0087] "Ori origin of replication" refers to the plasmid origin of replication.

[0088] "Rep protein" refers to a class of proteins that act on the plasmid replication origin site and control plasmid replication.

[0089] In a specific example, the nucleic acid fragment 1 that transcribes the toxic transcript of the present invention includes a UTR sequence (labeled as VecSeqA) and the toxic gene ccdB, the nucleic acid fragment 2 that transcribes the anti-toxic transcript is the anti-toxic gene ccdA, and the nucleic acid fragment 3 that transcribes the small RNA in the plasmid is labeled as VecSeqB. Taking this as an example, the resistance-free plasmid production system of the present invention is described in detail as follows:

[0090] (1) miniVec stable strain

[0091] (1) The ccdB / ccdA system was used to transform the E. coli genome. This involved inserting a ccdB toxic gene expression cassette and two tandem araBAD-araC / ccdA anti-toxic gene expression cassettes into a safe site in the E. coli genome. Without the addition of arabinose, the araC gene inhibited the activity of the araBAD promoter, preventing the ccdA anti-toxic gene from expressing normally while the ccdB toxic gene expressed normally, leading to the death of the strain. With the addition of arabinose, the anti-toxic gene ccdA was expressed normally, thereby inhibiting the ccdB toxic gene and enabling the strain to grow normally.

[0092] (2) A 5'UTR sequence (abbreviated as VecSeqA) is inserted before the ccdB start codon. This sequence can be bound by the small RNA inhibitor on miniVec, so that in the absence of arabinose, as long as the strain contains the miniVec plasmid and provides the small RNA inhibitor to inhibit the expression of the ccdB toxic gene, the strain can survive. Furthermore, a high-copy plasmid can be extracted from the surviving strain. (3) Optionally, depending on the type of replication initiation site Ori, a Rep protein can be introduced into the genome of the E. coli strain, such as the R6K plasmid replication essential protein R6KRep, which is suitable for the preparation of plasmids such as R6K that require auxiliary proteins for conditional replication. An example of a strain functional element (i.e., a host cell expression cassette) is shown in Figure 1.

[0093] (2) miniVec Plasmid non-resistant plasmid

[0094] The miniVec Plasmid contains only the Ori required for plasmid replication and a nucleic acid fragment 3 (named VecSeqB) that transcribes a small RNA inhibitor. The remaining sequence is the target gene expression cassette (Figure 2). The prokaryotic backbone region of traditional plasmids ranges from 2000 to 2500 bp. However, depending on the Ori selected, the proportion of the prokaryotic backbone region in the miniVec Plasmid can be significantly reduced, typically reducing the sequence length by 4-5 times.

[0095] The synergistic effects of VecSeqB with VecSeqA / ccdB and araBAD-araC / ccdA are shown in Figure 3. When the miniVec stable strain contains the miniVec Plasmid, VecSeqA binds to VecSeqB to form a complex, creating a steric hindrance that inhibits ccdB protein translation and allows bacterial survival. When the miniVec stable strain does not contain the miniVec Plasmid, the lack of arabinose in the culture medium prevents ccdA protein expression from being activated, and thus, the toxicity of ccdB cannot be inhibited. Consequently, bacteria without the miniVec Plasmid die during culture and are unable to continue dividing. The entire plasmid production process does not require the addition of antibiotics or other auxiliary inhibitors, eliminating the hazards of antibiotics and inhibitors to plasmids in gene therapy while also reducing the costs of plasmid preparation and purification.

[0096] The test materials used in the present invention are all common commercial products and can be purchased in the market.

[0097] The present invention will be further described below in conjunction with the embodiments:

[0098] Example 1

[0099] MiniVec stable strain transformation:

[0100] miniVec stable strain 1: A VecSeqA1 / ccdB protein expression cassette (i.e., toxic gene expression cassette), an araC / ccdA protein expression cassette (anti-toxic gene expression cassette), and an R6KRep protein expression cassette were inserted into the DH5α strain. The VecSeqA1 sequence was selected from repA of IncF.

[0101] miniVec stable strain 2: A VecSeqA1 / ccdB protein expression cassette, two tandem araC / ccdA protein expression cassettes, and an R6K Rep protein expression cassette were inserted into the DH5α strain.

[0102] Construction of an intermediate vector (containing homology arms HA that recombines with the bacterial genome):

[0103] Intermediate Vector 1: Expresses VecSeqA1 / ccdB protein (SEQ ID NO: 1, wherein 1-252 are UTR), one araC / ccdA protein, and R6K Rep protein, as shown in Figure 4;

[0104] Intermediate Vector 2: Expresses VecSeqA1 / ccdB protein (SEQ ID NO: 1, wherein 1-252 are UTR), two tandem araC / ccdA proteins, and R6K Rep protein, as shown in Figure 5 .

[0105] Construction of temperature-sensitive auxiliary plasmid (pHelper Vector):

[0106] pHelperVector1: A 30°C temperature-sensitive helper plasmid that expresses the pRed / ET homologous recombinase under rhamnose induction. This plasmid is Tet-resistant, as shown in Figure 6.

[0107] pHelperVector2: A 30°C temperature-sensitive helper plasmid that expresses the Flp / Frt site recombinase under arabinose induction. This plasmid is Tet-resistant, as shown in Figure 7.

[0108] Digest Intermediate Vector 1 and Intermediate Vector 2 with AarI and recover the recombinant fragments as follows:

[0109] Recombinant fragment 1: DH5αHA-VecSeqA1 / ccdB-araC / ccdA-Frt-Chl-Frt-R6K Rep-DH5αHA, 7195 bp;

[0110] Recombinant fragment 2: DH5αHA-VecSeqA1 / ccdB-2(araC / ccdA)-Frt-Chl-Frt-R6KRep-DH5αHA, 9033bp;

[0111] Transform 1 μg of pHelper vector 1 into the DH5α strain using electroporation, spread on Tet plates, and incubate overnight at 30°C. The next day, single colonies were picked and confirmed by colony PCR to confirm entry of the pHelper vector into the DH5α strain. Positive colonies were retained and designated the intermediate strain for the next step.

[0112] Preparation of Intermediate Strain electroporation competent cells:

[0113] 1 mL of Intermediate Strain bacterial solution was inoculated into 100 mL of Tet LB medium and cultured at 30°C until the OD600 reached 0.2-0.3. 2 mL of 10% rhamnose was added and cultured at 37°C for 45 min to induce pHelper Vecto1 to express the recombinase. At the same time, pHelper vector1 was removed and the Intermediate Strain electroporation competent cell was prepared according to the conventional steps.

[0114] 1 μg of recombinant fragment 1 and recombinant fragment 2 recovered in 1.3 were electroporated into Intermediate Strain competent cells, coated on Chl + Tet + 0.37% arabinose plates, and cultured overnight at 37°C. At this point, the DH5α genome homology arms on recombinant fragments 1 and 2 recombined with the DH5α genome under the action of recombinase, and VecSeqA1 / ccdB-araC / ccdA-Frt-Chl-Frt-R6K Rep or VecSeqA1 / ccdB-2(araC / ccdA)-Frt-Chl-Frt-R6K Rep was inserted into the DH5α genome, obtaining the initial strains: miniVec stable strain 1 and miniVec stable strain 2, respectively.

[0115] Colony PCR was performed to identify whether miniVec stable strain 1 and miniVec stable strain 2 had inserted the recombinant target fragment. The positive clones of colony PCR were sent for testing to confirm the integrity of the recombinant target fragment. The sequencing results are as follows:

[0116] MiniVec stable strain 1: VecSeqA1 / ccdB-araC / ccdA-Frt-Chl-Frt-R6K Rep. The target fragment contains a ccdB sequence mutation, rendering the ccdB protein non-toxic. This means that the ccdA protein expressed by a single araC / ccdA pair is insufficient to suppress the toxicity of the ccdB protein, leading to the strain's own mutation and resulting in viability. This is related to the use of a stronger promoter to mediate the expression of the toxic gene in Example 1.

[0117] MiniVec stable strain 2: VecSeqA1 / ccdB-2(araC / ccdA)-Frt-Chl-Frt-R6K Rep. The ccdB sequence in the target fragment is intact, with no mutations or deletions, and the ccdB protein maintains its toxicity. This indicates that the ccdA protein expressed by the two araC / ccdA residues is sufficient to inhibit the toxicity of the ccdB protein, allowing the strain to grow normally. This strain is now designated miniVec stable-Chl-A1.

[0118] MiniVec stable-Chl-A1 electroporation competent medium was prepared according to conventional procedures.

[0119] 1 μg of pHelper Vector2 containing Flp recombinase was electroporated into miniVec stable-Chl-A1 competent cells, coated on 0.37% arabinose plates, and cultured at 30°C overnight.

[0120] The strain with the Chl resistance gene deleted after Frt recombination was screened, and the positive clones were sent for testing to verify the integrity of the fragment VecSeqA1 / ccdB-2(araC / ccdA)-Frt-R6KRep. The pHelpervector2 was removed by culturing at 37°C. This strain was the miniVec stable-A1 strain.

[0121] miniVec Plasmid Construction

[0122] Multiple versions of VecSeqB1 were designed. According to the differences between Ori and VecSeqB, Gibson homologous recombination reaction was used to construct the different miniVec Plasmids shown in Table 1:

[0123] Table 1 Summary of different miniVec Plasmids

[0124] Among them, the length of the VecSeqB1 sequence in the miniVec Plasmid2 plasmid is 229 bp, and its sequence is shown in SEQ ID NO: 4, that is, the nucleotide sequence shown at positions 30 to 258 of SEQ ID NO: 3.

[0125] In the miniVec Plasmid 3 plasmid, the VecSeqB1 sequence is 181 bp in length, and its sequence is shown in SEQ ID NO: 5, namely, the nucleotide sequence shown at positions 30 to 210 of SEQ ID NO: 3.

[0126] In the miniVec Plasmid 4 plasmid, the VecSeqB1 sequence is 154 bp in length, and its sequence is shown in SEQ ID NO: 6, ie, the nucleotide sequence shown at positions 30 to 183 of SEQ ID NO: 3.

[0127] In the miniVec Plasmid 5 plasmid, the VecSeqB1 sequence is 123 bp in length, and its sequence is shown in SEQ ID NO: 7, ie, the nucleotide sequence shown at positions 61 to 183 of SEQ ID NO: 3.

[0128] Test of synergistic effect between VecSeqA1 / VecSeqB1 and miniVec stable-A1 strain

[0129] 100 ng of miniVec Plasmids 1-6 were electroporated into the miniVec stable-A1 strain, plated onto conventional antibiotic-free plates, and cultured overnight at 37°C. The results are shown in Table 2. As shown in Table 2, in Group 1, where the miniVec strain lacked miniVec Plasmid, the number of colonies on the antibiotic-free plates was 2.99E+05-fold lower than that on the plates containing arabinose. This indicates that in the absence of arabinose, araC can inhibit ccdA expression, causing the ccdB protein to kill the strain, resulting in a significant decrease in colony number. In Groups 2, 3, 4, and 7, where miniVec Plasmids were introduced into the miniVec strain, the number of colonies on the antibiotic-free plates increased by 2.07E+03-1.27E+04-fold compared to the antibiotic-free plates in Group 1. This indicates that the combination of VecSeqB1 (SEQ ID NOs: 3-5) and VecSeqA1 (SEQ ID NO: 1) can effectively inhibit ccdB protein expression. Groups 2, 3, 4, and 7 are miniVec Plasmids of different Ori, and the screening results are not much different, indicating that the miniVec strain is suitable for plasmid preparation of various types of Ori, such as R6K and pUC. In the data of Groups 5 and 6, miniVec Plasmids 4 and 5 were transformed into miniVec stable-A1 strains, and the number of colonies on the non-antibiotic plate increased by 1-5 times compared with the non-antibiotic plate of Group 1, indicating that different truncated versions of VecSeqB1 sequences have different binding efficiencies with VecSeqA1. 154 and VecSeqB1 / 123 The sequence is shorter and its efficiency in inhibiting the expression of ccdB toxic protein is lower than that of other sequences, but it still has a certain screening effect.

[0130] For ease of understanding, the screening effect multiples in Table 2 are converted into a more easily interpretable plasmid screening positive rate (see the rightmost column). The calculation formula is: Plasmid screening positive rate = (1-negative rate) x 100%, where negative rate = the number of non-resistant plate clones of the miniVec strain only / the number of non-resistant plate clones of the corresponding group. It can be seen from the results that without the addition of antibiotics and adjuvants, the plasmid screening positive rate ranges from 20% to 100%, reaching the level of conventional antibiotic plasmid screening. The plasmid screening positive rate of Group 1 is 0% because there is no miniVec Plasmid described in the present invention in the strain. The small RNA sequence on the Plasmid in Groups 3-6 is a step-by-step truncated version of the small RNA sequence of the Plasmid in Group 2, indicating that sequences with a certain similarity can also produce a steric effect and play a role in plasmid screening.

[0131] Table 2 Conversion effect data summary

[0132] ccdB gene sequencing verification:

[0133] The clones from Groups 2-4, as well as three clones each from the 0.37% arabinose plate and the non-antibiotic plate from Group 1, were sequenced to verify the integrity of the ccdB element. The sequencing results are summarized in Table 3:

[0134] Table 3 Summary of ccdB gene sequence integrity sequencing results

[0135] As shown in Table 3, the ccdB sequences of the non-resistant plate clones in Group 1 exhibited varying degrees of deletion or mutation, indicating that in the absence of arabinose, ccdA protein expression is incapable. Survival of the strain relies on mutation or deletion of the ccdB element, thereby eliminating ccdB toxicity. Sequencing results from the arabinose plate clones revealed normal ccdB sequences, indicating that arabinose can activate ccdA protein expression and enable strain survival. In Groups 2-4, the addition of miniVec Plasmid resulted in no mutations or deletions in the ccdB sequence, even in the absence of arabinose, indicating that the combination of VecSeqB1 and VecSeqA1 significantly and effectively inhibits ccdB protein expression.

[0136] MiniVec Plasmid plasmid extraction, comparison of different miniVec Plasmid plasmid yields:

[0137] Pick three clones from each of the Group 2-Group 7 non-antibiotic plates, inoculate into 5 mL of LB medium, and culture at 37°C overnight.

[0138] The yields are shown in Table 4, and the traditional strain DH5α+traditional resistance pAAV[Exp]-CMV>EGFP (https: / / www.vectorbuilder.cn / vector / VB191212-1252ttc.html) was used as a control.

[0139] Table 4 Summary of miniVec Plasmid production

[0140] As can be seen from Table 4, Group 5 and Group 6 cannot achieve an efficient screening effect due to the mutation of ccdB itself, which makes the strains survive, resulting in a significant decrease in the number of plasmid copies entering the corresponding strains. In contrast, compared with the yield of traditional resistance plasmids, the miniVec plasmid yield in Group 2-Group 4 and Group 7 can be increased by about 2-3 times, and the plasmid yield of miniVec Plasmids with different Ori (miniVec Plasmid 1 and miniVec Plasmid 6) is not much different, indicating that the miniVec strain can be applied to the preparation of various types of plasmids such as R6K and pUC plasmids. When expressing the same target gene, the total length of the traditional plasmid sequence is about 2 times longer than that of the miniVec Plasmid, that is, for the same weight of plasmid, the copy number of the miniVec Plasmid is twice that of the traditional plasmid, which means that the copy number of the target gene produced by some strains and plasmids of the present invention can be increased by 4-6 times.

[0141] As can be seen from the above examples, different clones have different sequence lengths, different steric hindrance efficiencies, and different toxic protein inhibition efficiencies, which in turn lead to different plasmid yields. However, no matter which group, it is possible to produce positive clones without resistance plasmids. Even if the individual yields are low, it can still solve the technical problem faced by the present invention, that is, to achieve the screening and production of non-resistance plasmids without the addition of antibiotics and other auxiliary agents. In other words, as long as the screening and production of non-resistance plasmids can be achieved without the addition of antibiotics and other auxiliary inhibitors, it will be possible to solve the bottleneck problem of the existing technology that requires the addition of antibiotics or other auxiliary inhibitors. The level of plasmid yield can be solved by further optimizing the specific small RNA sequence.

[0142] Example 2

[0143] The 5'UTR of the toxic gene was replaced with another sequence VecSeqA2 (the nucleic acid sequence shown in positions 1 to 64 of SEQ ID NO: 2, the sequence was selected from tnp of IS10), and the sequence on the miniVec vector was designed to be VecSeqB2 (SEQ ID NO: 8) to further test the effect.

[0144] MiniVec stable strain transformation:

[0145] miniVec stable strain 3: A VecSeqA2 / ccdB protein expression cassette, two araC / ccdA protein expression cassettes, and an R6K Rep protein expression cassette were inserted into the DH5α strain.

[0146] Construction of an intermediate vector (containing homology arms HA that recombines with the bacterial genome):

[0147] Intermediate Vector 3: expresses VecSeqA2 / ccdB protein (SEQ ID NO: 2), two araC / ccdA proteins, and R6K Rep protein, as shown in Figure 8;

[0148] Construction of temperature-sensitive auxiliary plasmid (pHelper Vector):

[0149] Same as 1.2 of Example 1.

[0150] Intermediate Vector 3 was digested with AarI and the recombinant fragment was recovered as follows:

[0151] Recombinant fragment 3: DH5αHA-VecSeqA2 / ccdB-araC / ccdA-Frt-Chl-Frt-R6K Rep-DH5αHA, 8704 bp, as shown in Figure 8;

[0152] Transform 1 μg of pHelper vector 1 into the DH5α strain using electroporation, spread on Tet plates, and incubate overnight at 30°C. The next day, single colonies were picked and confirmed by colony PCR to confirm entry of the pHelper vector into the DH5α strain. Positive colonies were retained and designated the intermediate strain for the next step.

[0153] Preparation of Intermediate Strain electroporation competent cells:

[0154] 1 mL of Intermediate Strain bacterial solution was inoculated into 100 mL of Tet LB medium and cultured at 30°C until the OD600 reached 0.2-0.3. 2 mL of 10% rhamnose was added and cultured at 37°C for 45 min to induce pHelper Vecto1 to express the recombinase. At the same time, pHelper vector1 was removed and the Intermediate Strain electroporation competent cell was prepared according to the conventional steps.

[0155] Electroporate 1 μg of recombinant fragment 3 recovered in 1.3 into an Intermediate Strain competent cell, plate onto a Chl + Tet + 0.37% arabinose plate, and incubate overnight at 37°C. The DH5α genome homology arms on recombinant fragment 3 recombined with the DH5α genome under the action of the recombinase, and the VecSeqA2 / ccdB-araC / ccdA-Frt-Chl-Frt-R6K Rep was inserted into the DH5α genome, generating the initial strain: miniVec stable strain 3.

[0156] Colony PCR was performed to identify whether miniVec stable strain 3 had inserted the recombinant target fragment. The positive clones from colony PCR were sent for testing to confirm the integrity of the recombinant target fragment. The sequencing results are as follows:

[0157] MiniVec stable strain 3: VecSeqA2 / ccdB-2(araC / ccdA)-Frt-Chl-Frt-R6K Rep. The ccdB sequence in the target fragment is intact, with no mutations or deletions, and the ccdB protein maintains its toxicity. This demonstrates that the ccdA protein expressed by the two araC / ccdA residues is sufficient to inhibit the toxicity of the ccdB protein, allowing normal growth of the strain. This strain is now designated miniVec stable-Chl-A2.

[0158] MiniVec stable-Chl-A2 electroporation competent medium was prepared according to conventional procedures.

[0159] 1 μg of pHelper Vector2 containing Flp recombinase was electroporated into miniVec stable-Chl-A2 competent cells, coated on 0.37% arabinose plates, and cultured at 30°C overnight.

[0160] The strain with the Chl resistance gene deleted after Frt recombination was screened, and the positive clones were sent for testing to verify the integrity of the fragment VecSeqA2 / ccdB-2(araC / ccdA)-Frt-R6KRep. The pHelpervector2 was removed by culturing at 37°C. This strain was the miniVec stable-A2 strain.

[0161] miniVec Plasmid Construction

[0162] MiniVec Plasmid 7 in Table 5 was constructed using the Gibson homologous recombination reaction:

[0163] Table 5 miniVec Plasmid 7 Information

[0164] Test of synergistic effect between VecSeqA2 / VecSeqB2 and miniVec stable-A2 strain

[0165] 100 ng of miniVec Plasmid 7 was electroporated into the miniVec stable-A2 strain, plated on conventional antibiotic-free plates, and cultured overnight at 37°C. The results are shown in Table 6. As can be seen from Table 6, the number of colonies on the antibiotic-free plates in Group 8, where the miniVec strain did not contain miniVec Plasmid, was 3.87E+05-fold lower than that on the plates supplemented with arabinose. This further demonstrates that in the absence of arabinose, araC can inhibit ccdA expression, causing the ccdB protein to kill the strain and significantly reducing the number of colonies. In Group 9, miniVec Plasmid 7 was transformed into the miniVec stable-A2 strain, and the number of colonies on the resistance-free plate increased by 1.48E+03 times compared with the resistance-free plate of Group 8, indicating that the partial complementary binding of VecSeqB2 (SEQ ID NO: 8) and VecSeqA2 (nucleic acid sequence shown in positions 1 to 64 of SEQ ID NO: 2) can effectively inhibit the expression of ccdB protein; the VecSeqA sequences of the miniVec-A2 strain and the miniVec-A1 strain are completely different, but as long as the VecSeqB sequence on the miniVec vector can produce a complementary sequence to it, the inhibition of ccdB is still effective. This shows that different sequence versions of the 5'UTR can be inserted before the ccdB start codon, and the design of a complementary VecSeqB can achieve the desired screening effect.

[0166] Table 6 Summary of miniVec Plasmid 7 transformation effect data

[0167] Three colonies were picked from each Group 9 non-resistant plate, inoculated into 5 mL of LB medium, and cultured overnight at 37°C. The yields are shown in Table 7.

[0168] Table 7 Summary of miniVec Plasmid 7 production

[0169] As can be seen from the yields in Table 7, the combination of VecSeqB2 and VecSeqA2 can inhibit the expression of ccdB protein and achieve plasmid screening; however, the yields of different clones are different.

[0170] The results showed that as long as the RNA sequence of the miniVec vector is partially or completely complementary to the toxic transcript, plasmid screening can be achieved.

[0171] Example 3 Testing of Various Nucleic Acid Fragments 1 and 3 Producing Steric Effects

[0172] The UTR sequence of nucleic acid fragment 1 was changed to VecSeqA3, VecSeqA4, and VecSeqA5, which were selected from rep of CoIE2, repC of pT181, and hok of IncFII, respectively. The sequence of nucleic acid fragment 3 was designed to be the corresponding VecSeqB3, VecSeqB4, and VecSeqB5. The specific sequences are as follows:

[0173] VecSeqA3

[0174] VecSeqB3:

[0175] VecSeqA4:

[0176] VecSeqB4:

[0177] VecSeqA5:

[0178] VecSeqB5:

[0179] 1. Strain Transformation

[0180] miniVec stable strain 4: A VecSeqA3 / ccdB protein expression cassette and an araC / ccdA protein expression cassette were inserted into the DH5α strain.

[0181] miniVec stable strain 5: A VecSeqA4 / ccdB protein expression cassette and two tandem araC / ccdA protein expression cassettes were inserted into the DH5α strain.

[0182] miniVec stable strain 6: A VecSeqA5 / ccdB protein expression cassette and two tandem araC / ccdA protein expression cassettes were inserted into the DH5α strain.

[0183] 1.1 Intermediate Vector Construction (The intermediate vector contains the homology arm HA that recombines with the bacterial genome):

[0184] Intermediate Vector 4: expresses VecSeqA3 / ccdB protein and one araC / ccdA protein, as shown in Figure 9;

[0185] Intermediate Vector 5: expresses VecSeqA4 / ccdB protein and two araC / ccdA proteins, as shown in Figure 10;

[0186] Intermediate Vector 6: expresses VecSeqA5 / ccdB protein and two araC / ccdA proteins, as shown in Figure 11;

[0187] 1.2 Digest Intermediate Vector 4, Intermediate Vector 5, and Intermediate Vector 6 with AarI and recover the recombinant fragments as follows:

[0188] Recombinant fragment 4: DH5αHA-VecSeqA3 / ccdB-(araC / ccdA)-Frt-Amp-Frt-DH5αHA, 8937 bp;

[0189] Recombinant fragment 5: DH5αHA-VecSeqA4 / ccdB-2(araC / ccdA)-Frt-Amp-Frt-DH5αHA, 9068 bp;

[0190] Recombinant fragment 6: DH5αHA-VecSeqA5 / ccdB-2(araC / ccdA)-Frt-Amp-Frt-DH5αHA, 8980 bp;

[0191] 1.3 Intermediate Strain Construction: 1.4 in Example 1 has been constructed as the strain for the next experiment.

[0192] 1.4 Preparation of Intermediate Strain Electroporation Competent Cells:

[0193] 1.5 Inoculate 1 mL of Intermediate Strain into 100 mL of Tet LB medium and culture at 30°C until the OD600 reaches 0.2-0.3. Add 2 mL of 10% rhamnose and culture at 37°C for 45 min to induce pHelperVecto1 to express the recombinase. Follow the standard procedure to prepare Intermediate Strain electroporation competent cells.

[0194] 1.6 Electroporate 1 μg of recombinant fragments 4, 5, and 6 recovered in 1.2 into competent intermediate strains. Plate the plates with Amp + 0.37% arabinose and incubate overnight at 37°C. The DH5α genome homology arms on the three recombinant fragments will recombine with the DH5α genome under the action of the recombinase, and the recombinant fragments will be inserted into the DH5α genome, resulting in the initial strains: miniVec stable-Amp strain 4, miniVec stable-Amp strain 5, and miniVec stable-Amp strain 6.

[0195] 1.7 Use colony PCR to identify whether the three miniVec stable strains in 1.5 have inserted the recombinant target fragment. Send the positive clones from colony PCR for testing to confirm the integrity of the recombinant target fragment.

[0196] 1.8 Prepare miniVec stable-Amp electroporation competent cells according to conventional procedures.

[0197] 1.9 1 μg of pHelper Vector 2 (constructed in Example 1) containing the Flp recombinase was electroporated into the competent cells of miniVec stable-Amp strain 4, miniVec stable-Amp strain 5 and miniVec stable-Amp strain 6, coated on 0.37% arabinose plates, and cultured at 30°C overnight.

[0198] 1.10 Screen the strains in which the Amp resistance gene was deleted after FRT recombination, and send the positive clones for testing to verify the sequence integrity of the inserted target fragment. The strains at this time are miniVec stable strain 4, miniVec stable strain 5, and miniVec stable strain 6.

[0199] 2.miniVec Plasmid Construction

[0200] 2.1 Based on the differences in VecSeqB, the different miniVec Plasmids listed in Table 8 were constructed using Gibson homologous recombination reactions:

[0201] Table 8 Summary of different miniVec Plasmids

[0202] 3. Test of synergistic effect of VecSeqA / VecSeqB and miniVec stable strains

[0203] 100 ng of miniVec Plasmid 8 was electroporated into miniVec stable strain 4, miniVec Plasmid 9 into miniVec stable strain 5, and miniVec Plasmid 10 into miniVec stable strain 6. The cells were plated onto conventional antibiotic-free plates and incubated overnight at 37°C. The results are shown in Table 9.

[0204] As shown in Table 9, the number of colonies on plates containing no arabinose in miniVec strains in Groups 1*, 3*, and 5* was over 2.86E+03 times lower than that on plates containing arabinose. This indicates that in the absence of arabinose, araC inhibits ccdA expression, causing lethality of the ccdB protein and a significant reduction in colony number. However, the different VecSeqA strains did not affect ccdB expression. In Groups 2*, 4*, and 6*, plasmid screening positive rates exceeded 59% when miniVec Plasmid was introduced into miniVec strains. This indicates that the different VecSeqB strains, in reverse complementation with VecSeqA, produce a steric hindrance that effectively inhibits toxic transcripts, thereby achieving effective plasmid screening.

[0205] Table 9 Conversion effect data summary

[0206] Three colonies were picked from each of the antibiotic-free plates in Group 2*, Group 4*, and Group 6*, inoculated into 5 mL of LB medium, and cultured overnight at 37°C to extract the miniVec Plasmid. Yields are summarized in Table 10.

[0207] As shown in Table 10, the reverse complementation of different VecSeqB and VecSeqA produces a steric hindrance effect, inhibiting the function of toxic transcripts and thus facilitating plasmid selection. Although the steric hindrance efficiency, toxic protein inhibition efficiency, and plasmid yield vary, all can be used for plasmid selection and production under conditions without antibiotics or auxiliary inhibitors.

[0208] Table 10 Summary of miniVec Plasmid Production

[0209] Example 4 Testing Various Toxicity and Anti-Toxicity Supporting Systems

[0210] The ccdB / ccdA toxicity and anti-toxicity genes were replaced with the Zeta / Epsilon gene and the Doc / Phd gene. The UTR of nucleic acid fragment 1 is VecSeqA1, and the specific sequence is as follows:

[0211] VecSeqA1 / Zeta:

[0212] Wherein UTR is 1-252, corresponding to 1-252 of SEQ ID NO:1

[0213] araBAD-araC / Epsilon:

[0214] VecSeqA1 / Doc:

[0215] Wherein UTR is 1-252, corresponding to 1-252 of SEQ ID NO:1

[0216] araBAD-araC / Phd:

[0217] 1. Strain Transformation

[0218] miniVec stable strain 7: A VecSeqA1 / zeta protein expression cassette, two tandem araBAD-araC / epsilon protein expression cassettes, and an R6K Rep protein expression cassette were inserted into the DH5α strain.

[0219] miniVec stable strain 8: A VecSeqA1 / Doc protein expression cassette, two tandem araBAD-araC / Phd protein expression cassettes, and an R6K Rep protein expression cassette were inserted into the DH5α strain.

[0220] 1.1 Intermediate Vector Construction (The intermediate vector contains the homology arm HA that recombines with the bacterial genome):

[0221] Intermediate Vector 7: Expresses VecSeqA1 / zeta protein, two araBAD-araC / epsilon proteins, and R6K Rep protein, as shown in Figure 12;

[0222] Intermediate Vector 8: Expresses VecSeqA1 / Doc protein, two araBAD-araC / Phd proteins, and R6K Rep protein, as shown in Figure 13;

[0223] 1.2 Digest Intermediate Vector 7 and Intermediate Vector 8 with AarI and recover the recombinant fragments as follows:

[0224] Recombinant fragment 7: DH5αHA-VecSeqA1 / zeta-2(araC / epsilon)-Frt-Amp-Frt-R6K Rep-DH5αHA, 9922 bp;

[0225] Recombinant fragment 8: DH5αHA-VecSeqA1 / Doc-2(araC / Phd)-Frt-Amp-Frt-R6K Rep-DH5αHA, 9337 bp.

[0226] 1.3 Intermediate Strain Construction: 1.4 in Example 1 has been constructed as the strain for the next experiment.

[0227] 1.4 Preparation of Intermediate Strain Electroporation Competent Cells:

[0228] 1.5 Inoculate 1 mL of Intermediate Strain into 100 mL of Tet LB medium and culture at 30°C until the OD600 reaches 0.2-0.3. Add 2 mL of 10% rhamnose and culture at 37°C for 45 min to induce pHelperVecto1 to express the recombinase. Follow the standard procedure to prepare Intermediate Strain electroporation competent cells.

[0229] 1.6 Electroporate 1 μg of recombinant fragment 7 and recombinant fragment 8 recovered in 1.2 into Intermediate Strain competent cells, spread onto Amp plates with 0.37% arabinose, and incubate overnight at 37°C. The DH5α genomic homology arms on the recombinant fragments will recombine with the DH5α genome under the action of the recombinase, and the recombinant fragments will be inserted into the DH5α genome, generating the initial strains: miniVec stable-Amp strain 7 and miniVec stable-Amp strain 8.

[0230] 1.7 Use colony PCR to identify whether the two miniVec stable strains in 1.6 have inserted the recombinant target fragment. Send the positive clones from colony PCR for testing to confirm the integrity of the recombinant target fragment.

[0231] 1.8 Prepare miniVec stable-Amp electroporation competent cells according to conventional procedures.

[0232] 1.9 1 μg of pHelper Vector 2 (constructed in Example 1) containing the Flp recombinase was electroporated into the competent cells of miniVec stable-Amp strain 7 and miniVec stable-Amp strain 8, coated on 0.37% arabinose plates, and cultured at 30°C overnight.

[0233] 1.10 Screen the strains with the Amp gene deleted after FRT recombination, send the positive clones for testing to verify the integrity of the insert sequence. The strains at this time are miniVec stable strain 7 and miniVec stable strain 8.

[0234] 2.miniVec Plasmid Construction

[0235] The miniVec Plasmids of different Ori species listed in Table 11 were constructed using Gibson homologous recombination reaction:

[0236] Table 11 Summary of different miniVec Plasmids

[0237] 3. Testing the effects of different toxicity and anti-toxicity systems in miniVec stable strains

[0238] 100 ng of miniVec Plasmid 11 and miniVec Plasmid 12 were electroporated with miniVec stable strain 7 and miniVec stable strain 8, respectively, and plated on conventional antibiotic-free plates and cultured overnight at 37°C. The results are shown in Table 12.

[0239] As shown in Table 12, the number of colonies on the non-resistant plates in Groups 1' and 4' without miniVec Plasmid was over 2.99E+03 times lower than that on the plates containing arabinose. This indicates that in the absence of arabinose, araC can inhibit the expression of different anti-toxin proteins (epsilon / Phd) and different toxicity proteins (zeta / Doc) that cause lethality, significantly reducing the number of colonies. Therefore, the use of different toxicity / anti-toxin systems is also applicable to the present invention. In Group 2', 3' and Group 5', 6', miniVec Plasmid was transferred into the miniVec strain, and the plasmid screening positive rates ranged from 48.6% to 100%, indicating that the combination of VecSeqB and VecSeqA can effectively inhibit the expression of different toxic proteins (zeta / Doc). Moreover, the screening results of Group 2', 3' and Group 5', 6', which are miniVec Plasmids of different Ori, are not much different, indicating that the miniVec strain is suitable for plasmid preparation of various types of Ori, such as R6K and pUC.

[0240] Table 12 Conversion effect data summary

[0241] Three colonies were selected from each of the antibiotic-free plates in Groups 2', 3', 5', and 6', inoculated into 5 mL of LB medium, and cultured overnight at 37°C to extract the miniVec Plasmid. The yields are summarized in Table 13. As shown in Table 13, while the plasmid yields varied among the different groups, the overall differences were not significant. All groups were able to produce antibiotic-free plasmids, demonstrating that the system of the present invention allows for flexible interchange of toxic and anti-toxic transcripts and is not limited to specific toxic and anti-toxic transcripts.

[0242] Table 13 Summary of miniVec Plasmid Production 2

[0243] It can be seen from all the provided embodiments that after the operon inducer is removed, the anti-toxic transcript cannot be expressed normally. However, if the host strain is transformed with the plasmid of the present invention, the small RNA expressed on the plasmid is reversely complementary to the toxic transcript, causing a steric effect, thereby inhibiting the function of the toxic transcript. This can achieve anti-resistant screening of the plasmid without adding antibiotics or auxiliary inhibitors, and has universal applicability.

[0244] The above are only preferred embodiments of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A plasmid production system, characterized in that including host cell expression cassettes and plasmids; The host cell expression cassette includes a toxic gene expression cassette and an anti-toxic gene expression cassette; The toxic gene expression cassette includes a promoter and a nucleic acid fragment 1 for transcribing a toxic transcript; The anti-toxic gene expression cassette includes a promoter, an operator and a nucleic acid fragment 2 for transcribing an anti-toxic transcript; the anti-toxic transcript has a resistance effect on the toxicity produced by the toxic transcript; The plasmid comprises a nucleic acid fragment 3 for transcribing small RNA and an Ori replication initiation site. The nucleic acid fragment 3 is reversely complementary to the nucleic acid fragment 1 to produce a steric effect, thereby inhibiting the function of the toxic transcript.

2. The plasmid production system according to claim 1, characterized in that The nucleic acid fragment 3 is completely complementary to the nucleic acid fragment 1 in reverse direction, or has at least 20% sequence reverse complementarity, or has at least 10 consecutive bp reverse complementarity.

3. The plasmid production system according to claim 1 or 2, characterized in that: The nucleic acid fragment 1 includes a UTR sequence and a toxic gene.

4. The plasmid production system according to any one of claims 1 to 3, characterized in that The toxic gene is selected from any one of ccdB, ParE, MazF, Kid, HicA, RelE, VapC, Doc, RatA, HipA, Zeta, ToxN, YeeV, CptA, GhoT, Hok, TisB, SymE, and PasA; the nucleic acid fragment 2 is an anti-toxic gene, and the anti-toxic gene is selected from any one of ccdA, ParD, MazE, Kis, HicB, RelB, VapB, Phd, RatB, HipB, Epsilon, ToxI, YeeU, CptB, GhoS, Sok, IstR-1, SymR, and PasB / C.

5. The plasmid production system according to any one of claims 1 to 4, characterized in that: The operon is any one of an arabinose operon, a Lac operon, a rhamnose catabolism operon, a tryptophan operon, a gab operon and a Gal operon.

6. The plasmid production system according to any one of claims 3 to 5, characterized in that: The small RNA transcribed from the nucleic acid fragment 3 is reverse complementary to the UTR sequence in the nucleic acid fragment 1 and produces a steric hindrance effect.

7. The plasmid production system according to any one of claims 3 to 6, characterized in that: The UTR sequence is fused with the toxic gene for expression.

8. The plasmid production system according to any one of claims 1 to 7, characterized in that: The number of the anti-toxic gene expression cassettes and the number of the toxic gene expression cassettes are both selected from integers ≥1.

9. The plasmid production system according to any one of claims 1 to 8, characterized in that: The host cell expression cassette also includes Rep protein.

10. The plasmid production system according to any one of claims 1 to 9, characterized in that: The Ori replication origin site is selected from ColE1, pBR322, pMB1, R6K, pUC, F1, p15A, 2μori or oriV.

11. The plasmid production system according to any one of claims 1 to 10, characterized in that: The plasmid also includes a gene of interest.

12. The plasmid production system according to any one of claims 1 to 11, characterized in that: The plasmid comprises a promoter, a target gene, an Ori replication initiation site and a nucleic acid fragment 3 for transcribing small RNA, which are sequentially connected.

13. The plasmid production system according to any one of claims 1 to 12, characterized in that: It also includes host bacteria or host cells, wherein the host bacteria and host cells are derived from Escherichia coli, Agrobacterium, Bacillus, Bacillus or yeast.

14. Use of the plasmid production system according to any one of claims 1 to 13 in producing plasmids or in preparing products for cell gene therapy.

15. A method for producing a plasmid using the plasmid production system according to any one of claims 1 to 13, characterized in that: The plasmid is transferred into a host cell into which the host cell expression cassette is integrated, and cultured.

Images