A plasmid backbone capable of enhancing the stability of polyadenylate tails

Abstract

The present invention relates to the field of in vitro transcription, and particularly to a plasmid backbone capable of improving the stability of polyadenylate tails. The present invention provides an expression cassette, which sequentially includes: the rop gene, the replicon ori, and the resistance gene; the rop gene, the replicon ori, and the resistance gene are connected by a linking fragment. The present invention has established a low-copy pmRVacSL and a medium-copy pmRVacM plasmid vector system through artificial modification. Since pmRVacSL is a low-copy vector, the yield of plasmid DNA is relatively low. The pmRVacM plasmid backbone solves the problem of relatively low plasmid DNA yield of pmRVacSL. This vector system can not only maintain the stable replication of polyA tail sequences of more than 150 bp, but also quickly add genes of interest and polyadenylate (polyA) sequences, and efficiently and rapidly obtain the DNA template for in vitro transcription of the target mRNA.

Description

Technical Field

[0001] The present invention relates to the field of in vitro transcription, and particularly to a plasmid backbone capable of improving the stability of polyadenylate tails. Background Art

[0002] mRNA vaccines inject synthetic mRNA sequences encoding protein antigens into the human body, guiding the body to express the corresponding proteins and inducing the body to trigger specific immune responses to achieve the purpose of disease prevention and treatment. Compared with traditional vaccines, they have multiple advantages such as strong immunogenicity, high safety, and easy research and production.

[0003] The polyA tail is an important feature of eukaryotic mRNA, consisting of multiple adenosine acids and located at the 3' end of mRNA. It plays an important role in basic expression regulation, especially in translation and controlling mRNA stability. The polyA tail can prevent the degradation of the mRNA 3' end by exonucleases and increase the stability of mRNA. In addition, during translation, the polyA tail can act as a "translation enhancer". Research shows that removing the polyA site can reduce the mRNA expression level by up to 10 times.

[0004] Currently, when synthesizing mRNA by IVT, there are mainly the following three methods for adding polyA tails: one is enzymatic synthesis, adding polyA polymerase derived from Escherichia coli after mRNA transcription is completed; the second is co-transcription, directly transcribing from the polyA sequence already present on the template plasmid DNA or PCR product; the third is using phi29 DNA polymerase to amplify the plasmid by in vitro rolling circle amplification (RCA) technology. Among them, co-transcriptional tailing can maintain the homogeneity of the final product, reduce process steps, and save costs, becoming a more preferred method. Some scientists have also found a new way and selected a segmented polyA co-transcription method, which can reduce the deletion of polyA.

[0005] The preparation of mRNA plasmid DNA templates usually chooses bacterial fermentation to scale up the production of plasmids. However, during the bacterial amplification process, the polyA tails of plasmids carrying long polyhomonucleotide sequences may shorten as the bacteria continue to amplify, and when the polyA sequence on the plasmid is greater than 100 bp, it is more likely to be unstable during cultivation and then undergo base loss and shortening problems, causing great difficulties in production. Summary of the Invention

[0006] In view of this, the present invention provides a plasmid backbone that can improve the stability of polyadenylate tails. The present invention has established a low-copy pmRVacSL and a medium-copy pmRVacM plasmid vector system through artificial modification. Since pmRVacSL is a low-copy vector, the yield of plasmid DNA is relatively low. The pmRVacM plasmid backbone solves the problem of low plasmid DNA yield of pmRVacSL. This vector system can not only maintain the stable replication of polyA tail sequences over 150 bp, but also quickly add genes of interest and polyadenylate (polyA) sequences, and efficiently obtain the DNA template for in vitro transcription of the target mRNA.

[0007] To achieve the above-mentioned invention objectives, the present invention provides the following technical solutions:

[0008] The present invention provides an expression cassette, which sequentially includes: rop gene, replicon ori, and resistance gene;

[0009] The rop gene, the replicon ori, and the resistance gene are connected by a linker fragment.

[0010] In some embodiments of the present invention, the above expression cassette, from the 5' end to the 3' end, sequentially includes: rop gene, replicon ori, and resistance gene;

[0011] The rop gene, the replicon ori, and the resistance gene are connected by a linker fragment.

[0012] In some embodiments of the present invention, in the above expression cassette, the rop gene includes wild-type or mutant type; the mutant type is obtained from the wild-type through any of the following:

[0013] (I), removing the start codon; or

[0014] (II), mutating the start codon to a stop codon.

[0015] In some embodiments of the present invention, in the above expression cassette, the rop gene has:

[0016] (1), a nucleotide sequence as shown in any of SEQ ID NO:1 to SEQ ID NO:3; or

[0017] (2), a nucleotide sequence obtained by substituting, deleting, or adding one or more bases to the nucleotide sequence as shown in (1), and having the same or similar function as the nucleotide sequence as shown in (1); or

[0018] (3), a nucleotide sequence having at least 80%, 85%, 90%, or 95% identity with the nucleotide sequence as shown in (1) or (2).

[0019] In some embodiments of the present invention, in the above expression cassette, the sequence of SEQ ID NO:1 is:

[0020]

[0021] In some embodiments of the present invention, in the above expression cassette, the sequence of SEQ ID NO:2 is:

[0022]

[0023] In some embodiments of the present invention, in the above expression cassette, the sequence of SEQ ID NO:3 is:

[0024]

[0025] In some embodiments of the present invention, in the above expression cassette, the replicon ori includes: wild type or mutant type; the mutant type is obtained by mutating the 442nd position of the nucleotide sequence of the wild type.

[0026] In some embodiments of the present invention, in the above expression cassette, the mutant type includes mutating the G at the 442nd position of the nucleotide sequence of the wild type to A.

[0027] In some embodiments of the present invention, in the above expression cassette, the replicon ori has:

[0028] (4), a nucleotide sequence as shown in SEQ ID NO:4 or SEQ ID NO:5; or

[0029] (5), a nucleotide sequence obtained by substituting, deleting or adding one or more bases to the nucleotide sequence as shown in (4), and a nucleotide sequence having the same or similar function as the nucleotide sequence as shown in (4); or

[0030] (6), a nucleotide sequence having at least 80%, 85%, 90% or 95% identity with the nucleotide sequence as shown in (4) or (5).

[0031] In some embodiments of the present invention, in the above expression cassette, the sequence of SEQ ID NO:4 is:

[0032]

[0033] In some embodiments of the present invention, in the above expression cassette, the sequence of SEQ ID NO:5 is:

[0034]

[0035] In some embodiments of the present invention, in the above expression cassette, the resistance gene includes: kanamycin resistance gene.

[0036] In some embodiments of the present invention, in the above expression cassette, the sequence of the kanamycin resistance gene is as shown in SEQ ID NO: 11:

[0037]

[0038] In some embodiments of the present invention, in the above expression cassette, the linker fragment has:

[0039] (7), a nucleotide sequence as shown in any one of SEQ ID NOs: 6 to 10; or

[0040] (8), a nucleotide sequence obtained by substituting, deleting or adding one or more bases to the nucleotide sequence as shown in (7), and a nucleotide sequence having the same or similar function as the nucleotide sequence as shown in (7); or

[0041] (9), a nucleotide sequence having at least 80%, 85%, 90% or 95% identity with the nucleotide sequence as shown in (7) or (8).

[0042] In some embodiments of the present invention, in the above expression cassette, the sequence of SEQ ID NO: 6 is:

[0043]

[0044] In some embodiments of the present invention, in the above expression cassette, the sequence of SEQ ID NO: 7 is:

[0045]

[0046] In some embodiments of the present invention, in the above expression cassette, the sequence of SEQ ID NO: 8 is:

[0047]

[0048] In some embodiments of the present invention, in the above expression cassette, the sequence of SEQ ID NO: 9 is:

[0049]

[0050] In some embodiments of the present invention, in the above expression cassette, the sequence of SEQ ID NO: 10 is:

[0051]

[0052] In some embodiments of the present invention, in the above expression cassette, the linking fragment includes: linking fragment 1 to linking fragment 4;

[0053] The linking fragment 1 is located at the 5' end of the rop gene;

[0054] The linking fragment 2 is located between the rop gene and the replicon ori;

[0055] The linking fragment 3 is located between the replicon ori and the resistance gene;

[0056] The linking fragment 4 is located at the 3' end of the resistance gene.

[0057] In some embodiments of the present invention, in the above expression cassette, the sequence of the linking fragment 1 is as shown in SEQ ID NO:6 or SEQ ID NO:7.

[0058] In some embodiments of the present invention, in the above expression cassette, the sequence of the linking fragment 2 is as shown in SEQ ID NO:8.

[0059] In some embodiments of the present invention, in the above expression cassette, the sequence of the linking fragment 3 is as shown in SEQ ID NO:9.

[0060] In some embodiments of the present invention, in the above expression cassette, the sequence of the linking fragment 4 is as shown in SEQ ID NO:10.

[0061] In some embodiments of the present invention, the above expression cassette has:

[0062] (10), a nucleotide sequence as shown in any of SEQ ID NO:12 to SEQ ID NO:16; or

[0063] (11), a nucleotide sequence obtained by substituting, deleting or adding one or more bases to the nucleotide sequence as shown in (10), and having the same or similar function as the nucleotide sequence as shown in (10); or

[0064] (12), a nucleotide sequence having at least 80%, 85%, 90% or 95% identity with the nucleotide sequence as shown in (10) or (11).

[0065] In some embodiments of the present invention, the sequence of SEQ ID NO:12 is:

[0066]

[0067]

[0068]

[0069] In some embodiments of the present invention, the sequence of SEQ ID NO:13 is:

[0070]

[0071] In some embodiments of the present invention, the sequence of SEQ ID NO:14 is:

[0072]

[0073] In some embodiments of the present invention, the sequence of SEQ ID NO:15 is:

[0074]

[0075]

[0076] In some embodiments of the present invention, the sequence of SEQ ID NO:16 is:

[0077]

[0078] The present invention also provides a recombinant plasmid, comprising: the above expression cassette and an acceptable genetic element.

[0079] In some embodiments of the present invention, in the above recombinant plasmid, the acceptable genetic element includes: one or more of polyA, 5'UTR, 3'UTR, promoter, multiple cloning site and restriction enzyme site.

[0080] In some embodiments of the present invention, in the above recombinant plasmid, a backbone plasmid is further included, and the backbone plasmid includes: any one of a low-copy backbone plasmid, a medium-copy backbone plasmid and a high-copy backbone plasmid.

[0081] In some embodiments of the present invention, in the above recombinant plasmid, a backbone plasmid is further included, and the backbone plasmid is: a low-copy backbone plasmid.

[0082] The present invention also provides a host for transforming and / or transfecting the above recombinant plasmid.

[0083] The present invention also provides the use of the above expression cassette, the above recombinant plasmid, and / or the above host in any of the following items;

[0084] (I), preparing mRNA; and / or

[0085] (II), preparing an mRNA drug, preparation, or drug combination; and / or

[0086] (III), improving the stability of the plasmid; and / or

[0087] (IV), increasing the yield of the plasmid.

[0088] The present invention also provides a product, comprising: the above expression cassette, the above recombinant plasmid, and / or the above host.

[0089] The present invention artificially modified and constructed a set of plasmid backbones, which can still maintain stable replication during bacterial amplification when inserting a polyA tail of more than 150 bp, and there will be no phenomenon of polyA tail shortening. BRIEF DESCRIPTION OF THE DRAWINGS

[0090] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for the description of the embodiments or the prior art.

[0091] Figure 1 TBE-PAGE gel diagram showing pmRVac plasmids with different lengths of polyA tails; from left to right are Ladder, 150A, 120A, 100A, 80A, 60A, and Ladder; where: Ladder from top to bottom are 300 bp, 200 bp, and 100 bp;

[0092] Figure 2 TBE-PAGE gel diagram showing pmRVacSL plasmids with different lengths of polyA tails; from left to right are Ladder, 65A, 80A, 100A, 120A, 150A, and Ladder; where: Ladder from top to bottom are 300 bp, 200 bp, and 100 bp;

[0093] Figure 3 Showing the comparison of the DNA yields of pmRVacSL and pmRVacM 4 mutant plasmids;

[0094] Figure 4TBE-PAGE gel images showing the polyA tails of pmRVacSL and pmRVacM; from left to right are Ladder, pmRVacSL-120A, pmRVacSL-150A, pmRVacM(G to A)-120A, pmRVacM(G to A)-150A, pmRVacM(remove GTG)-120A, pmRVacM(remove GTG)-150A, pmRVacM(GTG to TAA)-120A, pmRVacM(GTG to TAA)-150A, pmRVacM(remove GGAGG)-120A, pmRVacM(remove GGAGG)-150A and Ladder; where: the Ladder from top to bottom is 300bp, 200bp and 100bp;

[0095] Figure 5 Showing the correct proportion of polyA of pmRVacSL plasmid DNA;

[0096] Figure 6 Showing the plasmid backbone map of pmRVac;

[0097] Figure 7 Showing the plasmid backbone map of pmRVacSL;

[0098] Figure 8 Showing the plasmid backbone map of pmRVacM(G to A);

[0099] Figure 9 Showing the plasmid backbone map of pmRVacM(remove GTG);

[0100] Figure 10 Showing the plasmid backbone map of pmRVacM(GTG to TAA);

[0101] Figure 11 Showing the plasmid backbone map of pmRVacM(remove GGAGG). Detailed implementation manners

[0102] The present invention discloses a plasmid backbone that can improve the stability of polyadenylate tails.

[0103] It should be understood that the expression "one or more of..." individually includes each of the objects recited after the expression and various different combinations of two or more of the recited objects, unless otherwise understood from the context and usage. The expression "and / or" in combination with three or more recited objects should be understood to have the same meaning, unless otherwise understood from the context.

[0104] The terms "comprising", "having", or "including", including the use of their grammatical synonyms, should generally be understood as open-ended and non-restrictive, for example, not excluding other unrecited elements or steps, unless specifically stated otherwise or understood from the context otherwise.

[0105] It should be understood that as long as the present invention remains operable, the order of steps or the order of performing certain actions is not important. In addition, two or more steps or actions can be carried out simultaneously.

[0106] The use of any and all examples or exemplary language herein, such as "for example" or "including", is merely intended to better illustrate the present invention and does not limit the scope of the present invention unless a claim is made. No language in this specification should be construed as indicating that any unclaimed element is essential for the practice of the present invention.

[0107] In addition, the numerical ranges and parameters used to define the present invention are approximate values. The relevant values in the specific embodiments have been presented as precisely as possible herein. However, any value inherently and inevitably contains standard deviations due to individual test methods. Therefore, unless otherwise clearly stated, it should be understood that all ranges, amounts, values, and percentages used in this disclosure are modified by "about". Here, "about" generally means that the actual value is within plus or minus 10%, 5%, 1%, or 0.5% of a specific value or range.

[0108] Three vector backbones, namely pmRVac, pmRVaSL, and pmRVacM, were obtained by artificial modification in the present invention. pmRVac is a high-copy vector, and the replication origin of the plasmid backbone vector is pUC ori; pmRVacSL is a low-copy plasmid vector, and pmRVacM is a medium-copy plasmid vector. The replication origins of the plasmid backbone vectors of pmRVacSL and pmRVacM are both pBR322 ori. Among them, pmRVacM was obtained by base mutation and removal from the pmRVacSL backbone vector, which can significantly improve the yield of plasmid DNA.

[0109] The present invention will be further described below in conjunction with embodiments:

[0110] Example 1 Construction of pmRVac Vectors Containing Different Lengths of polyA Tails

[0111] (1) The pmRVac-EGFP plasmid was obtained by modifying the pUC57 high-copy vector.

[0112] (2) The pmRVac-EGFP plasmid was digested with restriction enzymes BsrGI and NotI, and the purified linearized pmRVac backbone containing the EGFP target gene was obtained by a gel recovery kit.

[0113] (3) Synthesize plasmids containing polyA tails of different lengths, digest the plasmids containing pure polyA tail fragments of different lengths with restriction enzymes BsrGI and NotI, and obtain purified fragments containing polyA tails of different lengths through a gel extraction kit.

[0114] (4) Use T4 ligase to ligate the purified linearized pmRVac backbone containing the EGFP target gene and the purified fragments containing polyA tails of different lengths to obtain a reaction system of pmRVac vectors containing the target gene EGFP with polyA tails of different lengths.

[0115] (5) Transfer the T4 ligase reaction system into VB UltraStable competent cells by chemical transformation. Transformation steps: Thaw the competent cells on ice for 30 min, add the T4 ligase reaction system to the competent cells, incubate on ice for 30 min, perform a hot water bath at 42 °C for 1 min, incubate on ice for 2 min, add LB medium and shake culture at 37 °C for 1 h. Then, take an appropriate amount of the bacterial solution and spread it on an LB solid plate containing kanamycin, and incubate it upside down at 37 °C for 16 h.

[0116] (6) Randomly pick several single colonies on the plate into a small amount of sterile water. Using this bacterial solution as a template, design specific forward and reverse primers and PCR enzymes for bacterial solution PCR reaction. Verify the bacterial solution PCR results by gel electrophoresis. Inoculate the clones with bacterial solution PCR results that meet the expectations into an LB liquid medium containing kanamycin and shake culture at 37 °C for 16 h. Take a part of the cultured bacterial solution and store it in glycerol, and extract the plasmid from the remaining bacterial solution using a plasmid extraction kit. Thus, pmRVac plasmids containing polyA tails of different lengths are obtained.

[0117] (7) Digest the plasmid with restriction enzyme BtgI to digest the DNA sequences at both ends of the polyA tails containing 60A, 80A, 100A, 120A, and 150A, and obtain fragments containing polyA tails with lengths of 111 bp, 131 bp, 151 bp, 171 bp, and 201 bp respectively.

[0118] (8) Identify the stability of the polyA tail through TBE-PAGE gel. It can be observed that when the pmRVac vector backbone carries a polyA tail of more than 100 bp, the phenomenon of recombination and replication instability appears (such as Figure 1 ). The pmRVac plasmid backbone map is as shown in Figure 6 .

[0119] Example 2 Construction of pmRVacSL Vectors Containing PolyA Tails of Different Lengths

[0120] (1) Using the pBR322 low-copy plasmid as the DNA template, specific forward and reverse primers and PCR enzymes were designed for PCR amplification. A purified linearized backbone with pBR322 Rop and pBR322 Ori was obtained through a gel extraction kit.

[0121] (2) The pmRVac plasmid in Example 1 was digested with the restriction enzymes DraI and NotI, and a purified DNA fragment containing Kanamycin resistance and the target gene EGFP was obtained through a gel extraction kit.

[0122] (3) The purified linearized backbone with pBR322 Rop and pBR322 Ori and the DNA fragment containing Kanamycin resistance and the target gene EGFP were ligated through Gibson reaction to obtain a reaction system for an in vitro transcription vector containing the target gene EGFP.

[0123] (4) Referring to steps (5) and (6) of Example 1, the pmRVacSL plasmid was obtained.

[0124] (5) Referring to steps (2) to (6) of Example 1, pmRVacSL plasmids with different lengths of polyA tails were obtained.

[0125] (6) The pmRVacSL plasmid was digested with the restriction enzyme BtgI, and the DNA sequences at both ends of the polyA tails containing 65A, 80A, 100A, 120A, and 150A were digested, respectively obtaining polyA tail-containing fragments with lengths of 116bp, 131bp, 151bp, 171bp, and 201bp.

[0126] (7) The stability of the polyA tails was identified by TBE-PAGE gel. It was observed that when the pmRVacSL vector backbone carried a polyA tail up to 150bp, it still maintained stable replication during bacterial amplification and did not undergo recombination (such as Figure 2 ). The pmRVacSL plasmid backbone map is as shown in Figure 7 .

[0127] Example 3 Construction of the pmRVacM Vector System

[0128] (1) The pmRVacM vector system was derived from the pmRVacSL vector system through base mutation and removal.

[0129] (2) It was found that the increase in the copy number of pUC was due to a point mutation in RNA II, which was inhibited by the Rom / Rop protein or by reducing the growth temperature to 30 °C. Therefore, a G to A mutation was introduced into the 112-nucleotide fragment of the RNA II transcript, which is adjacent to the complementary region of RNA I (-1 position on the RNA I transcript), to obtain the pmRVacM(G to A) plasmid vector. The pmRVacM(G to A) plasmid backbone is as Figure 8 shown.

[0130] (3) Since the Rop gene encoding the Rop protein can promote the conversion of the unstable RNA I-RNA II complex into a stable complex and is used to reduce the copy number (source - plasmid pMB1). Therefore, the start codon GTG was removed to prevent the expression of the Rop protein while retaining its DNA sequence, resulting in the pmRVacM(remove GTG) plasmid vector. The pmRVacM(remove GTG) plasmid backbone is as Figure 9 shown.

[0131] (4) By mutating the start codon GTG to TAA to prevent the expression of the Rop protein while retaining its DNA sequence, the pmRVacM(GTG to TAA) plasmid vector was obtained. The pmRVacM(GTG to TAA) plasmid backbone is as Figure 10 shown.

[0132] (5) By removing the ribosome binding site GGAGG of the Rop gene to prevent the expression of the Rop protein while retaining its DNA sequence, the pmRVacM(remove GGAGG) plasmid vector was obtained. The pmRVacM(remove GGAGG) plasmid backbone is as Figure 11 shown.

[0133] (6) The yields of the pmRVacSL plasmid with 120A and 150A tails containing the target gene EGFP were compared with those of the pmRVacM(G to A), pmRVacM(remove GTG), pmRVacM(GTG to TAA), and pmRVacM(remove GGAGG) plasmids with 120A and 150A containing the target gene EGFP.

[0134] (7) The plasmid DNA yield was obtained by inoculating 10 μL of monoclonal bacterial solution into 2 mL of LB medium, culturing it in a 15 mL shaking tube at 37 °C and 250 rpm in a shaker for 15 h, and then extracting it using a plasmid miniprep kit and measuring the concentration.

[0135] (8) It was found that mutating the start codon GTG of the Rop gene in the pmRVacSL plasmid backbone to TAA resulted in the most significant increase in plasmid DNA yield, with a 1.5-fold increase per mL of bacterial solution and a significant difference (as Figure 3 ).

[0136] (9) After the plasmid in step (6) was digested with the restriction endonuclease BtgI, TBE-PAGE was used for identification. The polyA tails of 120A and 150A of pmRVacSL and the four mutant pmRVacMs could still maintain stable replication (as Figure 4 ).

[0137] Example 4 Correct ratio of polyA tails of pmRVacSL plasmid

[0138] (1) The pmRVacSL plasmid was digested with the restriction endonucleases NcoI and SapI, and the purified linearized pmRVacSL backbone was obtained through a gel recovery kit.

[0139] (2) Plasmids containing polyA tails of different lengths were synthesized by gene synthesis. The plasmids containing pure polyA tail fragments of different lengths were digested with the restriction endonucleases NcoI and SapI, and the purified DNA fragments containing polyA tails of different lengths were obtained through a gel recovery kit.

[0140] (3) The purified linearized pmRVacSL backbone and the purified DNA fragments containing polyA tails of different lengths were ligated through a T4 ligase reaction to obtain a reaction system for in vitro transcription vectors containing polyA tails of different lengths.

[0141] (4) Referring to steps (5) and (6) of Example 1, pmRVacSL plasmids containing polyA tails of different lengths can be obtained.

[0142] (5) The correct ratio of the pmRVacSL plasmid DNA polyA tail was verified through TBE-PAGE gel and Sanger sequencing (as Figure 5 ).

[0143] The above are only the preferred embodiments of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and refinements can be made, and these improvements and refinements should also be regarded as the protection scope of the present invention.

Claims

1. Expression cassette, characterized in that, Including in order: rop gene, replicon ori and resistance gene; The rop gene, the replicon ori and the resistance gene are connected by a connecting fragment; The sequence of the rop gene: the nucleotide sequence shown in SEQ ID NO: 3; The sequence of the replicon ori: the nucleotide sequence shown in SEQ ID NO: 4; The resistance gene is: kanamycin resistance gene; The sequence of the connecting fragment: the nucleotide sequence shown in SEQ ID NO:6 and SEQ ID NO:8 to SEQ ID NO:10; The connecting fragments include: connecting fragment 1 to connecting fragment 4; The connecting fragment 1 is located at the 5' end of the rop gene; The connecting fragment 2 is located between the rop gene and the replicon ori; The connecting fragment 3 is located between the replicon ori and the resistance gene; The connecting fragment 4 is located at the 3' end of the resistance gene.

2. Recombinant plasmid, characterized in that, include: The expression cassette of claim 1 and acceptable genetic elements.

3. Host, characterized in that, Transform and / or transfect the recombinant plasmid as claimed in claim 2.

4. Use of the expression cassette according to claim 1, the recombinant plasmid according to claim 2 and / or the host according to claim 3 in improving the stability of the plasmid.

5. Product, characterized in that, include: The expression cassette according to claim 1, the recombinant plasmid according to claim 2 and / or the host according to claim 3.

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