CN117568348B - Gene for maintaining monomer supercoiled poly-A plasmid and application thereof - Google Patents
Gene for maintaining monomer supercoiled poly-A plasmid and application thereof Download PDFInfo
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- 239000013612 plasmid Substances 0.000 title claims abstract description 120
- 239000000178 monomer Substances 0.000 title claims abstract description 59
- 108090000623 proteins and genes Proteins 0.000 title claims abstract description 41
- 108020004999 messenger RNA Proteins 0.000 claims abstract description 23
- 230000008488 polyadenylation Effects 0.000 claims description 40
- 238000003780 insertion Methods 0.000 claims description 16
- 230000037431 insertion Effects 0.000 claims description 16
- 239000002773 nucleotide Substances 0.000 claims description 14
- 125000003729 nucleotide group Chemical group 0.000 claims description 14
- 230000000295 complement effect Effects 0.000 claims description 13
- 238000000034 method Methods 0.000 claims description 12
- 230000003115 biocidal effect Effects 0.000 claims description 8
- 238000012163 sequencing technique Methods 0.000 claims description 7
- 238000007480 sanger sequencing Methods 0.000 claims description 6
- 239000003242 anti bacterial agent Substances 0.000 claims description 4
- 238000012216 screening Methods 0.000 claims description 4
- 238000012258 culturing Methods 0.000 claims description 2
- 230000001131 transforming effect Effects 0.000 claims description 2
- 108091034057 RNA (poly(A)) Proteins 0.000 description 13
- 238000004519 manufacturing process Methods 0.000 description 8
- 238000011161 development Methods 0.000 description 7
- 230000000694 effects Effects 0.000 description 7
- 238000002560 therapeutic procedure Methods 0.000 description 6
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- 238000000246 agarose gel electrophoresis Methods 0.000 description 2
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- C12N15/64—General methods for preparing the vector, for introducing it into the cell or for selecting the vector-containing host
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Abstract
The invention relates to a gene for maintaining a monomer supercoiled poly A plasmid and application thereof. The sequence of the gene is one of SEQ ID No.3 to SEQ ID No. 8. The invention can obviously improve the supercoiled proportion of mRNA plasmid monomer with long PolyA tail, can reach the supercoiled proportion level of common plasmid monomer, and can also improve the yield of plasmid.
Description
Technical Field
The invention relates to a gene for maintaining a monomer supercoiled poly-A plasmid and application thereof, belonging to the field of biotechnology.
Background
Plasmids containing a polyadenylation (poly (A)) sequence (abbreviated as polyadenylation plasmids) are important raw materials for in vitro transcription to produce mRNA, however, poly (A) sequences present a series of problems, in addition to their low stability, which are prone to deletions, and also result in low plasmid yields, low monomer ratios, etc.
Current plasmid-aggregate problem-solving techniques focus mainly on strain improvement, but these techniques have limited effectiveness in treating mRNA plasmids with long polyA tails (about 60 to 150 nucleotides in length). Such a long polyA tail type plasmid is prone to form a polymer in bacteria, resulting in a problem that a polymer is easily formed during production, which is difficult to control effectively in the prior art. This problem is particularly acute in the development of mRNA therapies. The mRNA plasmid of the long polyA tail can encounter significant technical challenges in the research and reporting stages of biopharmaceutical enterprises, especially in the face of regulatory requirements imposed by the national drug administration drug review Center (CDE). The prior art fails to provide a stable, efficient solution to ensure the quality and consistency of such plasmids, thereby affecting the development of the whole biopharmaceutical field and the market admission speed of pharmaceuticals. Therefore, new technical means are urgently needed to solve this key problem in order to facilitate production and quality control of mRNA plasmids.
Disclosure of Invention
The main purpose of the invention is as follows: the gene can be used for remarkably improving the monomer supercoiled proportion of the polyadenylation plasmid. Also provides the application of the gene.
The technical scheme for solving the technical problems is as follows:
a gene for maintaining supercoiled polyadenylation plasmid monomer, wherein the sequence of the gene sequentially comprises a first public sequence SEQ ID No.1 and a second public sequence SEQ ID No.2 in the 5 'to 3' direction; in the first public sequence and the second public sequence, r is g or a, s is g or c, n is one of a, g, c, t, and y is t or c; the first public sequence and the second public sequence are separated by a nucleotide sequence of 6-8 bp.
The sequence of the gene is one of SEQ ID No.3 to SEQ ID No. 8.
The gene can effectively remove plasmid polymer, raise plasmid monomer supercoiled proportion and raise plasmid yield.
The invention also provides:
a method of increasing the supercoiled proportion of monomers of a polyadenylation plasmid, said method comprising the steps of:
firstly, introducing the sequence of the gene for maintaining the supercoiled monomer of the polyadenylation plasmid or the reverse complementary sequence thereof into the polyadenylation plasmid to obtain a recombinant plasmid;
and step two, transforming the recombinant plasmid obtained in the step one into competent cells, screening, culturing and sequencing to obtain correct clones.
Preferably, in the first step, the polyadenylation plasmid is an mRNA plasmid with a long polyA tail of 60 to 150 nucleotides in length.
Preferably, in the first step, the polyadenylation plasmid has an ori element, an insertion sequence site, and an antibiotic resistance gene element, the insertion sequence site being located between the ori element and the antibiotic resistance gene element; the insertion sequence site in the polyadenylation plasmid introduces the gene sequence for maintaining the supercoiled monomers of the polyadenylation plasmid or its reverse complement.
Preferably, in the second step, a medium containing an antibiotic corresponding to the antibiotic resistance gene element in the polyadenylation plasmid is used in the screening.
Preferably, in the second step, sanger sequencing is used for sequencing.
The method can effectively improve the supercoiled proportion of the monomer of the polyadenylation plasmid and the yield of the plasmid.
The invention also provides:
a method of constructing a recombinant plasmid, the method comprising: the recombinant plasmid is obtained by introducing the sequence of the gene for maintaining the supercoiled monomer of the polyadenylation plasmid or the reverse complement thereof into the polyadenylation plasmid.
The recombinant plasmid obtained by construction by the construction method of the recombinant plasmid is constructed.
The invention also provides:
the use of the genes described above for maintaining the monomer supercoiled form of a polyadenylation plasmid for recombining the polyadenylation plasmid to increase its monomer supercoiled ratio and plasmid yield.
Preferably, the polyadenylation plasmid is an mRNA plasmid with a long PolyA tail of 60 to 150 nucleotides in length.
Compared with the prior art, the invention can obviously improve the supercoiled proportion of mRNA plasmid monomer with long PolyA tail (60 to 150 nucleotides in length) to the level of supercoiled proportion (> 95%) of common plasmid monomer, and can also improve the yield of plasmid. The invention overcomes the limitation of the prior art, can promote the stability and consistency of the long PolyA tail mRNA plasmid, thereby providing key technical support for the research and development and production of mRNA therapy and greatly promoting the development of the biopharmaceutical field and the market admittance speed of related medicines.
Drawings
FIG. 1 is a plasmid map of pKGCT7-Fluc in example 1 of the present invention.
FIG. 2 is a plasmid map of pKGCT7X-Fluc in example 1 of the present invention.
FIG. 3 is an agarose gel electrophoresis chart of example 1 of the present invention.
Detailed Description
In specific implementation, the gene for maintaining the supercoiled polyadenylation plasmid monomer contains a first public sequence ggtrcsnayaa (SEQ ID No. 1) and a second public sequence ttatggtaay (SEQ ID No. 2) in sequence from 5 'to 3', wherein r is g or a, s is g or c, n is one of a, g, c, t, and y is t or c; and a nucleotide sequence of 6-8bp is arranged between the first public sequence and the second public sequence.
The gene is one of ColE cer minimum (SEQ ID No. 3), colA cer minimum (SEQ ID No. 4) and CDF cer minimum (SEQ ID No. 5). And (3) injection: the above three sequences are collectively referred to as truncated mer release sequences.
Alternatively, the gene is one of ColE cer (SEQ ID No. 6), colA cer (SEQ ID No. 7) and CDF cer (SEQ ID No. 8). And (3) injection: the above three sequences are collectively referred to as the mer release sequences.
It should be noted that: the ColE cer, the ColA cer and the CDF cer are respectively truncated and core areas are reserved to obtain ColE cer minimum, colA cer minimum and CDF cer minimum.
In ColE cer or ColE cer minimum, the first public sequence is ggtgcgtacaa, the second public sequence is ttatggtaaat, and the first public sequence and the second public sequence are separated by a nucleotide sequence ttaaggga of 8 bp.
In ColA cer or ColA cer minimum, the first public sequence is ggtgccgacaa, the second public sequence is ttatggtaaat, and a nucleotide sequence cgglatg of 6bp is spaced between the first public sequence and the second public sequence.
In the CDF cer or CDF cer minimum, the first public sequence is ggtaccgataa, the second public sequence is ttatggtaaat, and a nucleotide sequence gggatg of 6bp is arranged between the first public sequence and the second public sequence.
When in use, the gene sequence or the reverse complementary sequence thereof is introduced into any position and any direction of the Poly (A) plasmid, so that the supercoiled proportion and yield of plasmid monomers can be obviously improved.
The invention is described in further detail below with reference to the accompanying drawings in combination with embodiments. The invention is not limited to the examples given.
Example 1
This example is a poly (A) plasmid containing a mer release sequence was synthesized and transformed.
The specific contents of this embodiment are as follows:
the present invention is directed to a gene for maintaining supercoiled monomers of a polyadenylation plasmid, the core region of which belongs to the XerrCD binding sequence and which comprises several helper sequences. However, not all genes containing XerCD binding sequences gave the same effect, and this example was performed by setting the control group: as a control, the gene pSC101 psi (SEQ ID No. 9) containing the XerCD binding sequence was used for comparative verification.
The following five plasmids were synthesized by the applicant at their own discretion:
pKGCT7-Fluc: the plasmid map of the gene for maintaining the supercoiled polyadenylation plasmid monomer does not contain the gene for maintaining the supercoiled polyadenylation plasmid monomer, and the plasmid map is shown in figure 1, and the sequence is SEQ ID No.10, wherein the insertion sequence site is 4210-4211.
pKGCT7W-Fluc: the reverse complement of pSC101 psi (SEQ ID No. 9) was inserted at the site of the insertion sequence of pKGCT7-Fluc, SEQ ID No.11.
pKGCT7X-Fluc: the reverse complement of ColE cer (SEQ ID No. 6) was inserted at the site of the insertion sequence of pKGCT7-Fluc, SEQ ID No.12.
pKGCT7Y-Fluc: the reverse complement of CDF cer (SEQ ID No. 8) was inserted at the site of the insertion sequence of pKGCT7-Fluc, SEQ ID No.13.
pKGCT7Z-Fluc: the reverse complement of ColA cer (SEQ ID No. 7) SEQ ID No.14 was inserted at the site of the insertion sequence of pKGCT7-Fluc.
As an example, the plasmid map of pKGCT7X-Fluc is shown in FIG. 2.
The five plasmids above were transformed into NEB Stable competent (NEB C3040) according to the instructions, kanamycin LB plates at 30℃overnight. Spot picking, shaking in 2mL LB medium at 30 ℃ and Sanger sequencing to confirm that the poly (a) sequence length is between 110-120.
The sequencing primer sequences were: gtttcgccacctctgacttg, SEQ ID No.15. And (3) injection: sanger sequencing requires only one 5' primer to be designed.
Selecting four clones with correct sequence, detecting and calculating plasmid yield and monomer supercoiled proportion; wherein, the supercoiled proportion of the monomer is determined by agarose gel electrophoresis, and the electrophoresis chart is shown in figure 3. The results obtained are shown in the following table.
From the above results, pSC101 psi contained XerCD binding sequence, but did not increase the supercoiled ratio of the monomer of the poly (A) plasmid, but rather made the supercoiled ratio of the monomer worse.
The ColE cer, the CDF cer and the ColA cer can obviously improve the supercoiled proportion of the poly (A) plasmid monomer and even can be equivalent to the supercoiled proportion (> 95%) of the common plasmid monomer; at the same time, plasmid yield is also significantly improved.
Example 2
This example demonstrates the effect of truncated mer release sequences to increase the supercoiled proportion of monomer in the poly (A) plasmid.
The specific contents of this embodiment are as follows:
the ColE cer (SEQ ID No. 6), colA cer (SEQ ID No. 7) and CDF cer (SEQ ID No. 8) were truncated and the core region was retained to obtain ColE cer minimum (SEQ ID No. 3), colA cer minimum (SEQ ID No. 4) and CDF cer minimum (SEQ ID No. 5), respectively.
The following three plasmids were synthesized by the applicant at their own discretion:
pKGCT7-Fluc of example 1 was used.
pKGCT7Xm-Fluc: the reverse complement of ColE cer minimum (SEQ ID No. 3) was inserted at the site of the insertion sequence of pKGCT7-Fluc, SEQ ID No.16.
pKGCT7Ym-Fluc: the reverse complement of CDF cer minimum (SEQ ID No. 5) was inserted at the site of the insertion sequence of pKGCT7-Fluc, SEQ ID No.17.
pKGCT7Zm-Fluc: the reverse complement of ColA cer minimum (SEQ ID No. 4) was inserted at the site of the insertion sequence of pKGCT7-Fluc, SEQ ID No.18.
The above three plasmids were transformed into NEB Stable competent (NEB C3040) according to the instructions, kanamycin LB plates at 30℃overnight. Spot picking, shaking in 2mL LB medium at 30 ℃ and Sanger sequencing to confirm that the poly (a) sequence length is between 110-120. The sequence of the sequencing primer is SEQ ID No.15.
Four clones sequenced correctly were selected each, and plasmid yield and monomer supercoiled ratio were detected and calculated. The results obtained are shown in the following table.
From the above results, it is known that, although the effect of increasing the supercoiled ratio of the monomer is inferior to that of ColE cer, CDF cer and ColA cer, the supercoiled ratio of the monomer of the poly (A) plasmid can be increased significantly by ColE cer minimum, CDF cer minimum and ColA cer minimum; at the same time, the plasmid yield is also obviously improved.
Example 3
This example demonstrates the effect of the polymer release sequence on increasing the supercoiled proportion of GFP poly (A) plasmid monomers.
The specific contents of this embodiment are as follows:
the following plasmids were synthesized by the applicant at their own discretion:
GFP124A: the gene for maintaining the supercoiled polyadenylation plasmid monomer does not contain the gene of the invention; the sequence is SEQ ID No.19, wherein the insertion sequence site is 2189-2190.
GFP124A-X: colE cer (SEQ ID No. 6) was inserted at the insertion site of GFP 124A.
The above two plasmids were transformed into NEB Stable competent (NEB C3040) according to the instructions, kanamycin LB plates at 30℃overnight. Spot picking, shaking in 2mL LB medium at 30 ℃ and Sanger sequencing to confirm that the poly (a) sequence length is between 120-130.
The sequencing primer sequences were: cgtttcccgttgaatatggc, SEQ ID No.20.
Four clones sequenced correctly were selected each, and plasmid yield and monomer supercoiled ratio were detected and calculated. The results obtained are shown in the following table.
From the above results, colE cer can similarly promote monomer supercoiled ratio and plasmid yield for other poly (A) plasmids such as GFP 124A.
The main technical challenge of the present invention, combining the above examples, is to effectively solve the problem that long poly a tail mRNA plasmids are prone to form aggregates during production, while increasing their yield and monomer supercoiled ratio (also known as monomer ratio). Through systematic screening and comparative studies, applicants have discovered a specific set of sequences that are effective in removing plasmid aggregates while increasing the yield of long poly a tail mRNA plasmids. Furthermore, we have found that the core region of these sequences itself increases the proportion of plasmid monomers, although slightly less effective than the full length sequences used, with significant improvements. The invention provides a new strategy for solving the problem of long PolyA tail mRNA plasmid aggregate, and opens up a new way for improving the yield and purity of the plasmids.
The invention can obviously improve the monomer proportion of mRNA plasmid with long PolyA tail (60 to 150 nucleotides in length) to reach the level of supercoiled proportion (> 95%) of common plasmid monomer, and can also improve the yield of plasmid. The invention overcomes the limitation of the prior art, can promote the stability and consistency of the long PolyA tail mRNA plasmid, thereby providing key technical support for the research and development and production of mRNA therapy and greatly promoting the development of the biopharmaceutical field and the market admittance speed of related medicines.
Specifically:
1. in the technical effect aspect:
by introducing the gene for maintaining the monomer supercoiled of the poly A plasmid, the monomer supercoiled proportion of the long poly A tail mRNA plasmid can be remarkably improved. Experimental results show that compared with the existing vector, the invention can increase the supercoiled proportion of the monomer from 60% to 78% (truncated mer release sequence) or 91% (mer release sequence) on average. This significantly increased proportion of monomers is critical to ensure plasmid stability and functionality, particularly in mRNA therapies and other high precision biotechnology applications.
2. In the aspect of economic effect:
after the gene for maintaining the supercoiled monomer of the polyadenylation plasmid is introduced, the yield of the same PolyA plasmid is improved by more than 50 percent. The remarkable yield improvement not only reduces the production cost, but also improves the production efficiency, and brings remarkable economic benefit for biological pharmaceutical enterprises. In a highly competitive market environment, this cost-effective advantage is critical.
3. In terms of social effects:
with the rapid development and wide application of mRNA therapy, the technical innovation of the invention provides a high-efficiency and stable plasmid production method for the field. The quality and the yield of the long PolyA tail mRNA plasmid are improved, and the development and the marketing of new drugs are accelerated, so that the public health requirement is better met, and the health of the social group is positively influenced.
4. In terms of specific data:
(1) And (3) lifting the supercoiled proportion of the monomer: the average 60% rise from the prior art to over 78% (truncated mer release sequence) or 91% (mer release sequence).
(2) Plasmid yield improvement: the improvement is more than 50 percent.
In summary, the technical innovation of the invention not only provides a remarkable improvement on the technical level, but also has profound effects on the economic and social level. By increasing the proportion and yield of the single polymer of the plasmid, the invention not only improves the efficiency of the biopharmaceutical, but also is beneficial to the rapid development and wide application of new therapies, and finally benefits the masses of society.
In addition to the embodiments described above, other embodiments of the invention are possible. All technical schemes formed by equivalent substitution or equivalent transformation fall within the protection scope of the invention.
Claims (5)
1. A method for increasing the supercoiled proportion of monomers of a polyadenylation plasmid, said method comprising the steps of:
firstly, introducing a sequence of a gene for maintaining supercoiled monomer of a polyadenylation plasmid or a reverse complementary sequence thereof into the polyadenylation plasmid to obtain a recombinant plasmid;
the sequence of the gene for maintaining the supercoiled polyadenylation plasmid monomer sequentially comprises a first public sequence SEQ ID No.1 and a second public sequence SEQ ID No.2 in the 5 'to 3' direction; in the first public sequence and the second public sequence, r is g or a, s is g or c, n is one of a, g, c, t, and y is t or c; a nucleotide sequence of 6-8bp is arranged between the first public sequence and the second public sequence; the sequence of the gene for maintaining the supercoiled polyadenylation plasmid monomer is one of SEQ ID No.3 to SEQ ID No. 8;
the polyadenylation plasmid is an mRNA plasmid with a long PolyA tail of 60 to 150 nucleotides in length;
and step two, transforming the recombinant plasmid obtained in the step one into competent cells, screening, culturing and sequencing to obtain correct clones.
2. The method of claim 1, wherein in the first step, the polyadenylation plasmid has an ori element, an insertion sequence site, and an antibiotic resistance gene element, the insertion sequence site being located between the ori element and the antibiotic resistance gene element; the insertion sequence site in the polyadenylation plasmid introduces the gene sequence for maintaining the supercoiled monomers of the polyadenylation plasmid or its reverse complement.
3. The method for increasing the supercoiled proportion of a monomer of a polyadenylation plasmid according to claim 2, wherein in the second step, a medium containing an antibiotic corresponding to the antibiotic resistance gene element in the polyadenylation plasmid is used.
4. The method of claim 1, wherein in the second step, sanger sequencing is used for sequencing.
5. Use of a gene for maintaining the monomer supercoiled form of a polyadenylation plasmid for recombining the polyadenylation plasmid to increase its monomer supercoiled ratio and plasmid yield;
the sequence of the gene for maintaining the supercoiled polyadenylation plasmid monomer sequentially comprises a first public sequence SEQ ID No.1 and a second public sequence SEQ ID No.2 in the 5 'to 3' direction; in the first public sequence and the second public sequence, r is g or a, s is g or c, n is one of a, g, c, t, and y is t or c; a nucleotide sequence of 6-8bp is arranged between the first public sequence and the second public sequence; the sequence of the gene for maintaining the supercoiled polyadenylation plasmid monomer is one of SEQ ID No.3 to SEQ ID No. 8;
the polyadenylation plasmid is an mRNA plasmid with a long PolyA tail of 60 to 150 nucleotides in length.
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| CN110373421A (en) * | 2019-06-28 | 2019-10-25 | 浙江工业大学 | A kind of tyrosine phenol lyase gene recombination plasmid and application |
| CN111394378A (en) * | 2020-04-03 | 2020-07-10 | 深圳市新合生物医疗科技有限公司 | Plasmid vector for in vitro expression of mRNA and construction method and application thereof |
| CN116200480A (en) * | 2023-01-06 | 2023-06-02 | 深圳新合睿恩生物医疗科技有限公司 | Primers and method for detecting length of plasmid polyA tail |
| CN116555342A (en) * | 2023-05-15 | 2023-08-08 | 华南理工大学 | Modified pT7TS plasmid and application thereof |
| CN116716328A (en) * | 2023-08-04 | 2023-09-08 | 苏州左旋星生物科技有限公司 | Method for rapidly constructing polyadenylation polyA plasmid and corresponding plasmid |
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