CN116004682A - Method for rapidly preparing mRNA containing long poly adenine without trace and application - Google Patents

Abstract

The invention discloses a method for preparing mRNA containing long poly adenine rapidly without trace and application thereof, which comprises the steps of rapidly synthesizing and decomposing complex genes containing long poly (A) into simple target genes and connecting the simple target genes with a pre-prepared leading plasmid without trace through a modularized design and an IIS type restriction DNA endonuclease system. mRNA synthesized based on the method can carry poly (A) with fixed length without tail addition operation, and the preparation speed and efficiency of the template plasmid are greatly improved because the target gene synthesis does not involve long poly (A) complex fragments. The plasmid can be directly used as a linearization template for in vitro transcription synthesis of long poly A DNA poly (A) mRNA, and the IIS type restriction endonuclease system does not need to introduce an additional splicing sequence between a target gene and poly (A), so that the transcribed mRNA sequence completely accords with expectations.

Description

Method for rapidly preparing mRNA containing long poly adenine without trace and application

Technical Field

The invention discloses a preparation method of a plasmid, and belongs to the technical field of nucleic acid.

Background

Messenger ribonucleic acid (mRNA) is an important molecule that maintains the normal physiological functions of the body, mediating the transfer of genetic information from DNA to proteins by transcription and translation. mRNA as a single-stranded ribonucleic acid itself has inherent defects of easy degradation, high immunogenicity, difficult delivery and the like, and its clinical application is limited for a long time. In recent years, along with key technical innovations such as nucleoside chemical modification, liposome nanoparticle delivery and the like, development of novel vaccines and medicaments based on mRNA technology is continuously advanced. mRNA technology has the advantages of flexible design, rapid synthesis, large-scale preparation and the like, gradually becomes a general technical platform, and can be applied to the fields of infectious disease vaccines, cancer treatment, gene therapy, cell therapy and other biotechnology.

mRNA vaccine or medicine is a nucleic acid preparation, and its principle is that it enters into organism cell through delivery system, expresses target gene in vivo, stimulates specific immune response or produces functional target protein, and obtains immune protection or function regeneration. Linear mRNA biologicals can be divided into two major classes, self-amplifying and non-replicating, with the core elements including the T7 promoter, the 5 '-non-transcribed region (UTR), the protein sequence of interest, the stop codon, the 3' UTR, and the poly (a) fragment of the poly-a. Wherein the T7 promoter is used to mediate T7 RNA polymerase initiated transcription; UTR is derived from natural sequence or artificial designed sequence of human body, which has important influence on stability and expression quantity of mRNA molecule; the target protein sequence can improve the biological activity of mRNA by means of codon optimization, secondary structure optimization and the like; poly (a) fragments are capable of significantly enhancing the intracellular half-life of mRNA. The poly (A) length of the mRNA vaccine currently on the market is above 100nt (Moderna and Biontech mRNA vaccine). In vitro synthesis of mRNA generally involves transcription, capping, dephosphorylation, template DNA enzymatic hydrolysis, mRNA purification, and the like, using linearized DNA as a template. If the template plasmid itself does not contain poly (A) fragment, a single capping step is required after mRNA synthesis to add poly (A) to the 3' end of transcribed mRNA by enzymatic reaction, but this method results in difficulty in ensuring the length and uniformity of the final mRNA product.

To transcribe mRNA molecules with exact sequences, the current template plasmids for in vitro mRNA transcription usually already contain poly (A) fragments, which can be prepared by gene synthesis of the complete sequence comprising the T7 promoter, UTR, protein gene of interest and poly (A) and insertion into the plasmid. However, poly (A) is taken as a homopolymer, DNA comprising the sequence belongs to a complex sequence difficult to synthesize, for such gene synthesis, a gene company is difficult to provide technical services according to common standards, and particularly for poly (A) synthesis of more than 100nt, more than 3 times of standard services are required to be tried, the gene synthesis, clone screening and sequence verification are difficult, and finally whether successful delivery can be achieved or not has great randomness. In order to avoid the bottleneck that the poly (A) sequence is difficult to synthesize, a leader plasmid containing the poly (A) with the target length can be firstly prepared, a simple gene sequence only containing a T7 promoter, UTR and encoding target protein is independently synthesized, and then the leader plasmid and the synthesized gene are assembled and spliced by a restriction enzyme method and the like. However, current assembly and splicing methods often introduce additional cleavage sites at the junction of the synthetic gene and poly (A) sequence, which may adversely affect mRNA biological activity.

Disclosure of Invention

Aiming at the defects of the prior art, the invention aims to provide a method for preparing a long poly adenine mRNA template plasmid rapidly without trace and application thereof.

Based on the design of general leading plasmid, the invention is to clone the poly adenine (poly (A)) homopolymer which is difficult to synthesize into skeleton plasmid as fixing element, assemble the target gene which is easy to synthesize with it by splicing method, and combine into the template plasmid which can be used for mRNA in vitro transcription. The invention adopts the enzyme cutting site design of IIS type restriction enzyme system during splicing to ensure the traceless preparation of long poly adenine mRNA template plasmid, ensure the pure natural sequence expression of structural gene and maintain the due biological activity to the greatest extent.

Based on the above inventive concept, the present invention provides a method for rapidly preparing a long polyadenylation mRNA template plasmid without trace, the method comprising the steps of:

(1) Constructing a leader plasmid containing long polyadenylation, and inserting a DNA fragment formed by serially connecting a promoter, an IIS type restriction enzyme A reverse recognition DNA fragment, any interval fragment and an IIS type restriction enzyme A forward recognition DNA fragment, the long polyadenylation and the IIS type restriction enzyme B reverse recognition DNA fragment into a multiple cloning site of the leader plasmid in the forward direction or the reverse direction to obtain the leader plasmid containing the long polyadenylation; the insertion may be performed by cloning methods conventional in the art, such as a double-adaptor cloning method.

(2) Constructing a leader plasmid containing UTR and coding genes, inserting forward recognition fragments of forward recognition DNA fragments of IIS type restriction enzyme A, DNA fragments at the 3' end of a promoter, 5' UTR, coding genes, terminators, 3' UTR, a DNA fragment formed by serially connecting reverse recognition DNA fragments of polyadenylation fragments and IIS type restriction enzyme A into multiple cloning sites of the leader plasmid in forward or reverse direction to obtain the leader plasmid containing UTR and coding genes; the insertion may be performed by cloning methods conventional in the art, such as a double-adaptor cloning method.

(3) Cleaving the long polyadenylation-containing leader plasmid obtained in step (1) with a type IIS restriction enzyme a to obtain a linear fragment comprising the T7 promoter and long polyadenylation;

(4) Obtaining a linear fragment containing a 5'UTR, a terminator and a 3' UTR by cleaving the leader plasmid containing UTR and coding sequence obtained in step (2) with type IIS restriction endonuclease A;

(5) Ligating the linear fragment containing the promoter sequence and the long polyadenylation obtained in the step (3) with the linear fragment containing the 5'UTR, the terminator and the 3' UTR obtained in the step (4) to obtain the long polyadenylation mRNA template plasmid.

The IIS type restriction enzyme and the traditional restriction enzyme are unique in that the IIS type restriction enzyme and the traditional restriction enzyme are cut outside the recognition sequence, the sequence of a cutting site has no specific requirement, and can be any nucleotide combination, and after the cutting, a sticky end with specific design can be generated, and a plurality of base flanking overhangs are generated. Since these overhangs are not part of the recognition sequence, they can be tailored to direct the assembly of DNA fragments. If properly designed, recognition sites will not be present in the final construct, allowing for accurate, traceless cloning. Type IIS restriction enzymes include, but are not limited to AcuI, alwI, alw I, baeI, bbsI, bbvI, bccI, bceAI, bcgI, bciVI, bcoDI, bfuAI, bmrI, bmsI, bpmI, bpuEI, bsaXI, bseGI, bseRI, bsgI, bsmAI, bsmBI, bsmFI, bsmI, bspCNI, bspMI, bsrDI, bsrI, btgZI, btsCI, btsI, btsIMutI, cspCI, earI, eciI, eco31I, esp3I, fauI, fokI, hgaI, hphI, hpyAV, lguI, mboII, mlyI, mmeI, mnlI, nmeAIII, mva1269I, paqCI, pleI, sapI, sfaNI and the like.

In the present invention, the forward/reverse recognition fragments of type IIS restriction enzyme A are introduced into the long polyadenylation-containing precursor plasmid of step (1) and the UTR-and coding gene-containing precursor plasmid of step (2), respectively, in order to use type IIS restriction enzyme A for cleavage when linearizing the two precursor plasmids, and then to construct the long polyadenylation mRNA template plasmid of step (5) by ligation without introducing additional cleavage sites, thereby ensuring the traceless preparation of the template plasmid and the structural fidelity and stability of the mRNA involved.

In the present invention, the introduction of the type IIS restriction enzyme B reverse recognition DNA fragment into the long polyadenylation precursor plasmid containing long polyadenylation in step (1) is aimed at being used in linearizing the long polyadenylation mRNA template plasmid constructed in step (5) to ensure that no additional cleavage site is introduced as linearization product of mRNA transcription template, i.e. to ensure traceless preparation of template plasmid, and structural fidelity and stability of the mRNA involved. The object of the present invention can be achieved by using a type IIS restriction enzyme other than type IIS restriction enzyme A.

In a specific embodiment of the present invention, the type IIS restriction enzyme a is a DNA restriction enzyme BsaI, the type IIS restriction enzyme B is a DNA restriction enzyme BspQI, the sequence of the forward recognition DNA fragment of the type IIS restriction enzyme a in the step (1) is shown in SEQ ID No.1, the sequence of the reverse recognition DNA fragment of the type IIS restriction enzyme a is shown in SEQ ID No.2, and the sequence of the reverse recognition DNA fragment of the type IIS restriction enzyme B is shown in SEQ ID No. 3.

In a preferred embodiment, the long poly (a) adenine is 30-120 polyadenines.

In a specific embodiment of the invention, the long polyadenine has a length in the range of 110 polyadenines. In practical applications, the length of the long polyadenylation of the universal leader plasmid is not limited to 110A used in the examples of the present invention, and the specific length thereof may be adjusted within the range of 30-120nt according to the method provided by the present invention.

The type of the leader plasmid described in the present invention is not particularly limited, and any cloning vector commonly used in molecular biology can be used in the methods provided by the present invention, including, but not limited to, the PUC series, pcDNA3 series, pET series, pMAL series, pGEX series, and in one embodiment of the present invention the leader plasmid is a PUC57 plasmid.

The promoter, terminator, 5'UTR and 3' UTR are all functional elements conventionally used in genetic engineering and can be applied to the invention. In a specific embodiment of the present invention, the promoter fragment in step (2) is a T7 promoter, the sequence of the DNA fragment at the 3 'end of the promoter is shown in SEQ ID No.4, the first base N in SEQ ID No.4 may be any single nucleotide A, T, G or C, and the N appearing in the DNA fragment at the 3' end of the promoter appears only in the middle of splicing, and disappears after splicing is completed. The sequence of the T7 promoter is shown in SEQ ID NO. 13.

The sequence of the poly adenine fragment in the step (2) is shown as SEQ ID NO.6, the last base N in the sequence can be any single nucleotide A, T, G or C, and the N of the poly adenine fragment only appears in the middle of splicing, and disappears after the splicing is completed.

In a specific embodiment of the present invention, the terminator has a sequence shown in SEQ ID NO.5, the 5'UTR has a sequence shown in SEQ ID NO.8, and the 3' UTR has a sequence shown in SEQ ID NO. 10.

The sequence of any interval fragment is shown as SEQ ID NO.14, the any interval fragment is only used for spacing the enzyme cleavage sites of IIS type restriction enzyme A at two sides in the precursor plasmid, the sticky end is generated for subsequent enzyme cleavage connection, the method for preparing the long poly (A) mRNA template plasmid without trace is not limited, and a person skilled in the art can select other available interval fragments according to specific needs to realize the practical application of the invention.

In a preferred embodiment, the coding gene according to the invention is a gene which can be transcribed into mRNA which can be translated into a protein. GFP used in one embodiment of the invention is only exemplary and does not limit the encoding genes, and the practical application is not limited to GFP used in the examples of the invention, and other genes can be used to prepare mRNA transcription templates and to transcribe mRNA molecules with functional activity according to the same method.

Next, the present invention provides a long polyadenylation mRNA template plasmid prepared according to the above method.

In the method provided by the invention, the assembly connection between the leader plasmid containing elements such as long poly adenine and the target gene is mainly based on an IIS type restriction enzyme system, a forward or reverse recognition sequence is fused to the two ends of the leader plasmid and the gene to be synthesized, the elements such as a T7 promoter, UTR, the target gene and poly (A) can be orderly connected through common cohesive ends after enzyme digestion, and the combined long poly adenine mRNA template plasmid sequence does not contain additional enzyme digestion sites and belongs to traceless connection.

Finally, the invention provides application of the long poly adenine mRNA template plasmid in preparing mRNA vaccine, gene therapy medicine and cell therapy medicine.

By using the general method for constructing the long poly (A) mRNA template plasmid, the complex gene synthesis comprising the long poly (A) is decomposed into two steps of rapid synthesis of simple target genes and seamless connection with a pre-prepared precursor plasmid by a modularized design and IIS type restriction DNA endonuclease system, namely, on the basis that the precursor plasmid PUC57-T7-poly (A) is prepared, the plasmid PUC57-UTR-GFP is synthesized and constructed, and then the plasmid PUC57-T7-UTR-GFP-poly (A) large-lifting plasmid with the correct sequence is obtained by splicing and assembling, the time is only required to be up to 10 days at maximum (7 days for constructing and large-lifting the PUC57-UTR-GFP plasmid, 1 day for carrying out enzyme digestion and splicing assembly on the target plasmid PUC57-T7-UTR-GFP-poly (A), and 2 days for carrying out large-lifting sequence verification. As the leader plasmid containing long poly (A) is prepared in advance, the rest target gene fragments do not contain complex sequences, so the difficulty in the steps of gene synthesis, sequencing identification and the like is greatly reduced, and the preparation speed and efficiency are greatly improved compared with the existing method. The method and the prepared long poly (A) mRNA template plasmid can be applied to the directions of vaccines, gene therapy, cell therapy and the like based on mRNA technology.

Drawings

FIG. 1 is a schematic diagram of the structure of a T7-poly (A) fragment;

FIG. 2 shows a schematic diagram of the PUC 57;

FIG. 3A schematic diagram of the structure of a leader plasmid PUC57-T7-poly (A) comprising a T7 promoter and a long poly (A);

FIG. 4 shows a schematic structural diagram of UTR-GFP fragment;

FIG. 5A schematic representation of the structure of a leader plasmid PUC57-UTR-GFP comprising UTR and GFP genes of interest;

FIG. 6 shows the structure of a linear fragment of the pUC57-UTR-GFP plasmid after cleavage with BsaI;

FIG. 7 is a schematic diagram of the structure of a linear fragment of a pUC57-T7-poly (A) leader plasmid digested with BsaI;

FIG. 8 shows agarose gel electrophoresis patterns of BsaI digested PUC57-UTR-GFP plasmid, PUC57-T7-poly (A) plasmid and PUC57-T7-UTR-GFP-poly (A) plasmid;

FIG. 9A schematic representation of the structure of the mRNA transcription template plasmid PUC57-T7-UTR-GFP-poly (A) containing long poly (A);

FIG. 10 shows a BspQI cut PUC57-T7-UTR-GFP-poly (A) plasmid agarose gel electrophoresis pattern;

FIG. 11 is a diagram of a capillary electrophoresis detection of the molecular integrity of mRNA-GFP transcripts;

FIG. 12 shows a cell imaging assay for expression of the target protein 24 hours after mRNA-GFP transfection;

FIG. 13 shows a graph of quantitative analysis of average GFP fluorescence intensity of cells 24 hours after mRNA-GFP transfection.

Detailed Description

The invention will be further described with reference to specific embodiments, and advantages and features of the invention will become apparent from the description. These examples are only exemplary and do not limit the scope of the invention in any way, which is defined by the claims.

EXAMPLE 1 distributed traceless construction of mRNA transcription template plasmid PUC57-T7-UTR-GFP-poly (A) containing long poly (A)

The distributed traceless means that the constructed plasmid is not introduced with additional enzyme cutting sites, thereby ensuring the expression of the pure natural sequence of the structural gene and keeping the due biological activity to the greatest extent.

The IIS type restriction endonuclease disclosed by the invention cuts DNA at a specific position at the downstream of a recognition site, the sequence of the cutting site has no specific requirement, and the cutting site can be any nucleotide combination, and a sticky end which is specifically designed can be generated after the cutting. In one embodiment of the invention, bsaI is selected as a type IIS restriction enzyme A to construct an mRNA transcription template plasmid comprising long poly (A). It will be appreciated by those skilled in the art that the selection of other type IIS restriction enzymes will also accomplish the objects of the present invention, and therefore, the selection of BsaI should not be construed as limiting the type IIS restriction enzyme A.

Taking BsaI as an example, the recognition sequence and the cleavage site are not overlapped, the cleavage site is located at any nucleotide downstream of the 3 'end of the forward recognition sequence (5' -GGTCTC-3 '(SEQ ID NO. 1)) and before any 5 nucleotides upstream of the 5' end of the reverse recognition sequence (5 '-GAGACC-3' (SEQ ID NO. 2)), and 4 nt sticky ends are generated after cleavage, so that specific design can be performed according to requirements.

(1) Construction of the leader plasmid PUC57-T7-poly (A)

The T7 promoter sequence (5'-TAATACGACTCACTATAGG-3' (SEQ ID NO. 13)), bsaI enzyme reverse recognition sequence (5 '-GAGACC-3' (SEQ ID NO. 2)), any interval fragment ((5'-GCGTGGGGCCATGCTATGTCCCCTGCCCTCAA-3', SEQ ID NO. 14), any interval fragment is randomly designed and is only used for spacing the cleavage sites of two sides IIS type restriction enzyme A, sticky ends are conveniently generated, any interval fragment described in the embodiment is only used for the fragments designed for completing the implementation of the invention, can be replaced by any other interval fragment, and does not contain the any interval fragment in the constructed mRNA transcription template plasmid), bsaI enzyme forward recognition sequence (5 '-GGTCTC-3' (SEQ ID NO. 1)), 110 nt poly (A) sequence, bspQI enzyme reverse recognition sequence (5 '-GAAGAGC-3' (SEQ ID NO. 3)) are serially designed to obtain T7-poly (A) sequence (the sequence is shown as SEQ ID NO.7, the structure is shown in FIG. 1, the consignment gene synthesis company synthesizes, inserts the restriction enzyme forward recognition sequences (SEQ ID NO. 7) and the restriction enzyme forward recognition sequences (57) into the plasmid forward recognition sequences (57, 57-3) (SEQ ID NO. 57) of the restriction enzyme reverse recognition sequences of the restriction enzyme reverse recognition sequence (SEQ ID NO. 57) of the plasmid reverse recognition sequence of SEQ ID NO. 57, the plasmid reverse transcription template plasmid reverse recognition sequence of 3) (3) are inserted in the forward recognition sequence of the plasmid (57, the plasmid reverse recognition sequence of the plasmid reverse transcription sequence of 3-3) (SEQ ID NO. 57), the construction of the leader plasmid PUC57-T7-poly (A) was successful (the schematic structure is shown in FIG. 3).

In the above construction, bspQI was selected as type IIS restriction enzyme B to construct an mRNA transcription template plasmid containing long poly (A). It will be appreciated by those skilled in the art that the selection of other type IIS restriction enzymes other than type IIS restriction enzyme A will also accomplish the objects of the present invention, and therefore, the selection of BspQI should not be construed as limiting type IIS restriction enzyme B.

(2) Construction of the leader plasmid PUC57-UTR-GFP

BsaI enzyme forward recognition sequence (5 ' -GGTCTC-3' (SEQ ID NO. 1)), fragment containing T7 promoter 3' end (5 ' -NATAGG-3', N can be any single nucleotide A/T/G/C) shown in SEQ ID NO.4, which sequence can generate sticky end after BsaI enzyme cleavage, part of T7 promoter sequence in sticky end generated by PUC57-T7-poly (A) after BsaI enzyme cleavage can be complemented into complete T7 promoter sequence), 5' UTR sequence (shown in SEQ ID NO. 8), codon optimized GFP sequence (shown in SEQ ID NO. 9), terminator sequence (TGATAATAG (SEQ ID NO. 5)), 3' UTR sequence (shown in SEQ ID NO. 10), and polypeptide containing adenine (5 ' -AAAN-3 ' (SEQ ID NO. 6), N can be any single nucleotide A, T, G or C, can generate a sticky end after being cut by BsaI enzyme, can be complementary to a complete polyadenylation sequence with the sticky end generated by BsaI enzyme cutting of PUC57-T7-poly (A) and BsaI enzyme reverse recognition sequence (5 ' -GAGACC-3' (SEQ ID NO. 2)) to obtain UTR-GFP (the structure is shown in figure 4, the sequence is shown in SEQ ID NO. 11), the construction is carried out by a commission gene synthesis company, the enzyme cutting is reversely inserted into a polyclonal site of a PUC57 plasmid (GenScript SD 1176) through EcoRI and HindIII double enzyme cutting joints, the BsaI enzyme forward and reverse recognition sequence insertion positions are at the base positions of PUC57 plasmids 1291 and 408, the plasmid PUC57-UTR-GFP was successfully constructed (schematic structure is shown in FIG. 5).

The codon-optimized GFP sequences in this step are exemplary of the gene sequences encoding the target proteins of the present invention in one embodiment, and should not be construed as limiting the claimed embodiments of the present invention, and may be implemented in practice by selecting the specific coding sequences encoding the target proteins to be expressed.

(3) Construction of plasmid PUC57-T7-UTR-GFP-poly (A)

The pUC57-UTR-GFP plasmid and the pUC57-T7-poly (A) leader plasmid were digested with BsaI endonuclease, respectively, and reacted at 37℃for 1 h, the reaction system was as follows:

10. the Xs enzyme digestion Buffer is 5 mu l,

BsaI (20 U/μl) 2ul，

the amount of the plasmid DNA was 10ug,

RNase-free ddH ₂ o was made up to 50. Mu.l.

A schematic structure of a linear fragment of the pUC57-UTR-GFP plasmid digested with BsaI is shown in FIG. 6, at positions 408 and 1291; a schematic structure of a linear fragment of the PUC57-T7-poly (A) precursor plasmid digested with BsaI is shown in FIG. 7, where the digestion sites are located at 513 and 563.

As shown by agarose gel electrophoresis (FIG. 8), bsaI digested with UTR-GFP and linearized PUC57-T7-poly (A) target fragments of about 900bp and 2800bp, respectively. Linearized plasmid PUC57-T7-poly (A) and digested fragment UTR-GFP were recovered separately using a commercial gel recovery kit, assembled and spliced based on BsaI cohesive end ligation (cohesive ends 5'-CTAT-3' and 5'-AAAA-3', respectively) as follows, overnight reaction at 16 ℃):

10 × Ligase Buffer 1μl，

insert UTR-GFP 0.3 pmol,

the vector DNA PUC57-T7-poly (A) was used in an amount of 0.03 pmol,

T4 DNA Ligase (400 U/μl) 1μl ，

RNase-free ddH ₂ o was made up to 10. Mu.l.

Subsequently, 10. Mu.l of the ligation product was transformed into competent cells according to the instructions of commercial E.coli competent cell protocol, gently smeared on plates with the correct resistance using sterile smears, and cultured in an incubator at 37℃in an inverted manner for 12-16 h until monoclonal colonies were grown. Positive clones were identified by colony PCR method, the reaction system was as follows:

2 × Rapid Taq Master Mix 10μl，

M13 Primer Mix2μl，

1 mu l of the bacterial liquid is used for preparing the bacterial liquid,

ddH ₂ o was made up to 20. Mu.l.

The PCR reaction procedure was as follows:

95℃for 3min, (95℃for 15s, 55℃for 15s,72℃for 30 min) 35 cycles, 72℃for 5min.

After the positive monoclonal is picked and the sequence is verified to be correct by sequencing, the positive clone plasmid is extracted according to the operation shown by the commercial plasmid big extraction kit, and the mRNA transcription template plasmid PUC57-T7-UTR-GFP-poly (A) is successfully constructed (the structural schematic diagram is shown in figure 9).

The coding sequence of the T7-UTR-GFP-poly (A) target gene is shown as SEQ ID NO.11, and the sequencing result is completely consistent with the coding sequence, which shows that aiming at the bottleneck that a long poly (A) gene fragment is difficult to synthesize, the method for constructing a leader plasmid step by step and splicing based on BsaI enzyme digestion/sticky ends provided by the embodiment can splice fixed elements (T7 promoter and poly (A) fragment) and variable target fragments (UTR and coding gene) required by mRNA synthesis in a seamless manner through an IIS type restriction enzyme digestion system, and redundant sequences are not introduced after splicing. Further, it was confirmed by enzyme assay that the pUC57-T7-UTR-GFP-poly (A) plasmid did not contain BsaI cleavage sites and other additional sequences beyond the non-desired coding sequence introduced during preparation, and that the agarose gel electrophoresis band position was unchanged after cleavage (FIG. 8), indicating that the construction method did not introduce additional cleavage sites at the junctions of the synthetic gene UTR-GFP with the T7 promoter and poly (A) sequence.

Using the construction method of this example, on the basis that the leading plasmid PUC57-T7-poly (A) has been prepared, the plasmid PUC57-UTR-GFP was genetically synthesized and constructed, and then the plasmid PUC57-T7-UTR-GFP-poly (A) large-size plasmid with the correct sequence was obtained by splicing and assembling, only 10 days at the most required time (wherein the PUC57-UTR-GFP plasmid was constructed and largely extracted for 7 days, the PUC57-T7-poly (A) was digested with the PUC57-UTR-GFP, spliced and assembled for 1 day, the objective plasmid PUC57-T7-UTR-GFP-poly (A) was transformed, large-size extracted and sequence verified for 2 days). As the leader plasmid containing long poly (A) is prepared in advance, the rest target gene fragments do not contain complex sequences, so the difficulty in the steps of gene synthesis, sequencing identification and the like is greatly reduced, and the preparation speed and efficiency are greatly improved compared with the existing method.

Example 2 construction of mRNA transcription template plasmid PUC57-T7-UTR-GFP-poly (A) containing long poly (A) in one step in tandem (schematic identical to FIG. 9, only construction method is different)

The T7 promoter (SEQ ID NO. 13), the 5'UTR sequence (SEQ ID NO. 8), the codon optimized GFP sequence (SEQ ID NO. 9), the terminator sequence (TGATAATAG (SEQ ID NO. 5)), the 3' UTR sequence (SEQ ID NO. 10), and the sequence obtained by tandem connection of 110A were attempted to be shown in SEQ ID NO.12, and the gene synthesis company was commissioned to conduct gene synthesis by double digestion with EcoRI and HindIII, which was inserted in reverse to the multicloning site of the PUC57 plasmid (GenScript SD 1176) to construct the PUC57-T7-UTR-GFP-poly (A) plasmid, and the plasmids were proposed. In the period of about 2 months, the number of poly (A) of the prepared 4 constructed clones is seriously lost, the number of the final missing can reach 30, 42, 51 and 60, the deletion range can reach 30-60 compared with the embodiment 1, the objective plasmid of the expected sequence can not be prepared, and the large-scale plasmid of the expected sequence can not be obtained.

In contrast to example 1, the synthetic method of example 2 is limited by the high overall complexity of the sequence, the difficulty of conventional one-step tandem synthesis, and the high uncertainty, and the plasmid template of the expected target sequence cannot be obtained through multiple attempts.

Example 3 in vitro transcription of mRNA-GFP encoding GFP

For the PUC57-T7-UTR-GFP-poly (A) constructed in example 1, bspQI cleavage sites were located at 409 and 1658, respectively introduced for the self-priming and construction of the PUC57 plasmid, and the BspQI cleavage followed by cleavage of the linearized sequence of 409-1658 was used as a template for mRNA in vitro transcription.

(1) Linearization and purification of plasmid templates

The reaction system was configured as follows:

PUC57-T7-UTR-GFP-poly (A) plasmid 20. Mu.g,

BspQI（10U/μl） 10μl，

10×BspQ I Buffer 20μl，

Nuclease-Free H ₂ o is added up to 200ul.

After 1 hour of reaction at 50℃agarose gel electrophoresis showed cleavage by BspQI, yielding a linearized template of the expected size (around 1200 bp) (lower band in FIG. 10). Extracting and purifying the linearized plasmid by phenol-chloroform, adding an equal volume of phenol-chloroform (Tris saturated phenol: chloroform: isoamyl alcohol=25:24:1) into the DNA solution, and fully and uniformly mixing; centrifuging at room temperature for 10min at 12000g, carefully sucking the upper aqueous phase, adding an equal volume of chloroform solution (chloroform: isoamyl alcohol=24:1), and mixing thoroughly; after centrifugation, the supernatant was carefully aspirated and the DNA concentration was measured.

(2) mRNA in vitro transcription and purification

The reaction system was configured as follows:

linearized PUC57-T7-UTR-GFP-poly (A) plasmid 5. Mu.g,

T7 RNA Polymerase（50U/μl）10μl，

inorganic pyrophosphatase (0.1U/. Mu.l) 5. Mu.l,

RNase Inhibitor（40U/μl） 5μl，

10×Reaction buffer 10μl，

ATP（100mM） 10μl，

GTP（100mM）10μl，

m1ψ/UTP（100mM）10μl，

CTP（100mM） 10μl，

Nuclease-Free H ₂ o is added to 100ul.

After the mixture was uniformly mixed, the mixture was reacted at 37℃for 2 hours, and then 5. Mu.l of DNase I (1U/. Mu.l) was added to the reaction system, and the reaction was carried out at 37℃for 15 minutes, whereby the transcribed DNA template was removed. mRNA transcripts were purified as above using phenol chloroform.

(3) mRNA capping and purification

The reaction system was configured as follows:

the mRNA was transcribed at 200ug,

10 x Capping Reaction buffer 50μl，

GTP（10 mM） 25μl，

SAM（4 mM） 25μl，

Vaccinia Capping Enzyme（10U/μl）25μl，

2'-O-Methyltransferase（50U/μl） 25μl，

Nuclease-free H ₂ o is added to 100ul.

After mixing well, the reaction was carried out at 37℃for 1 hour, and the mRNA transcript was purified as above using phenol chloroform.

The molecular integrity of the mRNA transcripts was checked by capillary electrophoresis (Qsep 100 Advacne) (FIG. 11), which showed that the mRNA-GFP product size was as expected (about 1000 nt) and purity was over 90%.

Example 4 cell transfection and protein expression validation of in vitro transcribed mRNA-GFP

HeLa cells (3X 10) were cultured in 96-well plates overnight ⁴ Personal/hole), use ofTransIT poly-mRNA (Mirus) transfection reagent mRNA-GFP transcribed in example 3 above (100 ng/well), control wells were added with PBS, nuclei were stained 24 hours after transfection with hoechst33342, and DAPI and GFP channel signal intensities were detected using a cell imaging multifunctional detection system (Cystation 1, bioTek).

Qualitative analysis of the imaging results showed that the transcript mRNA-GFP was able to highly express GFP green fluorescent protein, whereas the control cells had no green fluorescent signal (FIG. 12). The mean fluorescence intensity of individual cells was quantitatively analyzed, and the mean GFP signal generated by mRNA-GFP transfection was 25164, which was 8.5 times that of control cells (mean signal 2957) (FIG. 13). The above results confirm that mRNA molecules containing long polyadenylation prepared rapidly tracelessly using example 1 and example 3 have the expected functional activity using GFP as target gene.

Claims (11)

1. A method for rapid traceless production of mRNA containing long poly-adenine, the method comprising the steps of:

(1) Constructing a precursor plasmid containing long polyadenine, and inserting a DNA fragment formed by connecting a promoter, an IIS type restriction enzyme A reverse recognition DNA fragment, any interval fragment, an IIS type restriction enzyme A forward recognition DNA fragment, the long polyadenine and an IIS type restriction enzyme B reverse recognition DNA fragment in series into a multiple cloning site of the precursor plasmid to obtain the precursor plasmid containing the long polyadenine;

(2) Constructing a leader plasmid containing UTR and coding genes, and inserting a forward recognition fragment of a forward recognition DNA fragment of IIS type restriction enzyme A, a DNA fragment at the 3' end of a promoter, a 5' UTR, the coding genes, a terminator, a 3' UTR, a polyadenylation fragment and a DNA fragment formed by tandem connection of reverse recognition DNA fragments of IIS type restriction enzyme A into a multiple cloning site of the leader plasmid to obtain the leader plasmid containing UTR and coding genes;

(3) Cleaving the long polyadenylation-containing leader plasmid obtained in step (1) with a type IIS restriction enzyme a to obtain a linear fragment comprising the T7 promoter and long polyadenylation;

(4) Obtaining a linear fragment containing a 5'UTR, a terminator and a 3' UTR by cleaving the leader plasmid containing UTR and coding sequence obtained in step (2) with type IIS restriction endonuclease A;

(5) Ligating the linear fragment containing the promoter sequence and the long polyadenylation obtained in the step (3) with the linear fragment containing the 5'UTR, the terminator and the 3' UTR obtained in the step (4) to obtain the long polyadenylation mRNA template plasmid.

2. The method according to claim 1, wherein the type IIS restriction enzyme a is a DNA restriction enzyme BsaI, the type IIS restriction enzyme B is a DNA restriction enzyme BspQI, the sequence of the forward recognition DNA fragment of the type IIS restriction enzyme a in step (1) is shown in SEQ ID No.1, the sequence of the reverse recognition DNA fragment of the type IIS restriction enzyme a is shown in SEQ ID No.2, and the sequence of the reverse recognition DNA fragment of the type IIS restriction enzyme B is shown in SEQ ID No. 3.

3. The method of claim 1, wherein the long polyadenylation in step (1) is 30-120 polyadenines.

4. The method of claim 1, wherein the leader plasmid is PUC series, pcDNA3 series, pET series, pMAL series, or pGEX series.

5. The method of claim 4, wherein the leader plasmid is PUC57.

6. The method of claim 1, wherein the promoter fragments in steps (1) and (2) are T7 promoters, and the sequence of the DNA fragment at the 3' end of the promoter is shown in SEQ ID NO. 4.

7. The method of claim 1, wherein the poly adenine fragment of step (2) has the sequence shown in SEQ ID NO. 6.

8. The method of claim 1, wherein the terminator sequence is set forth in SEQ ID No.5, the 5'utr is set forth in SEQ ID No.8, and the 3' utr is set forth in SEQ ID No. 10.

9. The method of claim 1, wherein the coding gene of step (1) is a gene that is transcribed into mRNA and translated into protein.

10. A long polyadenylation mRNA template plasmid prepared according to the method of any one of claims 1-9.

11. Use of the long poly adenine mRNA template plasmid of claim 10 in the preparation of mRNA vaccine, gene therapy drug, cell therapy drug.

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