A method for rapid scarless preparation of long polyadenylated mRNA-containing mRNA and application

Abstract

The application discloses a method for rapidly and non-traceably preparing mRNA containing long polyadenine and application thereof. Through modular design and an IIS type restriction DNA endonuclease system, complex gene synthesis containing long poly(A) is decomposed into two steps of rapid synthesis of simple target genes and non-traceable connection with a previously prepared leading plasmid. The mRNA synthesized based on the method can carry a fixed length of poly(A) without tailing operation, and since the target gene synthesis does not involve a long poly(A) complex fragment, the speed and efficiency of the template plasmid preparation are greatly improved. The plasmid can be directly used as a linearized template for in vitro transcription synthesis of long polyadenosine deoxyribonucleic acid poly(A) mRNA, the IIS type restriction DNA endonuclease system does not need to introduce an additional splicing sequence between the target gene and poly(A), and the transcribed mRNA sequence completely meets the expectation.

Description

TECHNICAL FIELD

[0001] The application discloses a plasmid preparation method and belongs to the technical field of nucleic acids. BACKGROUND

[0002] Messenger ribonucleic acid (mRNA) is an important molecule for maintaining normal physiological functions of the body, and mediates the transmission of genetic information from DNA to protein through transcription and translation. As a single-stranded ribonucleic acid, mRNA itself has inherent defects such as easy degradation, high immunogenicity, and difficult delivery, and its clinical application has been limited for a long time. In recent years, with the innovation of key technologies such as nucleoside chemical modification and liposome nanoparticle delivery, new vaccines and drugs based on mRNA technology have continuously made breakthrough progress. mRNA technology has the advantages of flexible design, rapid synthesis, and large-scale preparation, and is gradually becoming a general technology platform that can be applied to various biotechnology fields such as infectious disease vaccines, cancer treatment, gene therapy, and cell therapy.

[0003] An mRNA vaccine or drug is a nucleic acid preparation, and its principle is to enter the body cells through a delivery system, express a target gene in the body, stimulate a specific immune response or produce a functional target protein, and obtain immune protection or functional regeneration. Linear mRNA biological products can be divided into two categories: self-amplifying and non-replicating, and the core elements of both include a T7 promoter, a 5'-untranslated region (UTR), a target protein sequence, a stop codon, a 3'UTR, and a poly(A) segment. Among them, the T7 promoter is used to mediate the initiation of transcription by T7 RNA polymerase; the UTR is derived from a natural sequence or an artificially designed sequence of the human body, and has an important influence on the stability and expression amount of the mRNA molecule; the target protein sequence can be optimized by codon optimization and secondary structure optimization to improve the biological activity of the mRNA; and the poly(A) segment can significantly enhance the intracellular half-life of the mRNA. The poly(A) length of the currently marketed mRNA vaccines is more than 100 nt (Moderna and BioNTech mRNA vaccines). The in vitro synthesis of mRNA usually includes the steps of transcription with a linearized DNA as a template, capping, dephosphorylation, template DNA digestion, and mRNA purification. If the template plasmid itself does not contain a poly(A) segment, a capping operation is still needed after the synthesis of mRNA, and a poly(A) is added to the 3' end of the transcribed mRNA through an enzymatic reaction, but this method makes it difficult to guarantee the length and uniformity of the mRNA end product.

[0004] In order to transcribe mRNA molecules with exact sequences, the template plasmids currently used for in vitro transcription of mRNA usually contain a poly(A) segment, which can be prepared by synthesizing the complete sequence of the T7 promoter, UTR, protein gene of interest and poly(A) by gene synthesis and inserting it into the plasmid. However, as a homopolymer, the DNA containing the sequence of poly(A) belongs to a complex sequence that is difficult to synthesize, and for such gene synthesis, the gene company is difficult to provide technical services according to the usual standard, especially for the synthesis of poly(A) longer than 100 nt, which often requires more than 3 times the standard service time to try, and the gene synthesis, cloning screening and sequence verification are all difficult, and there is a great randomness in the final success or not. In order to avoid the difficulty of synthesizing the poly(A) sequence, a leading plasmid containing a poly(A) of a target length can be prepared first, and a simple gene sequence containing only a T7 promoter, a UTR and a coding protein of interest can be synthesized separately, and then the leading plasmid and the synthesized gene are assembled and spliced by restriction enzymes. However, the current assembly and splicing method usually introduces additional enzyme cutting sites at the joint of the synthesized gene and the poly(A) sequence, which may have an adverse effect on the biological activity of mRNA. SUMMARY

[0005] In view of the defects of the prior art, the purpose of the present application is to provide a method for quickly and tracelessly preparing a long poly(A) mRNA template plasmid and an application thereof.

[0006] Based on the design of a general leading plasmid, the concept of the present application is to pre-clone the difficult-to-synthesize poly(A) homopolymer as a fixed element into the backbone plasmid, and then assemble and splice the easy-to-synthesize target gene with it by splicing method, and combine it into a template plasmid that can be used for in vitro transcription of mRNA. In the splicing process, the enzyme cutting site of the IIS type restriction enzyme system is used to ensure the traceless preparation of the long poly(A) mRNA template plasmid, to ensure the expression of the pure natural sequence of the structural gene, and to maximize the biological activity thereof.

[0007] Based on the above inventive concept, the present application first provides a method for quickly and tracelessly preparing a long poly(A) mRNA template plasmid, which comprises the following steps:

[0008] (1) Constructing a leading plasmid containing a long poly(A), inserting a DNA fragment composed of a promoter, a reverse recognition DNA fragment of IIS type restriction enzyme A, an arbitrary spacer, a forward recognition DNA fragment of IIS type restriction enzyme A, a long poly(A) and a reverse recognition DNA fragment of IIS type restriction enzyme B in series into the multiple cloning site of the leading plasmid in forward or reverse direction to obtain a leading plasmid containing a long poly(A); the insertion can be carried out by using conventional cloning methods in the art, such as double enzyme digestion and adapter cloning method.

[0009] (2) Constructing a plasmid containing UTR and coding gene, a DNA fragment containing IIS type restriction enzyme A forward recognition DNA fragment, 3' end of promoter DNA fragment, 5' UTR, coding gene, terminator, 3' UTR, poly A fragment and IIS type restriction enzyme A reverse recognition DNA fragment are connected in series, and the DNA fragment is inserted into the multiple cloning site of the plasmid in forward or reverse direction to obtain a plasmid containing UTR and coding gene; the insertion can use the conventional cloning method in the art, for example, double enzyme digestion adapter cloning method.

[0010] (3) Cutting the plasmid containing long poly A obtained in step (1) with IIS type restriction enzyme A to obtain a linear fragment containing T7 promoter and long poly A;

[0011] (4) Cutting the plasmid containing UTR and coding sequence obtained in step (2) with IIS type restriction enzyme A to obtain a linear fragment containing 5' UTR, terminator and 3' UTR;

[0012] (5) Connecting the linear fragment containing promoter sequence and long poly A obtained in step (3) and the linear fragment containing 5' UTR, terminator and 3' UTR obtained in step (4) to obtain a long poly A mRNA template plasmid.

[0013] The IIS-type restriction enzymes described in the present application are unique from "traditional" restriction enzymes in that they cut outside of the recognition sequence, the sequence of the cut site is not specific and can be any combination of nucleotides, and the enzyme cut can produce a specially designed sticky end, producing several base overhangs. Since these overhangs are not part of the recognition sequence, they can be tailored to direct the assembly of DNA fragments. If designed correctly, the recognition site will not be present in the final construct, allowing for precise, scarless cloning. IIS-type restriction enzymes include, but are not limited to, Acul, Alwl, Alw26l, Baell, Bbsl, BbvI, BccI, BceAI, BcgI, BciVI, BcoDI, BfuAI, Bmrl, Bmsl, Bpml, BpuEI, BsaXI, BseGI, BseRI, BsgI, BsmAI, BsmBI, BsmFI, BsmI, BspCNI, BspMI, BsrDI, BsrI, BtgZI, BtsCI, BtsI, BtsIMutI, CspCI, EarI, EciI, Eco31I, Esp3I, FauI, FokI, Hgal, HphI, HpyAV, LguI, MboII, MlyI, Mmel, MnlI, NmeAIII, Mva1269I, PaqCI, PleI, SapI, SfaNI, and the like.

[0014] In the present application, the purpose of introducing the forward / reverse recognition fragment of IIS-type restriction enzyme A into the long poly A-containing leading plasmid in step (1) and the UTR- and gene-encoding leading plasmid in step (2) is to use IIS-type restriction enzyme A cleavage when linearizing the two leading plasmids, and to not introduce additional cleavage sites in the process of connecting the cleavage products to construct the long poly A mRNA template plasmid in step (5), to ensure scarless preparation of the template plasmid, as well as structural fidelity and stability of the mRNA involved.

[0015] In the present application, the purpose of introducing the reverse recognition DNA fragment of IIS-type restriction enzyme B into the long poly A-containing leading plasmid in step (1) is to use it when linearizing the long poly A mRNA template plasmid constructed in step (5), to ensure that the linearized product as an mRNA transcription template does not introduce additional cleavage sites, i.e., to ensure scarless preparation of the template plasmid, as well as structural fidelity and stability of the mRNA involved. Using other IIS-type restriction enzymes different from IIS-type restriction enzyme A can also achieve the purpose of the present application.

[0016] In one specific embodiment of the present application, the IIS-type restriction enzyme A is DNA endonuclease Bsal, the IIS-type restriction enzyme B is DNA endonuclease BspQI, the sequence of the forward recognition DNA fragment of the IIS-type restriction enzyme A in step (1) is shown in SEQ ID NO. 1, the sequence of the reverse recognition DNA fragment of the IIS-type restriction enzyme A is shown in SEQ ID NO. 2, and the sequence of the reverse recognition DNA fragment of the IIS-type restriction enzyme B is shown in SEQ ID NO. 3.

[0017] In one preferred embodiment, the long poly(A) is 30-120 poly(A).

[0018] In one specific embodiment of the present application, the length of the long poly(A) is 110 poly(A). In practical applications, the length of the long poly(A) of the universal type of the leading plasmid is not limited to 110 A used in the examples of the present application, and the specific length can be adjusted within the range of 30-120 nt according to the method provided by the present application.

[0019] The type of the leading plasmid in the present application is not particularly limited, and the cloning vectors commonly used in molecular biology can be applied in the method provided by the present application, including but not limited to PUC series, pcDNA3 series, pET series, pMAL series, pGEX series. In one specific embodiment of the present application, the leading plasmid is PUC57 plasmid.

[0020] The promoter, terminator, 5'UTR and 3'UTR in the present application are all functional elements commonly used in genetic engineering, and can be applied in the present application. In one specific embodiment of the present application, the promoter fragment in step (2) is 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 can be any single nucleotide A, T, G or C, and the N appearing in the DNA fragment at the 3' end of the promoter only appears in the middle process of splicing and disappears after splicing. The sequence of the T7 promoter is shown in SEQ ID NO. 13.

[0021] The sequence of the poly(A) fragment in step (2) of the present application is shown in SEQ ID NO. 6, and the last base N in the sequence can be any single nucleotide A, T, G or C. The N appearing in the poly(A) fragment only appears in the middle process of splicing and disappears after splicing.

[0022] In one specific embodiment of the present application, the sequence of the terminator is as shown in SEQ ID NO. 5, the sequence of the 5' UTR is as shown in SEQ ID NO. 8, and the sequence of the 3' UTR is as shown in SEQ ID NO. 10.

[0023] Any spacer fragment described in the present application has a sequence as shown in SEQ ID NO. 14, which is only used to space the cleavage sites of the IIS type restriction enzyme A in the leading plasmid, to generate sticky ends for subsequent cleavage and ligation, and should not be considered as a limitation on the method for preparing a long poly(A) mRNA template plasmid without trace. Those skilled in the art can select other available spacer fragments according to specific needs, and all of them can achieve the practical application of the present application.

[0024] In a preferred embodiment, the coding gene described in the present application is a gene that can be transcribed into mRNA, which can be translated into a protein. In one specific embodiment of the present application, GFP is only used as a demonstration, and does not constitute a limitation on the coding gene. In practical applications, it is not limited to the GFP used in the examples of the present application. Other genes can be prepared into mRNA transcription templates according to the same method, and functional mRNA molecules can be transcribed and synthesized.

[0025] Secondly, the present application provides a long poly(A) mRNA template plasmid prepared according to the above-mentioned method.

[0026] In the above-mentioned method provided by the present application, the assembly and ligation between the leading plasmid containing long poly(A) and other elements and the target gene are mainly based on the IIS type restriction enzyme system. The forward or reverse recognition sequence is fused to the two ends of the leading plasmid and the gene to be synthesized. After cleavage, the T7 promoter, UTR, target gene and poly(A) and other elements can be sequentially connected through the common sticky end. The combined long poly(A) mRNA template plasmid sequence does not contain additional cleavage sites, and belongs to traceless ligation.

[0027] Finally, the present application provides the application of the above-mentioned long poly(A) mRNA template plasmid in the preparation of mRNA vaccines, gene therapy drugs and cell therapy drugs.

[0028] The universal method for constructing long poly(A) mRNA template plasmid of the application, by modular design and IIS type restriction DNA endonuclease system, decomposes complex gene synthesis containing long poly(A) into two steps of rapid synthesis of simple target gene and scarless ligation with pre-prepared leading plasmid, i.e., on the basis of having prepared the leading plasmid PUC57-T7-poly(A), the plasmid PUC57-UTR-GFP is synthesized and constructed, and then splicing assembly is performed to obtain the sequence-corrected PUC57-T7-UTR-GFP-poly(A) large extraction plasmid, which only takes no more than 10 days at the fastest (7 days for PUC57-UTR-GFP plasmid construction and large extraction, 1 day for PUC57-T7-poly(A) and PUC57-UTR-GFP enzyme cutting, splicing assembly, 2 days for target plasmid PUC57-T7-UTR-GFP-poly(A) transformation, large extraction and sequence verification). Since the leading plasmid containing long poly(A) has been prepared in advance, the remaining target gene fragments do not contain complex sequences, so the difficulty in gene synthesis, sequencing identification and other steps 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 direction of mRNA technology-based vaccine, gene therapy, cell therapy, etc. BRIEF DESCRIPTION OF DRAWINGS

[0029] Figure 1 .T7-poly(A) fragment structure diagram;

[0030] Figure 2 . PUC57 structure diagram;

[0031] Figure 3 . Leading plasmid PUC57-T7-poly(A) structure diagram containing T7 promoter and long poly(A);

[0032] Figure 4 . UTR-GFP fragment structure diagram;

[0033] Figure 5 . PUC57-UTR-GFP structure diagram of the leading plasmid containing UTR and GFP target gene;

[0034] Figure 6 . Structure diagram of the linear fragment of the PUC57-UTR-GFP plasmid after BsaI enzyme cutting;

[0035] Figure 7 . Structure diagram of the linear fragment of the PUC57-T7-poly(A) leading plasmid after BsaI enzyme cutting;

[0036] Figure 8. Agarose gel electrophoresis map of BsaI digestion of PUC57-UTR-GFP plasmid, PUC57-T7-poly(A) plasmid and PUC57-T7-UTR-GFP-poly(A) plasmid;

[0037] Figure 9 . Structure diagram of mRNA transcription template plasmid PUC57-T7-UTR-GFP-poly(A) containing long poly(A);

[0038] Figure 10 . Agarose gel electrophoresis map of BspQI digestion of PUC57-T7-UTR-GFP-poly(A) plasmid;

[0039] Figure 11 . Capillary electrophoresis detection map of mRNA-GFP transcription product molecular integrity;

[0040] Figure 12 . Cell imaging detection map of target protein expression after mRNA-GFP transfection for 24 hours;

[0041] Figure 13 . Quantitative analysis map of cell average GFP fluorescence intensity after mRNA-GFP transfection for 24 hours. DETAILED DESCRIPTION

[0042] The present application will be further described below in conjunction with specific examples, and the advantages and features of the present application will become more apparent as the description proceeds. However, these examples are only exemplary and do not constitute any limitation on the protection scope defined by the claims of the present application.

[0043] Example 1. Distribution scarless construction of mRNA transcription template plasmid PUC57-T7-UTR-GFP-poly(A) containing long poly(A)

[0044] The distribution scarless of the present application refers to that the constructed plasmid does not introduce additional enzyme digestion sites, which ensures the expression of the pure natural sequence of the structural gene and maximally maintains its inherent biological activity.

[0045] The IIS-type restriction DNA endonuclease selected in the present application cuts DNA at a specific position downstream of the recognition site, and the sequence of the cutting site has no specific requirement and can be any combination of nucleotides, and the enzyme digestion can produce a specially designed sticky end. In a specific embodiment of the present application, BsaI is selected as the IIS-type restriction endonuclease A to construct the mRNA transcription template plasmid containing long poly(A). Those skilled in the art can understand that other IIS-type restriction DNA endonucleases can also achieve the purpose of the present application, and therefore the selection of BsaI should not be regarded as a limitation on the IIS-type restriction DNA endonuclease A.

[0046] Take BsaI as an example, the recognition sequence does not overlap with the enzyme cutting position, the enzyme cutting position is respectively located after any one 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 after enzyme cutting, a 4 nt sticky end is generated, which can be specifically designed according to needs.

[0047] (1) Construction of the leading plasmid PUC57-T7-poly(A)

[0048] The T7 promoter sequence (5'-TAATACGACTCACTATAGG-3' (SEQ ID NO. 13)), the BsaI enzyme reverse recognition sequence (5'-GAGACC-3' (SEQ ID NO. 2)), the arbitrary spacer fragment (5'-GCGTGGGGCCATGCTATGTCCCCTGCCCTCAA-3', SEQ ID NO. 14), the arbitrary spacer fragment is randomly designed, only used to separate the enzyme cutting sites of the IIS type restriction enzyme A on both sides, to facilitate the generation of a sticky end, the arbitrary spacer fragment described in the embodiment can be replaced by any other spacer fragment, and the arbitrary spacer fragment is not contained in the mRNA transcription template plasmid constructed, the BsaI enzyme forward recognition sequence (5'-GGTCTC-3' (SEQ ID NO. 1)), the 110 nt poly(A) sequence, and the BspQI enzyme reverse recognition sequence (5'-GAAGAGC-3' (SEQ ID NO. 3) are connected in series to obtain the T7-poly(A) sequence (the sequence is shown as SEQ ID NO. 7, the structural schematic diagram is shown as Figure 1 The above series of sequences (SEQ ID NO. 7) are synthesized by a gene synthesis company, and the series of sequences are reversely inserted into the multiple cloning site of the PUC57 plasmid (GenScript SD1176) by double enzyme cutting of EcoRI and HindIII, the BsaI enzyme reverse recognition sequence, the BsaI enzyme forward recognition sequence, and the BspQI enzyme reverse recognition sequence are inserted at the positions of 563, 513, and 409 bases of the PUC57 plasmid (the structural schematic diagram of the PUC57 plasmid is shown as Figure 2 The leading plasmid PUC57-T7-poly(A) (the structural schematic diagram is shown as Figure 3 ).

[0049] In the above construction, BspQI is selected as the IIS-type restriction endonuclease B to construct the mRNA transcription template plasmid containing long poly(A). Those skilled in the art can understand that other IIS-type restriction endonucleases different from IIS-type restriction endonuclease A can also achieve the purpose of the present application, and therefore the selection of BspQI should not be regarded as a limitation of IIS-type restriction endonuclease B.

[0050] (2) Construction of the leading plasmid PUC57-UTR-GFP

[0051] The BsaI enzyme forward recognition sequence (5'-GGTCTC-3' (SEQ ID NO. 1)), a fragment containing the 3' end of the T7 promoter (5'-NATAGG-3', N can be any single nucleotide A / T / G / C, which can be complementary to part of the T7 promoter sequence in the sticky end generated by BsaI enzyme digestion of PUC57-T7-poly(A) to form a complete T7 promoter sequence after BsaI enzyme digestion), a 5' UTR sequence (the sequence is shown in SEQ ID NO. 8), a codon-optimized GFP sequence (the sequence is shown in SEQ ID NO. 9), a terminator sequence (TGATAATAG (SEQ ID NO. 5)), a 3' UTR sequence (the sequence is shown in SEQ ID NO. 10), a poly(A) sequence (5'-AAAAAN-3' (SEQ ID NO. 6), N can be any single nucleotide A, T, G or C, which can be complementary to the complete poly(A) sequence in the sticky end generated by BsaI enzyme digestion of PUC57-T7-poly(A)) and the BsaI enzyme reverse recognition sequence (5'-GAGACC-3' (SEQ ID NO. 2)) are connected in series to obtain UTR-GFP (the structure is shown in Figure 4 The sequence is shown in SEQ ID NO. 11), and a gene synthesis company is commissioned to synthesize it. The UTR-GFP is inserted into the multiple cloning site of the PUC57 plasmid (GenScript SD1176) by double enzyme digestion of EcoRI and HindIII, and the BsaI enzyme forward and reverse recognition sequences are inserted at the 1291 and 408 bases of the PUC57 plasmid. The structure is shown in Figure 5 ).

[0052] The codon-optimized GFP sequence in this step is an example of the coding sequence of the target gene in a specific embodiment of the present application, and should not be regarded as a limitation of the technical solution claimed in the present application. In actual application, the specific coding sequence encoding the protein of interest can be selected according to the desired expressed protein.

[0053] (3) Constructing plasmid PUC57-T7-UTR-GFP-poly(A)

[0054] Using Bsal endonuclease, respectively, PUC57-UTR-GFP plasmid and PUC57-T7-poly(A) leading plasmid were digested, 37℃ reaction 1 h, the reaction system as follows:

[0055] 10 × enzyme Buffer 5μl,

[0056] Bsal (20 U / μl) 2ul,

[0057] Plasmid DNA 10ug,

[0058] RNase-free ddH2O to 50μl.

[0059] The structure of linear fragment of PUC57-UTR-GFP plasmid after Bsal digestion is shown in Figure 6 , the enzyme cutting position is located at 408 and 1291; The structure of linear fragment of PUC57-T7-poly(A) leading plasmid after Bsal digestion is shown in Figure 7 , the enzyme cutting position is located at 513 and 563.

[0060] The results of agarose gel electrophoresis show that Figure 8 ), Bsal enzyme cutting out about 900bp and 2800bp UTR-GFP and linear PUC57-T7-poly(A) target fragments. Using commercialized gel recovery kit, respectively, linearized plasmid PUC57-T7-poly(A) and enzyme cutting fragment UTR-GFP were recovered, according to the following system, based on Bsal sticky end connection (sticky ends are 5'-CTAT-3' and 5'-AAAA-3', respectively), 16℃ overnight reaction:

[0061] 10 × Ligase Buffer 1μl,

[0062] Insertion fragment UTR-GFP 0.3 pmol,

[0063] Vector DNA PUC57-T7-poly(A) 0.03 pmol,

[0064] T4 DNA Ligase (400 U / μl) 1μl,

[0065] RNase-free ddH2O to 10μl.

[0066] Subsequently, 10 μl of the ligation product was transformed into competent cells according to the instructions of the commercial E. coli competent cells, and spread on a plate containing the correct resistance with a sterile spreader, and incubated in an inverted position at 37°C for 12-16 h to grow single colonies. Positive clones were identified by colony PCR, and the reaction system was as follows:

[0067] 2 × Rapid Taq Master Mix 10 μl,

[0068] M13 Primer Mix 2 μl,

[0069] Bacterial solution 1 μl,

[0070] ddH2O to 20 μl.

[0071] The PCR reaction program was as follows:

[0072] 95°C for 3 min, 35 cycles of (95°C for 15 s, 55°C for 15 s, 72°C for 30 s), and 72°C for 5 min.

[0073] After picking positive single colonies and verifying the sequence by sequencing, the positive clone plasmid was extracted according to the operation shown in the commercial plasmid extraction kit, and the mRNA transcription template plasmid PUC57-T7-UTR-GFP-poly(A) (structure schematic diagram as shown in Figure 9 ) was successfully constructed.

[0074] The coding sequence of the target gene T7-UTR-GFP-poly(A) is shown in SEQ ID NO. 11, and the sequencing result is completely consistent with it, indicating that it is difficult to synthesize the long poly(A) gene fragment. The method provided in the embodiment for constructing the leading plasmid in steps and based on BsaI enzyme digestion / sticky end splicing can seamlessly splice the fixed elements (T7 promoter and poly(A) fragment) and variable target fragments (UTR and coding gene) required for mRNA synthesis through the IIS type restriction enzyme digestion system. No extra sequence is introduced after splicing. Further verification by enzyme digestion shows that the PUC57-T7-UTR-GFP-poly(A) plasmid no longer contains the BsaI enzyme digestion site and other additional sequences introduced in preparation except for the non-intended coding sequence, and the band position of the agarose gel electrophoresis after enzyme digestion is unchanged Figure 8 ), indicating that the construction method does not introduce additional enzyme digestion sites at the junction of the synthesized gene UTR-GFP and T7 promoter and poly(A) sequence.

[0075] Using the construction method of the present embodiment, on the basis of having prepared the leading plasmid PUC57-T7-poly(A), the plasmid PUC57-UTR-GFP is genetically synthesized and constructed, and then spliced and assembled to obtain the sequence-corrected PUC57-T7-UTR-GFP-poly(A) large extraction plasmid, which can be prepared in no more than 10 days at the fastest (7 days for construction and large extraction of the PUC57-UTR-GFP plasmid, 1 day for enzyme digestion and splicing assembly of the PUC57-T7-poly(A) and the PUC57-UTR-GFP, and 2 days for transformation, large extraction, and sequence verification of the target plasmid PUC57-T7-UTR-GFP-poly(A)). Since the leading plasmid containing the long poly(A) has been prepared in advance, the remaining target gene fragments do not contain complex sequences, so the difficulty in the steps of genetic synthesis, sequence identification, etc. is greatly reduced, and the preparation speed and efficiency are greatly improved compared with existing methods.

[0076] Example 2. One-step tandem construction of mRNA transcription template plasmid PUC57-T7-UTR-GFP-poly(A) containing long poly(A) (schematic diagram is the same as Figure 9

[0077] The sequence obtained by concatenating 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 110 A is shown in SEQ ID NO. 12. A genetic synthesis company was commissioned to perform genetic synthesis. The sequence was reversely inserted into the multiple cloning site of the PUC57 plasmid (GenScript SD1176) by EcoRI and HindIII double enzyme digestion to construct the PUC57-T7-UTR-GFP-poly(A) plasmid, and the plasmid was large extracted. Within a period of about 2 months, the number of poly(A) in the 4 construction clones prepared was severely deleted, and the final number of deletions reached 30, 42, 51, and 60. The deletion range reached 30-60 compared with Example 1, and the target plasmid with the expected sequence could not be prepared, and the large extraction plasmid with the expected sequence could not be obtained.

[0078] In comparison with Example 1, the synthesis method of Example 2 is limited by the high overall complexity of the sequence, and the difficulty and uncertainty of conventional one-step concatenation synthesis are high. After several attempts, the plasmid template with the expected sequence could not be obtained.

[0079] Example 3. In vitro transcription of mRNA-GFP encoding GFP

[0080] ​For PUC57-T7-UTR-GFP-poly(A) constructed in Example 1, BspQI enzyme cutting site is located at 409 and 1658, respectively, which is self-provided by PUC57 plasmid and introduced by construction, and the linearized sequence of 409-1658 will be cut out after BspQI enzyme cutting, which is used as template for mRNA in vitro transcription.

[0081] (1) Linearization and purification of plasmid template

[0082] The reaction system is configured as follows:

[0083] PUC57-T7-UTR-GFP-poly(A) plasmid 20 μg,

[0084] BspQI (10 U / μl) 10 μl,

[0085] 10 x BspQ I Buffer 20 μl,

[0086] Nuclease-Free H2O to 200 ul.

[0087] After 50°C reaction for 1 hour, agarose gel electrophoresis shows that the linearized template of expected size (about 1200 bp) is obtained after BspQI enzyme cutting (middle and lower bands). Figure 10 The linearized plasmid is purified by phenol chloroform extraction. An equal volume of phenol chloroform (Tris saturated phenol: chloroform: isopropyl alcohol = 25:24:1) is added to the DNA solution and mixed well; centrifuge at 12000g for 10 min at room temperature, carefully aspirate the upper aqueous phase, add an equal volume of chloroform solution (chloroform: isopropyl alcohol = 24:1), mix well; after centrifugation as above, carefully aspirate the supernatant, and detect the DNA concentration.

[0088] (2) mRNA in vitro transcription and purification

[0089] The reaction system is configured as follows:

[0090] Linearized PUC57-T7-UTR-GFP-poly(A) plasmid 5 μg,

[0091] T7 RNA Polymerase (50 U / μl) 10 μl,

[0092] Inorganic pyrophosphatase (0.1 U / μl) 5 μl,

[0093] RNase Inhibitor (40 U / μl) 5 μl,

[0094] 10 x Reaction buffer 10 μl,

[0095] ATP (100 mM) 10 μl,

[0096] GTP (100 mM) 10 μl,

[0097] m1 ψ / UTP (100 mM) 10 μl,

[0098] CTP (100 mM) 10 μl,

[0099] Nuclease-Free H2O up to 100 ul.

[0100] After mixed well, the reaction was carried out at 37°C for 2 hours, then 5 μl DNase I (1 U / μl) was added into the reaction system, and the reaction was carried out at 37°C for 15 min to remove the DNA template of transcription. The mRNA transcription product was purified by phenol-chloroform as above.

[0101] (3) Capping and purification of mRNA

[0102] The reaction system was configured as follows:

[0103] Transcription of mRNA 200 ug,

[0104] 10 x Capping Reaction buffer 50 μl,

[0105] GTP (10 mM) 25 μl,

[0106] SAM (4 mM) 25 μl,

[0107] Vaccinia Capping Enzyme (10 U / μl) 25 μl,

[0108] 2'-O-Methyltransferase (50 U / μl) 25 μl,

[0109] Nuclease-free H2O up to 100 ul.

[0110] After mixed well, the reaction was carried out at 37°C for 1 hour, and the mRNA transcription product was purified by phenol-chloroform as above.

[0111] The molecular integrity of the mRNA transcription product was detected by capillary electrophoresis (Qsep100 Advacne) (Figure 2). Figure 11 ), and the results showed that the size of mRNA-GFP product was as expected (about 1000 nt), and the purity reached more than 90%.

[0112] Example 4. Cell transfection and protein expression verification of in vitro transcribed mRNA-GFP

[0113] HeLa cells were cultured overnight in 96-well plates (3 × 10⁻⁶ cells / well). 4 (per hole), using Trans The mRNA-GFP transcribed in Example 3 above was transfected with IT®-mRNA (Mirus) transfection reagent (100 ng / well). PBS was added to the control cell wells. The cell nuclei were stained with hoechst33342 24 hours after transfection, and the signal intensity of DAPI and GFP channels was detected using a multifunctional cell imaging detection system (Cytation 1, BioTek).

[0114] Qualitative analysis of the imaging results indicates that ( Figure 12 The transcription product mRNA-GFP highly expressed GFP (green fluorescent protein), while control cells showed no green fluorescence signal. Quantitative analysis of the average fluorescence intensity of individual cells showed that the average GFP signal generated by mRNA-GFP transfection was 25164, which was 8.5 times that of the control cells (average signal value 2957). Figure 13 The above results confirm that the mRNA molecules containing long polyadenine, prepared rapidly and tracelessly using GFP as the target gene in Examples 1 and 3, have the expected functional activity.

Claims

1. A method for rapid scarless production of long poly(A)-containing mRNA, characterized in that, The method comprises the following steps: (1) Constructing a leading plasmid containing long poly A, inserting a DNA fragment in which a promoter, a reverse recognition DNA fragment of type IIS restriction enzyme A, an arbitrary spacer, a forward recognition DNA fragment of type IIS restriction enzyme A, long poly A, a reverse recognition DNA fragment of type IIS restriction enzyme B are connected in series into a multiple cloning site of the leading plasmid to obtain the leading plasmid containing long poly A, wherein the type IIS restriction enzyme A is DNA endonuclease Bsal, and the type IIS restriction enzyme B is DNA endonuclease BspQI; (2) Constructing a leading plasmid containing UTR and a coding gene, inserting a DNA fragment in which a forward recognition DNA fragment of type IIS restriction enzyme A, a DNA fragment at the 3' end of a promoter, a 5' UTR, a coding gene, a terminator, a 3' UTR, a poly A fragment and a reverse recognition DNA fragment of type IIS restriction enzyme A are connected in series into a multiple cloning site of the leading plasmid to obtain the leading plasmid containing UTR and the coding gene; (3) Cutting the leading plasmid containing long poly A obtained in step (1) by type IIS restriction enzyme A to obtain a linear fragment containing a T7 promoter and long poly A; (4) Cutting the leading plasmid containing UTR and a coding sequence obtained in step (2) by type IIS restriction enzyme A to obtain a linear fragment containing a 5' UTR, a coding gene, a terminator and a 3' UTR; (5) Connecting the linear fragment containing a promoter sequence and long poly A obtained in step (3) and the linear fragment containing a 5' UTR, a coding gene, a terminator and a 3' UTR obtained in step (4) to obtain a long poly A mRNA template plasmid.

2. The method of claim 1, wherein, The sequence of the forward recognition DNA fragment of type IIS restriction enzyme A in step (1) is shown in SEQ ID NO. 1, the sequence of the reverse recognition DNA fragment of type IIS restriction enzyme A is shown in SEQ ID NO. 2, and the sequence of the reverse recognition DNA fragment of type IIS restriction enzyme B is shown in SEQ ID NO.

3.

3. The method of claim 1, wherein, The long poly A in step (1) is 30-120 poly A.

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

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

6. The method of claim 1, wherein, The promoter fragment in steps (1) and (2) is a T7 promoter, 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 sequence of the poly A fragment in step (2) is shown in SEQ ID NO.

6.

8. The method of claim 1, wherein, The sequence of the terminator is shown in SEQ ID NO. 5, the sequence of the 5' UTR is shown in SEQ ID NO. 8, and the sequence of the 3' UTR is shown in SEQ ID NO.

10.

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

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