Multi-segmented PolyA polynucleotide stably existing in host cell and application of multi-segmented PolyA polynucleotide

Abstract

The invention relates to a multi-segment PolyA polynucleotide stably existing in a host cell and application of the multi-segment PolyA polynucleotide. Firstly, a multi-segment PolyA polynucleotide is provided, the multi-segment PolyA polynucleotide comprises more than two segments of PolyA sequence fragments, adjacent PolyA sequence fragments are connected through linkers, each PolyA sequence fragment is composed of a plurality of continuous A, and each linker is composed of nucleotide residues which are 1-24 nt and are not all A. The multi-segment PolyA polynucleotide can stably exist in a host cell such as an escherichia coli passage process, mRNA stability and protein output can be improved, and compared with a traditional unit polyA sequence, the multi-segment PolyA polynucleotide can greatly reduce the recombination rate of fermentation in escherichia coli so as to more easily optimize and amplify a process; the method can be better applied to plasmid fermentation related to transcription template preparation in mRNA vaccine or drug production.

Description

Multi-segmented PolyA polynucleotides stably present in host cells and their applications Technical Field

[0001] The present invention relates to a polynucleotide containing PolyA and its application, and in particular to a polynucleotide with a multi-segmented PolyA sequence that can stably exist in host cells such as Escherichia coli and its application, belonging to the field of molecular biology technology. Background Art

[0002] Among various options for combating the spread of the novel coronavirus, vaccines are a highly effective, large-scale measure to prevent further spread. mRNA vaccines, in particular, have rapidly gained popularity due to their short development and production cycles and hold enormous potential for future pharmaceutical development. Currently, mRNA production is primarily achieved through in vitro transcription, which requires capping and tailing to function in vivo.

[0003] Currently, there are two main methods for mRNA tailing: enzymatic and template methods. The enzymatic method involves tailing the in vitro transcription product using PolyA polymerase. This is a tailing mechanism that exists in natural cells, but it has certain limitations during research or production. The additional tailing reaction makes the production process more complicated, and the number of tails can only be controlled within a range and cannot be precise. In addition, the difference in the length of the mRNA tails produced is also a limiting factor. The template method is to construct a certain number of PolyA sequences onto the transcription template, so that tailing can be added during the transcription process, which simplifies the process while avoiding the problem of not being able to determine the specific amount of PolyA.

[0004] The existing technology adds a certain number of PolyA tails to the transcription template. There are two common methods for different scales: PCR method and plasmid method. The PCR method refers to adding PolyA to the transcription template through PCR using primers. The final transcription template is a PCR product. Due to the limitations of the PCR system, this method is suitable for small-scale production of mRNA. The plasmid method is to directly construct the designed PolyA sequence onto the plasmid. The plasmid contains a T7 promoter, 5'UTR, target gene sequence, 3'UTR and constructed PolyA sequence. Through the rapid replication of the plasmid in host cells such as Escherichia coli, a large amount of transcription templates can be produced in a short time, so that the production of mRNA reaches an industrial scale. However, after the PolyA sequence is constructed onto the plasmid, the plasmid is prone to recombination when replicating in the host cell, causing the PolyA sequence to change, thereby making the PolyA tail sequence carried by the produced mRNA uncontrollable, which directly affects the efficacy of mRNA drugs or vaccines.

[0005] In order to improve the stability of the PolyA sequence in the host cell, the current methods mainly use strains that can reduce recombination to culture plasmids or modify the PolyA sequence to stabilize the PolyA sequence during replication.

[0006] Summary of the Invention

[0007] One object of the present invention is to provide a polynucleotide having a PolyA sequence that can stably replicate in a host cell.

[0008] Another object of the present invention is to provide related applications of the polynucleotide.

[0009] The present invention mainly inserts different non-PolyA sequences (linkers) of a certain length into different positions of the PolyA sequence, so that the PolyA sequence is multi-segmented, and a polynucleotide with a multi-segmented PolyA sequence is obtained (referred to as multi-segmented PolyA polynucleotide in the present invention). The present invention transfers the plasmid with the multi-segmented PolyA polynucleotide into a host cell such as Escherichia coli and amplifies the fermentation, and can still maintain a high stability level, that is, no recombination occurs during the passage of the host cell, which can improve the stability of the PolyA sequence in the host cell such as Escherichia coli, and the stable multi-segmented PolyA polynucleotides of different effective lengths can meet the different expression requirements of different target genes. The multi-segmented PolyA polynucleotide provided by the present invention can significantly reduce the difficulty of plasmid construction, and can be better applied to the plasmid fermentation involved in the preparation of transcription templates in the production of mRNA vaccines or drugs. Stable multi-segmented PolyA polynucleotides can greatly improve the loss of PolyA sequences during plasmid fermentation, thereby ensuring the efficacy of mRNA vaccines or drugs.

[0010] Specifically, on the one hand, the present invention provides a multi-segmented PolyA polynucleotide, which includes two or more PolyA sequence fragments, and adjacent PolyA sequence fragments are connected by linkers, wherein each PolyA sequence fragment is composed of multiple consecutive A's, and each linker is composed of 1-24nt nucleotide residues that are not all A's.

[0011] According to a specific embodiment of the present invention, the multi-segmented PolyA polynucleotide of the present invention comprises two, three, four or five PolyA sequence segments.

[0012] According to a specific embodiment of the present invention, the multi-segmented PolyA polynucleotide of the present invention is selected from one or more polynucleotides having the following structures:

[0013] PolyA sequence fragment 1-linker 1-PolyA sequence fragment 2;

[0014] PolyA sequence fragment 1-linker 1-PolyA sequence fragment 2-linker 2-PolyA sequence fragment 3;

[0015] PolyA sequence fragment 1-linker 1-PolyA sequence fragment 2-linker 2-PolyA sequence fragment 3-linker 3-PolyA sequence fragment 4.

[0016] According to a specific embodiment of the present invention, in the multi-segmented PolyA polynucleotide of the present invention, each PolyA sequence fragment is independently composed of 10-100 consecutive A's.

[0017] According to a specific embodiment of the present invention, in the multi-segmented PolyA polynucleotide of the present invention, each PolyA sequence fragment is independently composed of 30-100, such as 30, 40, 50, 60, 70, 80, 90 or 100 consecutive A's.

[0018] Those skilled in the art will understand that, unless otherwise specified, the number of base A's in the Poly A sequence fragments described in the present invention is an approximate number. In particular, when the number of base A's in the Poly A sequence fragment is a multiple of 10, an error of about ±5, preferably ±3, more preferably ±2 or ±1 base A's is allowed on the basis of the described numerical value. For example, a Poly A sequence fragment consisting of 30 consecutive A's means that it includes Poly A sequence fragments that actually consist of 25, 26, 27, 28, 29, 30, 31, 32, 33 and / or 34 consecutive A's; for example, a Poly A sequence fragment consisting of 70 consecutive A's means that it includes Poly A sequence fragments that actually consist of 65, 66, 67, 68, 69, 70, 71, 72, 73 and / or 74 consecutive A's; and so on.

[0019] According to a specific embodiment of the present invention, in the multi-segmented PolyA polynucleotide of the present invention, the PolyA sequence segment 1 consists of 30-60 consecutive A's.

[0020] According to a specific embodiment of the present invention, in the multi-segmented PolyA polynucleotide of the present invention, the total number of A in all PolyA sequence fragments is 30-300, preferably 60-240, and more preferably 100-200.

[0021] According to a specific embodiment of the present invention, in the multi-segmented PolyA polynucleotide of the present invention, each linker is independently composed of 1-18 nt, preferably 1-12 nt, nucleotide residues that are not all A.

[0022] According to a specific embodiment of the present invention, in the multi-segmented PolyA polynucleotide of the present invention, each linker is independently composed of G, or the G content is 20%-33% of the total bases in the linker sequence.

[0023] According to a specific embodiment of the present invention, in the multi-segmented PolyA polynucleotide of the present invention, each linker is independently selected from the nucleotides shown in the following sequence:

[0024] According to a specific embodiment of the present invention, in the multi-segmented PolyA polynucleotide of the present invention, the sum of the lengths of all linkers is 1-48 nt, more preferably 2-24 nt.

[0025] According to a specific embodiment of the present invention, in the multi-segmented PolyA polynucleotide of the present invention, the sum of the lengths of all linkers accounts for less than 20% of the total length of the polynucleotide, preferably less than 15%, more preferably less than 10%, and even more preferably less than 5%.

[0026] According to a specific embodiment of the present invention, the multi-segmented PolyA polynucleotide of the present invention includes a sequence of SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4, SEQ ID NO.5, SEQ ID NO.6, SEQ ID NO.7, SEQ ID NO.8, SEQ ID NO.9, SEQ ID NO.10, SEQ ID NO.11, SEQ ID NO.12, SEQ ID NO.13, SEQ ID NO.14, SEQ ID NO.15, SEQ ID NO.16, SEQ ID NO.17, SEQ ID NO.18, SEQ ID NO.19, SEQ ID NO.20, SEQ ID NO.21, SEQ ID NO.22, SEQ ID NO.23, SEQ ID NO.24, SEQ ID NO.25, SEQ ID NO.26, SEQ ID NO.27, SEQ ID NO.28, SEQ ID NO.29, SEQ ID NO.30, SEQ ID NO.31, SEQ ID NO.32, SEQ ID NO.33, SEQ ID NO. One or more polynucleotides of any one of the sequences shown in SEQ ID NO.34, SEQ ID NO.35, SEQ ID NO.36, SEQ ID NO.37, SEQ ID NO.38, SEQ ID NO.39, SEQ ID NO.40, SEQ ID NO.41, SEQ ID NO.42, SEQ ID NO.43, SEQ ID NO.44, SEQ ID NO.45, SEQ ID NO.46, SEQ ID NO.47, SEQ ID NO.48, SEQ ID NO.49, SEQ ID NO.50, SEQ ID NO.51, SEQ ID NO.52, SEQ ID NO.77, SEQ ID NO.78, SEQ ID NO.79, SEQ ID NO.80, and SEQ ID NO.81.

[0027] In another aspect, the present invention further provides a vector comprising the multi-segmented PolyA polynucleotide described herein. The multi-segmented PolyA polynucleotide described herein can be inserted into a plasmid vector using any method known in the art, thereby constructing a vector containing the multi-segmented PolyA polynucleotide. During construction, restriction sites can be added to the ends of the multi-segmented PolyA polynucleotide as needed.

[0028] On the other hand, the present invention also provides a host cell comprising the multi-segmented PolyA polynucleotide of the present invention or the vector of the present invention (a vector into which the multi-segmented PolyA polynucleotide is inserted).

[0029] In another aspect, the present invention further provides the use of the multi-segmented PolyA polynucleotide, the vector, and the host cell in in vitro transcription to produce target mRNA. The target mRNA carries a poly A tail formed by transcription of the multi-segmented PolyA polynucleotide of the present invention.

[0030] Therefore, the present invention also provides an mRNA comprising a poly A tail formed by in vitro transcription of the multi-segmented Poly A polynucleotide of the present invention. The poly A tail corresponds to the mRNA sequence of the multi-segmented Poly A polynucleotide of the present invention.

[0031] According to a specific embodiment of the present invention, the target mRNA is used to produce an mRNA vaccine or drug. The multi-segmented PolyA polynucleotide provided by the present invention can be better applied to the plasmid fermentation involved in preparing the transcription template in the production of mRNA vaccines or drugs. The stable multi-segmented PolyA polynucleotide can greatly reduce the loss of PolyA sequences during the plasmid fermentation process, thereby ensuring the efficacy of the mRNA vaccine or drug.

[0032] On the other hand, the present invention also provides a method for identifying the stability of a multi-segmented PolyA polynucleotide in a host cell, the method comprising:

[0033] 1) cloning the multi-segmented PolyA polynucleotide of the present invention into a universal plasmid vector to obtain a plasmid vector into which the multi-segmented PolyA polynucleotide is inserted;

[0034] 2) Transforming the plasmid vector containing the multi-segmented PolyA polynucleotide into a host cell, such as Escherichia coli, to obtain recombinant cells, plating the recombinant cells on a solid plate to obtain single colonies, picking the single colonies for preliminary PCR identification, and screening for stable colonies that have not undergone recombination; preferably, the PCR products are identified by 2%-3% agarose gel electrophoresis;

[0035] 3) The stable bacteria that have not undergone recombination and were screened out in the preliminary identification were amplified for fermentation culture. The culture solution was gradient diluted and then spread on a solid plate. Colony PCR was performed to identify the stability of the passage.

[0036] In some specific embodiments of the present invention, the method for identifying the stability of a multi-segmented PolyA polynucleotide in a host cell comprises:

[0037] 1) Prepare a gene fragment with the above sequence by gene synthesis, construct it into the pUC-GW-kana universal plasmid, and synthesize the corresponding universal primers;

[0038] 2) Using the plasmid as a template, perform gradient annealing PCR to identify the optimal annealing temperature;

[0039] 3) The plasmid was transformed into E. coli (including but not limited to DH5α, stbl2, stbl3, stable) by conventional E. coli transformation. After transformation, the plasmid was spread on LB plates with different kana resistance (50 μg / ml final concentration, and the kana resistance mentioned hereafter was referred to at this concentration and will not be repeated here). The plates were cultured at 30-37°C for 12-20 hours.

[0040] 4) Pick a single colony from each plate and add it to 20 μl of Kana-resistant liquid culture medium and mix thoroughly to prepare a colony suspension;

[0041] 5) preparing a colony PCR reaction system for identifying the stability of the sequence, wherein the colony PCR system (20 μl) includes the following components: 7 μl of RNA-free water; 0.5 μl each of primer 1 and primer 2; 10 μl of enzyme mix; and 2 μl of colony solution;

[0042] 6) Identify the PCR product obtained in step 5) by electrophoresis on a 2-3% agarose gel. Analyze the electrophoresis results to determine the stability of the corresponding PolyA sequence in E. coli.

[0043] 7) When the recombination rate is lower than 15%, select the bacterial solution that has not undergone recombination, take 5 μl of the suspended bacterial solution and transfer it to 100 ml of kana-resistant culture medium. Incubate in a shaker at 30°C and 220 rpm for 12-16 hours and then dilute 10 4 Spread onto a kana-resistant solid plate, incubate the plate upside down in a 30°C incubator for 20 h, and repeat steps 4) to 6) to further verify the stability of the PolyA sequence during the amplification process;

[0044] 8) Transfer the single clone obtained from the plate during further verification to 3 ml of Kana resistance medium and culture in a shaker at 30-37°C for 12-20 hours before performing plasmid mini-extraction. The mini-extracted plasmid is then commissioned to an authoritative biological company for sequencing. Sequence comparison analysis based on the sequencing results can further serve as evidence of sequence stability.

[0045] Specific experiments of the present invention show that the multi-segmented PolyA polynucleotide provided by the present invention can significantly reduce the phenomenon of PolyA sequence changes or losses due to homologous recombination during the passage of E. coli, thereby greatly improving the quality of the produced mRNA. PolyA sequences of different effective lengths can meet the expression requirements of different target genes. Stable passage in E. coli also provides the possibility of plasmid amplification production and fermentation, which greatly facilitates the simplification of production processes and the increase in yield. The multi-segmented PolyA polynucleotide provided by the present invention can be better applied to the plasmid fermentation involved in the preparation of transcription templates in the production of mRNA vaccines or drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0046] FIG1 is a schematic diagram of the design structure of the multi-segmented PolyA polynucleotide of the present invention.

[0047] Figure 2 is the plasmid map of pUC-GW-kana.

[0048] Figure 3 shows the results of exploring the optimal annealing temperatures of primers cgacggccagtgaattgac (SEQ ID No. 65) and cagctatgaccatgctcgag (SEQ ID No. 66) using the universal plasmid pUC-GW-kana having the sequence shown in SEQ ID No. 1 as a template.

[0049] FIG4 shows the comparative results of the stability of the partially fragmented PolyA polynucleotides of the present invention in E. coli stbl3.

[0050] FIG5 shows the comparative results of the stability of the partially fragmented PolyA polynucleotides of the present invention in Escherichia coli stbl2.

[0051] FIG6 shows the sequencing comparison results of plasmid miniprep of a partially fragmented PolyA polynucleotide of the present invention after fermentation and amplification in Escherichia coli stbl2.

[0052] FIG7 shows the correlation analysis results between the linker number, position, length, and G, C, and T contents and the stability of the multiplexed PolyA polynucleotide based on the data described in FIG4 and FIG5 .

[0053] FIG8 shows the fluorescence microscopy results of transfecting 293T / 17 cells after adding the partially fragmented PolyA polynucleotide of the present invention to a DNA template encoding GFP expression by plasmid or PCR methods and performing IVT to obtain the corresponding mRNA.

[0054] FIG9 shows the results of flow cytometric analysis of the cell samples treated in FIG8 (the table in the figure shows specific test data).

[0055] FIG10 shows the detection results of the expression levels of the polyA mRNAs represented by SEQ ID No. 77 to SEQ ID No. 81.

[0056] FIG11 shows the stability test data of A30G70 and A30L70 after extreme amplification. DETAILED DESCRIPTION

[0057] To provide a clearer understanding of the technical features, objectives, and beneficial effects of the present invention, the following detailed description of the technical solutions of the present invention is provided. However, this description is not intended to limit the scope of the present invention. All raw materials and reagents used in the experiments below are commercially available. Experimental methods without specific conditions are conventional methods and conditions well known in the art, or follow the conditions recommended by the instrument manufacturer.

[0058] Unless specifically defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the relevant art.

[0059] The present invention provides a multi-segmented PolyA polynucleotide, the structural design of which is shown in Figure 1. Specifically, the present invention provides the multi-segmented PolyA polynucleotides shown in SEQ ID No. 1 to SEQ ID No. 52 and SEQ ID No. 77 to SEQ ID No. 81 in Table 1 (wherein, the number of base A in the PolyA sequence segments shown in SEQ ID No. 1 to SEQ ID No. 52 has an error of ±1 base A, while the number of base A in the PolyA sequence segments shown in SEQ ID No. 77 to SEQ ID No. 81 is an exact value).

[0060] Table 1

[0061] In the present invention, the multi-segmented PolyA polynucleotide needs to be constructed onto a universal vector, and then transformed and identified. The present invention has no special limitation on the universal vector, and preferably uses the universal plasmid pUC-WG-kana for kana resistance. In the present invention, the pUC-GW-kana plasmid map is shown in Figure 2. The multi-segmented PolyA polynucleotide needs to be constructed into the EcoRVⅠ insertion site on the universal vector. Synthetic plasmids are generally stored and transported in a dry powder state, and are preferably dissolved in ultrapure water when used. The concentration of the plasmid after water dissolution is preferably 10 ng / μl, more preferably 1 ng / μl. The present invention has no special limitation on the ultrapure water, and conventional laboratory ultrapure water can be used.

[0062] After obtaining the synthetic plasmid, the present invention transfers the synthetic plasmid into different host cells to obtain recombinant cells, and for different recombinant cells, colony PCR is performed to test the stability of the corresponding sequence of the synthetic plasmid. In the present invention, the different host cells are preferably Escherichia coli competent cells (including but not limited to DH5α, stbl2, stbl3, stable); the present invention has no special restrictions on the method of transfer, and the conventional transfer method in the art can be used. After obtaining the recombinant cells, the present invention preferably performs stable sequence identification of positive recombinant cells. In the present invention, the screening of the positive recombinant cells is preferably carried out on a kana-resistant solid culture medium. In the present invention, a single colony on the kana-resistant solid culture medium is selected and fully dissolved in a certain volume of culture medium to obtain a dilution of a single colony. The present invention has no special restrictions on the method of dissolving the single colony, and the conventional bacterial transfer operation in the art can be used. After obtaining the colony dilution, a small amount of bacterial liquid is taken for colony PCR. When the number of bands that match the target band exceeds 75%, the bacterial liquid whose amplified band matches the target band is selected and inoculated into 100 ml of kana-resistant liquid culture medium for further subculture. In the present invention, the sequence of the primer F of the colony PCR is: cgacggccagt gaattgac (SEQ ID No. 65); the sequence of the primer R of the colony PCR is: cagctatgaccatgctcgag (SEQ ID No. 66). In the present invention, the amplification procedure of the colony PCR is preferably as follows: pre-denaturation at 98°C for 10 min; denaturation at 98°C for 300 s, annealing at 66.5°C for 15 s, extension at 72°C for 30 s, 30 cycles; final extension at 72°C for 5 min. In the present invention, the system of the colony PCR is preferably as follows: 2× HotStart PCR Genotyping Master Mix (With Dye) 10 μl; Primer F 0.5 μl; Primer R 0.5 μl; Bacterial solution 2 μl; Water 7 μl.

[0063] In the present invention, the initial concentration of the primer F and primer R is preferably 10 μmol / L. HotStart PCR Genotyping Master Mix (With Dye) contains the following components: DNA Polymerase, dNTPs, Mg 2+and nucleic acid dyes. After the PCR amplification is completed, the present invention preferably identifies and analyzes single colonies that match the size of the target band by agarose gel electrophoresis. The present invention has no special restrictions on the agarose gel electrophoresis, and the conventional gel preparation and electrophoresis methods in this field can be used. Preferably, 3% agarose gel is used, and 130V is used for running for 30 minutes. When the proportion of bands that match the size of the target band exceeds 50%, any strain that matches the size of the target band is selected and transferred to 100ml of kana-resistant liquid culture medium for strain culture. The present invention has no special restrictions on the kana-resistant liquid culture medium, and the conventional kana-resistant liquid culture medium in this field can be used. The present invention has no special restrictions on the strain transfer culture method, and the conventional strain transfer culture method in this field can be used. It is preferred to use a shaking environment of 30°C and 220rpm for culture, and preferably culture for 16h. After the culture is completed, 5μl of bacterial solution is taken for gradient dilution, and diluted to 10 4 , 10 5 , 10 6 After the dilution is doubled, it is spread on a kana-resistant solid culture medium and cultured overnight in a 30°C incubator. The present invention does not specifically limit the dilution of the bacterial solution, and the conventional bacterial solution gradient dilution method in the art can be used. After overnight culture, a plate with 100-300 single colonies is selected for the next step of colony PCR identification. The identification method steps are the same as described above, namely, picking bacteria, diluting the bacterial solution, preparing the PCR system, reacting according to a specific procedure, and identifying by agarose gel electrophoresis. The primers, procedures, and electrophoresis methods used are the same as above. In addition, a single clone is picked from a suitable plate and cultured in 3 ml of Kana-resistant culture medium for plasmid mini-extraction sequencing. The comparison of sequencing results can also be used as another stability analysis evidence. Based on the identification results, the recombination rate of the stable sequence plasmid during the amplification culture in Escherichia coli can be calculated. The recombination rate calculation formula is: recombination rate = 1-the number of bands that match the size of the target band / total sample volume.

[0064] After obtaining all the results from the aforementioned detection experiments, the present invention selectively selects a portion of the polyA sequence with an extremely low recombination rate, i.e., a highly stable segmented polyA sequence, and then incorporates it into a vector capable of expressing eGFP. Alternatively, primers containing the corresponding stable segmented polyA sequence are synthesized, and the polyA sequence is added to the sequence encoding eGFP using PCR. The corresponding mRNA is then prepared by in vitro transcription and transfected into 293T / 17 cells for translation verification at the cellular level. Qualitative analysis using an inverted fluorescence microscope and quantification of fluorescence intensity by flow cytometry are then performed to analyze the effects of different polyA sequences on translational capacity.

[0065] The technical solutions provided by the present invention are described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0066] Example 1

[0067] Design and synthesis of multiplexed PolyA polynucleotides:

[0068] 1) Synthesize target DNA fragments of genes SEQ ID No. 1-SEQ ID No. 52 and SEQ ID No. 77-SEQ ID No. 81. The above sequence composition design diagram is shown in Figure 1 and constructed into the EcoRV I site of the universal vector pUC-GW-kana. The universal vector map is shown in Figure 2.

[0069] 2) Detection of the optimal annealing temperature of universal primers for colony PCR;

[0070] 3) Transform the target gene into competent E. coli STBL2 or STBL3 and culture in Kana-resistant liquid medium;

[0071] 4) Preliminary identification by colony PCR;

[0072] 5) The bacterial solution that meets the requirements is transferred to culture and graded diluted and plated for identification and analysis;

[0073] 6) Sequencing analysis of the target plasmid in the amplified strain;

[0074] 7) Count and analyze the stability data of all sequences.

[0075] The optimal annealing temperature detection steps are as follows:

[0076] Remove the enzyme, primers, and plasmid template from -20°C and place them on ice-water mixture to thaw until all materials are completely thawed and no solid ice is visible. Begin preparing the PCR reaction system, which is as follows:

[0077] In the present invention, the initial concentrations of primers F and R are 10 μmol / L; the concentration of the DNA template (a plasmid containing the sequence shown in SEQ ID No. 1) is 1 ng / μl. In the present invention, the sequence of primer F is as follows: cgacggccagtgaattgac (SEQ ID No. 65); the sequence of primer R is as follows: cagctatgaccatgctcgag (SEQ ID No. 66). After the reaction is prepared, the mixture is divided into 19 different PCR tubes, with 20 μl per tube. The different tubes are placed in a uniform row on a PCR instrument, and a gradient annealing PCR program is set to explore and detect the optimal annealing temperature.

[0078] In the present invention, the amplification program for the gradient annealing PCR is preferably as follows: initial denaturation at 98°C for 10 minutes; 30 cycles of denaturation at 98°C for 30 seconds, annealing at 55.7-66.5°C for 15 seconds, and extension at 72°C for 30 seconds; and a final extension at 72°C for 5 minutes. This program allows each well in a row of the PCR instrument to correspond to a different annealing temperature, while maintaining the same temperature profile. This allows for rapid analysis of product differences in the same system, with only the annealing temperature being varied, thereby determining the optimal annealing temperature for the primers.

[0079] After the above procedures were completed, a 3% agarose gel was prepared (weighing 3g of agarose and adding it to 100ml of TAE solution). 0.5μl of bromophenol blue was added to the PCR tube where the PCR reaction had completed, and the mixture was shaken. Samples were then spotted sequentially at different temperatures. The electrophoresis program was set to 130V and run for 30 minutes before color development. The results are shown in Figure 3. To minimize the impact of nonspecific bands on the test results, the annealing temperature of 66.5°C, which minimizes nonspecific bands, was selected for subsequent experimental testing.

[0080] The steps for plasmid transformation are as follows:

[0081] Place the corresponding number of clean 1.5ml EP tubes in an EP tube rack and label them with the corresponding plasmids to be transfected. Remove the synthetic gene powder (containing a synthetic gene plasmid containing one sequence from SEQ ID No. 1-SEQ ID No. 52 or SEQ ID No. 77-SEQ ID No. 81) from the tube and centrifuge at 10,000g for 1 minute in a high-speed centrifuge. Dilute the solution to 10 ng / μl with enzyme-free water on the workbench and pre-chill on ice for 5 minutes. Simultaneously, remove the competent cells, STBL2 and STBL3, and thaw them on ice. Once the competent cells are free of ice chips, aliquot the solution into the prepared 1.5ml EP tubes, 50 μl per tube. Add 1 ng of the corresponding pre-chilled synthetic plasmid to the competent cells. Gently pipette to mix thoroughly and incubate on ice for 30 minutes. Then, heat shock the tubes in a pre-heated 42°C water bath, ensuring that the cells are fully submerged in the water. After 45 seconds of heat shock, remove the plate and gently insert it below ice. Incubate for 2 minutes, then add 950 μl of LB liquid medium and shake at 220 rpm at 30°C for 1 hour to recover. Spread 50 μl, 100 μl, and 150 μl of the bacterial cells onto a Kana-resistant solid plate. Incubate in a 30°C incubator for 20-24 hours. Select plates with approximately 100-300 distinct single colonies for identification.

[0082] The steps for colony PCR are as follows:

[0083] Picking a single colony: Remove the PCR tube rack and arrange the corresponding number of eight-tube strips. Pour LB medium into the pipette slot. Then, use a pipette to add 50μl of LB medium to each of the eight-tube strips. Use forceps to pick a single colony from the plate with a sterilized white pipette tip and place it into the eight-tube strip. Gently shake the PCR tube rack to thoroughly mix the bacteria on the pipette tip with the medium. Remove the white pipette tip, cap the eight-tube strip, and mark the cap with an alcohol-resistant marker. Incubate the tube strips at 30°C in a shaker for 2 hours. Record the mixture in a notebook (date, serial number, etc.). Remove from the shaker, centrifuge briefly in a handheld centrifuge, gently flick to remove any bubbles, centrifuge again for 15 seconds, and store at 4°C for later use.

[0084] Colony PCR reaction: Prepare the following PCR reaction system, add the prepared reaction solution to a 96-well plate, and use a dispenser to add 1μl of bacterial solution. In addition, you need to add a set of reactions using the original gene plasmid as a template as a positive control, and a reaction using water as a template as a negative control. Amplify according to the PCR procedure:

[0085] In the present invention, the initial concentrations of primers F and R are 10 μmol / L; the concentration of the DNA template is 1 ng / μl. In the present invention, the sequence of primer F is as follows: cgacggccagtgaattgac (SEQ ID No. 65); the sequence of primer R is as follows: cagctatgaccatgctcgag (SEQ ID No. 66). In the present invention, the PCR amplification procedure is preferably as follows: initial denaturation at 98°C for 10 minutes; 30 cycles of denaturation at 98°C for 30 seconds, annealing at 66.5°C for 15 seconds, and extension at 72°C for 30 seconds; and a final extension at 72°C for 5 minutes.

[0086] Agarose gel electrophoresis: First prepare 3% agarose gel (weigh 3g agarose and add it to 100ml TAE solution), add 0.5μl bromophenol blue to the eight-tube strip where the PCR reaction is completed, shake and mix, then apply the sample, take a picture of the electrophoresed agarose gel and save it.

[0087] Recombination rate definition and analysis: The recombination rate of stable sequences is determined based on agarose gel electrophoresis band patterns. Colonies with PCR bands comparable in size to the positive control (PCR template using the corresponding plasmid) and no other bands are considered non-recombinant strains; otherwise, they are considered recombinant strains. Recombination rate = number of recombinant strains counted / total number of strains tested.

[0088] After the recombination rate analysis, the stability test of the selected sequence plasmid after amplification: Based on the above screening, the partial sequences with the recombination rate lower than 15% (including SEQ ID No.44, SEQ ID No.45, SEQ ID No.46, SEQ ID No.47, and SEQ ID No.51) were screened for further amplification testing. The stable strains identified by colony PCR were selected, and 10 μl of the bacterial solution was transferred to 100 ml of kana-resistant LB liquid medium and cultured at 30°C / 220 rpm for 16 hours. Dilute 10 4 , 10 5 , 10 6 Spread the solution onto a Kana-resistant solid culture medium. Incubate in a 30°C incubator for 20 hours. Select a plate with relatively uniform colonies and pick a few single colonies for stability testing using the same method as above.

[0089] Figure 4 shows the recombination rate test results for some plasmids transformed into the stbl3 strain, and Figure 5 shows the test results for some plasmids transformed into the stbl2 strain, as well as the test results after amplification and fermentation of the stable strain. To more accurately determine sequence stability, the amplified strains were sequenced outsourced, and the sequencing alignment results are shown in Figure 6. Based on the extensive test results in Figures 4 and 5, and to more intuitively analyze the relationship between polyadenylation stability and sequence properties, matplotlib.pyplot was used for data processing. Sequence features were broken down into linker number, position, length, and G, C, and T content, and correlation analysis was performed with polyadenylation stability. The results are shown in Figure 7.

[0090] Example 2

[0091] Translation verification of partial polyA sequences:

[0092] 1) Obtaining a linearized vector backbone and a poly(A) sequence with homologous ends to the backbone by PCR;

[0093] 2) ligating the linearized backbone to the polyA sequence via homologous recombination;

[0094] 3) The ligation product was transformed into Escherichia coli stbl2 and plated on kana-resistant LB solid screening medium. The cells were cultured in a 30°C incubator overnight to perform preliminary recombinant screening.

[0095] 4) Confirm the final correct recombinant by sequencing;

[0096] 5) Linearize the recombinant and tail it by PCR to obtain the corresponding transcription template;

[0097] 6) Preparation of corresponding mRNA by in vitro transcription;

[0098] 7) Cellular translation verification.

[0099] The steps for backbone linearization and polyA sequence acquisition by PCR are as follows:

[0100] PCR reaction: Gene synthesis was used to obtain a kana-resistant plasmid containing a T7 promoter, 5'UTR, eGFP coding region, and 3'UTR sequence, which served as the backbone for the above-mentioned destination vector construction. Linearization was performed by PCR, and the reaction system was as follows: Max DNA Polymerase 10 μl; Primer F 0.5 μl; Primer R 0.5 μl; Plasmid template 0.5 μl; Water 8.5 μl.

[0101] In the present invention, the initial concentrations of primers F and R were 10 μmol / L; the concentration of the DNA template was 1 ng / μl. In the present invention, the sequence of primer F, the linearized vector backbone, was as follows: TGAGGGTCTAGAACTAGTGTCG (SEQ ID No. 67); the sequence of primer R was as follows: CTTCCTACTCAGGCTTTATTCAAAG (SEQ ID No. 68); and the plasmid template used was a synthetic gene containing the aforementioned backbone sequence.

[0102] The PCR system used to amplify polyA fragments containing backbone homology sequences was the same as above. The sequence of primer F used was as follows: CTTTGAATAAAGCCTGAGTAGGAAG (SEQ ID No. 69); the sequence of primer R used was as follows: CGACACTAGTTCTAGACCCTCA (SEQ ID No. 70). The plasmid template used was a synthetic gene containing a multi-segmented stable polyA sequence, including backbone homology sequences at both ends of the gene, 5'-end flanking sequences: CTTTGAATAAAGCCTGAGTAGGAAG (SEQ ID No. 71), and 3'-end flanking sequences: CGACACTAGTTCTAGACCCTCA (SEQ ID No. 72). The multi-segmented polyA sequences selected for construction included A30L70, A30L170, A30L270, A30G70, and A40L80.

[0103] In the present invention, the PCR amplification program is preferably as follows: pre-denaturation at 98°C for 10 min; denaturation at 98°C for 30 s, annealing at 55°C for 15 s, extension at 72°C for 30 s, 30 cycles; and final extension at 72°C for 5 min.

[0104] Agarose gel electrophoresis: First prepare 1% agarose gel (weigh 1g agarose and add it to 100ml TAE solution), add 0.5μl bromophenol blue to the PCR tube where the PCR reaction is completed, shake and mix, then apply the sample, apply a DNA marker to the lane near the sample, take a picture of the electrophoresed agarose gel and save it.

[0105] Analysis of target band: Develop the gel and compare the target band with a marker band of the same size to confirm that the target band is clear and the correct size. If the band size is incorrect or no band is detected, repeat the above steps.

[0106] The in vitro reconstitution steps for the target band are as follows:

[0107] Preparation of the recombination system: Use the above PCR products to prepare an in vitro recombination system: 2 μl of backbone PCR product, 2 μl of polyA PCR product, 0.1 μl of T5 Exonuclease, 0.5 μl of 4-buffer, and 0.4 μl of ddH2O. The entire system preparation should be performed on ice. After preparation, gently mix with a pipette and incubate on ice for 5 minutes. After incubation, add 50 μl of competent E. coli cells, preferably STBL2 cells. After gentle pipetting, incubate on ice for an additional 30 minutes. Heat shock the cells in a 42°C water bath for 45 seconds, then return them to ice for 2 minutes. Remove the cells, add 950 μl of liquid LB medium, and resuspend them in a shaker at 30°C for 1 hour. Plate the cells onto solid LB medium containing Kana-resistant cells and incubate them in a 30°C incubator for 20 hours.

[0108] Screening of the final correct recombinants: After culturing the above-mentioned coated culture medium for 20 hours, pick several single colonies and transfer them to 3 ml of kana-resistant liquid LB culture medium and culture at 30°C for 16 hours to extract the plasmids, and sequence them. The sequencing results are compared with the designed sequence. The plasmids with correct comparison results are numbered and packaged for storage.

[0109] The steps for preparing transcription template using the plasmid method are as follows:

[0110] Linearization of recombinant plasmid: The constructed recombinant plasmid is linearized using the designed ⅡS restriction endonuclease cleavage site. In the present invention, BspQⅠ is preferably used for enzyme cleavage. The enzyme cleavage system is as follows: 100 μg of recombinant plasmid (1 μg / μl), NEBuffer TM Mix 500 μl of r3.1, 30 μl of BspQⅠ (10,000 units / ml), and 4.37 ml of sterile water, then incubate in a 50°C metal bath for 1 h. After incubation, purify using the Cycle Pure Kit. Refer to the kit instructions for the operating instructions.

[0111] Detection of linearized product: After purification, remove 2 μl of the linearized product and measure its concentration using a nanodrop micro-spectrophotometer. Record the product concentration and analyze 400 ng by mixing with 10× DNA loading buffer for agarose gel electrophoresis. If a single, clear band of the correct size is present, label it and store at -20°C or use it for the next step of in vitro transcription.

[0112] The steps for preparing transcription template by PCR are as follows:

[0113] Preparation of PCR system: A kana-resistant plasmid containing a T7 promoter, 5'UTR, eGFP coding region, and 3'UTR sequence was obtained by gene synthesis. The above continuous sequence was tailed with a polyA tail by PCR. The reaction system was as follows:

[0114] In the present invention, the initial concentrations of the primer F and the primer R are 10 μmol / L; the concentration of the DNA template is 1 ng / μl. In the present invention, the sequence of the primer F is as follows: GTACAGAAGCTAATACGACTCAC (SEQ ID No.73); the sequence of the primer R is as follows: TTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTCTTCCTACTCAGGCTTTATTCAAAG (SEQ ID No.74) or TTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTCCTACTCAGGCTTTATTCAAAG (SEQ ID No.75) No.75) or TTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTAGTCATATGCTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTCTTCCTACTCAGGCTTTATT (SEQ ID No.76); the plasmid template used is the synthetic gene having T7 promoter, 5'UTR, eGFP coding region and 3'UTR sequence.

[0115] In the present invention, the PCR amplification program is preferably as follows: pre-denaturation at 98°C for 10 min; denaturation at 98°C for 30 s, annealing at 55°C for 15 s, extension at 72°C for 30 s, 30 cycles; and final extension at 72°C for 5 min.

[0116] Purification of PCR products: Weigh 0.5g agarose in 50ml 1×TAE, heat in a microwave until no obvious solid is visible, then add 5μl nucleic acid dye and mix thoroughly. Pour into the prepared gel caster to prepare a 400μl recovery gel. Let it stand at room temperature for 30 minutes until the gel is completely solidified. Pull out the comb, place it in the electrophoresis instrument, and add all the PCR products mixed with DNA loading buffer. After running at 130V for 30 minutes, transfer the gel to the blue light analyzer to analyze the target band. After confirming that the band is clear and the size is correct, take out the knife, disinfect it with alcohol, cut off the target band, and place it in an EP tube. Purify with Gel Extraction Kit. For specific operations, refer to the kit instructions.

[0117] Detection of Purified Product: After purification, remove 2 μl of the PCR product and measure its concentration using a nanodrop micro-spectrophotometer. Record the product concentration and analyze 400 ng by mixing with 10× DNA loading buffer for agarose gel electrophoresis. If a single, clear band of the correct size is present, label it and store at -20°C or use it for in vitro transcription.

[0118] Preparation of mRNA by in vitro transcription:

[0119] Prepare transcription system: Prepare different templates into in vitro transcription system for mRNA preparation. The specific reaction system is as follows:

[0120] After the above system was prepared, it was placed in a PCR instrument and set to react at 37°C for 4 hours. 2 μl DNAse I was added to the reaction system and the reaction was continued at 37°C for 45 minutes. After that, the reaction system was taken out and the MEGAclean kit was used. TM The specific purification steps can be found in the kit instructions.

[0121] mRNA Detection: Measure the concentration of 2 μl of the purified mRNA using a nanodrop spectrophotometer. Record the concentration and analyze 400 ng of the mRNA by mixing it with 10× RNA loading buffer for agarose gel electrophoresis. If a single, clear band of the correct size is present, label it and store it at -20°C or use it for further analysis.

[0122] Verification of cellular translation of GFP expressed by mRNA with different polyA sequences:

[0123] Preparation of cells and transfection reagents:

[0124] The host cells used for transfection are 293T cells. Cells are seeded into 24-well plates in advance to reach 70-90% confluency at the time of transfection. 0.5-4 hours before transfection, the medium is replaced with 5% serum Opti-MEM™ medium. Subsequent transfection requires the use of the transfection kit Lipofectamine. TM 3000, the specific steps are as follows:

[0125] Use Opti-MEM™ culture medium to dilute mRNA and transfection reagent Lipofectamine at a certain ratio. TM 3000, then dilute Lipofectamine TM 3000 and mRNA in a 1:1 ratio. The dilution system is as follows: 1) 25 μl of Opti-MEM™ medium, Lipofectamine TM 3000 1.5 μl; 2) 50 μl of Opti-MEM™ medium, corresponding to 1 μg of mRNA. After incubation for 15 minutes, drop the mixture into the prepared well plate containing host cells. Incubate in a CO2 incubator for 6-24 hours, then replace the medium with complete medium and perform the corresponding assays.

[0126] Microscopic examination using an inverted fluorescence microscope: After 24 hours of culture, the cell culture plate was removed from the CO2 incubator and placed under the lens of an inverted fluorescence microscope. The corresponding channels and detection parameters were set. First, a low-power microscope was used to observe the fluorescence in the field of view. When the lens stayed in a field of view with uniform and strong cell fluorescence, a high-power microscope was used for observation and photography. The images were saved, named, stored, and recorded. The test results are shown in Figure 8.

[0127] Flow cytometry: Cells that passed the microscopic examination were digested with trypsin at room temperature for 1 minute. The digestion reaction was terminated with complete medium and pipetted into a 1.5 ml conical EP tube. The cells were centrifuged at 1000 g. The supernatant was removed and the cells were washed twice with PBS. The samples were analyzed by flow cytometry, with mean fluorescence intensity measured using the FITC channel. Blank cells (non-mRNA transfected) served as a negative control. The results were compared and the fluorescence intensity of the cells expressing different mRNAs was analyzed. The results are shown in Figure 9. The expression levels of the poly A mRNAs represented by SEQ ID No. 77 to SEQ ID No. 81 are shown in Figure 10.

[0128] It can be seen from the above examples that the multi-segmented PolyA polynucleotide provided by the present invention can be stably present during the passage of E. coli.

[0129] Comparison of stability between A30G70 and A30L70 after extreme amplification

[0130] (1) Transformation of plasmid containing target gene

[0131] The plasmids carrying A30G70 and A30L70 were re-transformed into Escherichia coli competent cells. The specific operation steps were as follows: dilute the synthetic gene with enzyme-free water to 1 ng / μl, take the Escherichia coli competent cells stbl2 out of the -80℃ refrigerator and place them on an ice-water mixture for 5 minutes. The competent cells were completely thawed. Sterile 1.5ml EP tubes were marked with corresponding labels, and 50μl of competent cells were added to each tube. Then, 1μl of plasmid was added in sequence according to the label on the tube. Gently pipette 5 times to mix the competent cells and plasmid. The operation process did not leave the ice-water mixture to ensure a low temperature environment, and the technique needed to be as gentle as possible to reduce the impact on the competent efficiency. After thoroughly mixing the competent cells and plasmid, incubate them on an ice-water mixture. After 30 minutes, heat shock the cells by incubating them in a 42°C water bath for 45 seconds. Immediately remove the cells and continue to incubate them on an ice-water mixture for 2 minutes. In a sterile operating table, add 950 μl of LB liquid medium to each tube. Place the EP tube in a 30°C shaking incubator and incubate for 1 hour. After the incubation period, remove the tube from the shaking incubator and pipette 100 μl of each tube for coating. Select kana resistance on a solid plate. After evenly coating, turn the plate upside down and incubate it in a 30°C incubator for 20 hours. Acceptable criteria are the presence of approximately 100-300 distinct single colonies on the plate, allowing for further identification.

[0132] (2) Fermentation stability verification

[0133] Remove the PCR tube rack and arrange the corresponding number of eight-tube strips. Pour LB medium into the pipette slot. Then, use the pipette to add 120μl of LB medium to the eight-tube strip. Use tweezers to pick up a single colony from the plate with a sterilized white pipette tip and place it into the eight-tube strip. Gently shake the PCR tube rack to thoroughly mix the bacteria on the pipette tip with the medium. Remove the white pipette tip and cap the eight-tube strip. Mark the cap with an alcohol-resistant marker. Place the tube strip in a 30℃ shaker and incubate for 2 hours. Record the mixture in a notebook (date, serial number, etc.). Remove from the shaker, centrifuge briefly on a handheld centrifuge, gently flick off bubbles with your fingers, centrifuge again for 15 seconds, and store at 4℃ for later use.

[0134] Colony PCR reaction: Prepare the following PCR reaction system, add the prepared reaction solution to a 96-well plate, and use a dispenser to add 1μl of bacterial solution. In addition, you need to add a set of reactions using the original gene plasmid as a template as a positive control, and a reaction using water as a template as a negative control. Amplify according to the PCR procedure:

[0135] In the present invention, the initial concentrations of primers F and R are 10 μmol / L; the concentration of the DNA template is 1 ng / μl. In the present invention, the sequence of primer F is as follows: 5'-gtaaaacgacggccagt-3' (SEQ ID No. 82); the sequence of primer R is as follows: 5'-caggaaacagctatgac-3' (SEQ ID No. 83). In the present invention, the PCR amplification procedure is preferably as follows: initial denaturation at 98°C for 10 minutes; 30 cycles of denaturation at 98°C for 30 seconds, annealing at 60°C for 15 seconds, and extension at 72°C for 30 seconds; and a final extension at 72°C for 5 minutes.

[0136] Agarose gel electrophoresis: first prepare 3% agarose gel (weigh 3g agarose and add it to 100ml TAE solution), add 0.5μl bromophenol blue to the eight-tube strip where the PCR reaction is completed, shake to mix, then spot the sample, take a picture of the electrophoresed agarose gel and save it. The recombination rate of the stable sequence is judged according to the agarose gel electrophoresis band pattern. When the size of the colony PCR band is equivalent to that of the positive control and there are no mixed bands, it is defined as no recombination. Otherwise, it is judged as recombination. Select the strain that has not undergone recombination and amplify it with a volume ratio of 1:1000. Cultivate it at 30℃ for 24h and define it as one generation. After the cultivation is completed, take 100μl and transfer it to 100ml fresh culture medium for continuous cultivation. Cultivate continuously for 20 generations and perform stability tests on the intermediate and final generations. The detection method is the same as above.

[0137] The test results are shown in Figure 11. Analysis shows that after extreme amplification, A30G70 shows obvious advantages. After 20 generations of extreme amplification fermentation, the recombination rate of A30G70 is less than 10, which is almost only one-third of A30L70. This indicates that after the process is scaled up to a certain extent, the quality of the mRNA product brought by A30G70 will be taken to a higher level.

[0138] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as within the scope of protection of the present invention.

Claims

1. A multi-segmented PolyA polynucleotide comprising two or more PolyA sequence segments, wherein adjacent PolyA sequence segments are connected by a linker, wherein: Each PolyA sequence segment consists of multiple consecutive A residues, and each linker consists of 1-24 nt nucleotide residues that are not all A residues.

2. The multi-segmented PolyA polynucleotide according to claim 1, which is selected from one or more polynucleotides having the following structures: PolyA sequence fragment 1-linker 1-PolyA sequence fragment 2; PolyA sequence fragment 1-linker 1-PolyA sequence fragment 2-linker 2-PolyA sequence fragment 3; PolyA sequence fragment 1-linker 1-PolyA sequence fragment 2-linker 2-PolyA sequence fragment 3-linker 3-PolyA sequence fragment 4.

3. The multi-segmented PolyA polynucleotide according to claim 1 or 2, wherein Each PolyA sequence fragment independently consists of 10-100 consecutive A's; Preferably, the PolyA sequence fragment 1 consists of 30-60 consecutive A's; Preferably, each PolyA sequence fragment is independently composed of 30, 40, 50, 60, 70, 80, 90 or 100 consecutive A's; Preferably, the total number of A in all PolyA sequence fragments in the multi-segmented PolyA polynucleotide is 30-300, preferably 60-240, more preferably 100-200.

4. The multi-segmented PolyA polynucleotide according to claim 1, wherein Each linker consists of 1-12 nt of nucleotide residues that are not all A; Preferably, each linker consists of G, or the content of G accounts for 20%-33% of the total bases in the linker sequence.

5. The multi-segmented PolyA polynucleotide according to any one of claims 1 to 3, wherein: Each linker is independently selected from the nucleotide sequence shown below: G； C； GGGGGG; ATGCAT; GCATATGACT; CAGTAATGAC; AGTCATATGC; GTCATTACTG; TGTCAGATAC; GCTCATATGC; GCATATATGC; CGCCATTAGAGG; ATGCATGATATC; TGCAACATCGAT; CCTCTAATGGCG; CAACCCCTGATTGTGTCCGCATCT; Preferably, in each multiplexed PolyA polynucleotide, the sum of the lengths of all linkers is 1-48 nt, more preferably 2-24 nt.

6. The multi-segmented PolyA polynucleotide according to any one of claims 1 to 5, comprising a sequence of SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4, SEQ ID NO.5, SEQ ID NO.6, SEQ ID NO.7, SEQ ID NO.8, SEQ ID NO.9, SEQ ID NO.10, SEQ ID NO.11, SEQ ID NO.12, SEQ ID NO.13, SEQ ID NO.14, SEQ ID NO.15, SEQ ID NO.16, SEQ ID NO.17, SEQ ID NO.18, SEQ ID NO.19, SEQ ID NO.20, SEQ ID NO.21, SEQ ID NO.22, SEQ ID NO.23, SEQ ID NO.24, SEQ ID NO.25, SEQ ID NO.26, SEQ ID NO.27, SEQ ID NO.28, SEQ ID NO.29, SEQ ID NO.30, SEQ ID NO.31, SEQ ID NO.32, SEQ ID NO.33, SEQ ID NO.34 One or more polynucleotides of any one of the sequences shown in SEQ ID NO.34, SEQ ID NO.35, SEQ ID NO.36, SEQ ID NO.37, SEQ ID NO.38, SEQ ID NO.39, SEQ ID NO.40, SEQ ID NO.41, SEQ ID NO.42, SEQ ID NO.43, SEQ ID NO.44, SEQ ID NO.45, SEQ ID NO.46, SEQ ID NO.47, SEQ ID NO.48, SEQ ID NO.49, SEQ ID NO.50, SEQ ID NO.51, SEQ ID NO.52, SEQ ID NO.77, SEQ ID NO.78, SEQ ID NO.79, SEQ ID NO.80, and SEQ ID NO.

81. A vector comprising the multi-segmented PolyA polynucleotide according to any one of claims 1 to 6.

8. A host cell comprising the multi-segmented PolyA polynucleotide according to any one of claims 1 to 6 or the vector according to claim 7.

9. Use of the multi-segmented PolyA polynucleotide according to any one of claims 1 to 6, the vector according to claim 7, and the host cell according to claim 8 in in vitro transcription to produce target mRNA; preferably, the target mRNA is used to produce an mRNA vaccine or drug.

10. An mRNA comprising a poly A tail formed by in vitro transcription of the multi-segmented Poly A polynucleotide according to any one of claims 1 to 6.

11. A method for identifying the stability of a multi-segmented PolyA polynucleotide in a host cell, the method comprising: 1) cloning the multi-segmented PolyA polynucleotide according to any one of claims 1 to 6 into a universal plasmid vector to obtain a plasmid vector into which the multi-segmented PolyA polynucleotide is inserted; 2) Transforming the plasmid vector containing the multi-segmented PolyA polynucleotide into a host cell, such as Escherichia coli, to obtain recombinant cells, plating the recombinant cells on a solid plate to obtain single colonies, picking the single colonies for preliminary PCR identification, and screening for stable colonies that have not undergone recombination; preferably, the PCR products are identified by 2%-3% agarose gel electrophoresis; 3) The stable bacteria that have not undergone recombination and were screened out in the preliminary identification were amplified for fermentation culture. The culture solution was gradient diluted and then spread on a solid plate. Colony PCR was performed to identify the stability of the passage.

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