Polyelement polyA polynucleotide for improving mRNA stability and protein expression and application thereof

Abstract

The invention provides a multielement polyA polynucleotide for improving mRNA (messenger ribonucleic acid) stability and protein expression and application of the multielement polyA polynucleotide. The invention firstly provides a polynucleotide, the polynucleotide comprises more than two sections of polyA sequence fragments, the adjacent polyA sequence fragments are connected through Linkers, each polyA sequence fragment is composed of one or more continuous A, and each Linker is composed of C and / or G. The polynucleotide provided by the invention can improve mRNA stability and protein expression, and compared with a traditional unit poly-A sequence, the polynucleotide provided by the invention can greatly reduce the recombination rate of fermentation in escherichia coli so as to more easily optimize and amplify the process, and can be better applied to plasmid fermentation related to transcription template preparation in mRNA vaccine or drug production.

Description

Technical Field

[0001] The present invention relates to a polynucleotide containing polyA and its application, and in particular to a polyvalent polyA polynucleotide capable of improving mRNA stability and protein expression and its application, belonging to the technical field of genetic engineering and cell biology. Background Art

[0002] While the spread of the novel coronavirus has brought disasters to the economic life of all mankind, it has also promoted the development of a series of mRNA vaccine or drug-related industries, mainly mRNA novel coronavirus vaccines. However, the preparation of mRNA into vaccines or drugs requires the use of in vitro transcription to prepare mRNA, and in vitro transcribed mRNA vaccines or drugs always face the problem of reduced efficacy due to the easy degradation of mRNA or low translation efficiency. For mature mRNA of eukaryotic organisms (histone replaces polyA sequence with stemloop structure), polyA is the basic functional element to maintain its stability and ensure translation. The degradation of mRNA in the body generally starts with the "shortening" of polyA. Under the synergistic action of multiple deadenylases, polyA of different lengths will also be shortened to a considerable extent, and then the polymerase will add a polyU tag, and then the mRNA will be decapped and tailed, and finally completely degraded by hydrolases. In this process, another important member, polyA binding protein (PABP), is involved. During the shortening of polyA, PABP can resist the action of some deadenylases, but this resistance is not absolute, because there is another protein in the body that specifically recognizes the polyA sequence protected by PABP and releases the binding of PABP, thereby achieving further shortening of polyA. In addition to participating in the degradation protection of mRNA, PABP also participates in the translation process of mRNA.

[0003] The translation process of eukaryotic mRNA is mediated by the cap structure. The cap recruits the eukaryotic translation initiation factor eIF4E and further recruits eIF4G and eIF4A to form a translation initiation complex. Among them, eIF4A mainly acts as a helicase, opening the 5' secondary structure of the mRNA before the start of translation to facilitate the ribosome scanning of the subsequent translation process, while eIF4G mainly plays a supporting role, maintaining the stability of the overall structure of the complex. At the same time, it can also bind to PABP, and at this time PABP binds to polyA, so that the mRNA to be translated forms a closed loop structure, forming information exchange between the head and the tail, thereby helping to further improve the translation efficiency.

[0004] It can be seen that polyA plays a very important role in the entire life cycle of mRNA and the execution of physiological functions. In the preparation of mRNA products, the addition of polyA is an important aspect. Similar to in vitro transcription, the tailing mechanism in vivo is taken out for in vitro use. Enzymatic tailing seems to be the easiest to think of, but the tail length added by enzymatic tailing can only be ensured within a range, which brings certain challenges to the homogenization of product quality and quality inspection. Co-transcriptional tailing is a solution proposed for some defects of enzymatic tailing. Based on different scales, co-transcriptional tailing is divided into PCR method and plasmid method. The PCR method is to use primers to bring polyA, and add polyA sequence to the transcription template through PCR, but it is limited by the PCR process and cannot be amplified and is only used for laboratory scientific research level. The plasmid method is to construct the polyA sequence onto a vector with a transcription template, and obtain a large number of transcription templates efficiently and at low cost by fermenting the plasmid in Escherichia coli. This method is easy to amplify, but the plasmid containing the polyA sequence is prone to recombination and loss during the fermentation process in Escherichia coli.

[0005] At this stage, more technical solutions are still needed to solve problems such as mRNA products being easily degraded, having low translation efficiency and being difficult to scale up through processes. Summary of the invention

[0006] One object of the present invention is to provide a polynucleotide having a polyA sequence.

[0007] Another object of the present invention is to provide related applications of the polynucleotide.

[0008] The present invention mainly inserts non-A bases on the basis of the traditional polyA sequence, so that the polyA sequence is multi-segmented, and a polynucleotide with a multi-segmented polyA sequence is obtained (hereinafter referred to as multi-segmented polyA polynucleotide, or poly-polyA polynucleotide). The present invention transfers the plasmid with the multi-segmented polyA polynucleotide into the host cell, which can improve the stability of mRNA and protein expression, and compared with the traditional unit poly A sequence, it can greatly reduce its recombination rate in Escherichia coli fermentation to make it easier to optimize the amplification process, and can be better applied to the plasmid fermentation involved in the preparation of transcription templates in the production of mRNA vaccines or drugs, and can reduce or avoid the loss of polyA sequences during plasmid fermentation, thereby ensuring the efficacy of mRNA vaccines or drugs.

[0009] Specifically, on the one hand, the present invention provides a polynucleotide comprising two or more polyA sequence fragments, adjacent polyA sequence fragments being connected by linkers, wherein each polyA sequence fragment is composed of one or more consecutive A's, and each linker is composed of C and / or G.

[0010] According to a specific embodiment of the present invention, the number of linkers in the polynucleotide of the present invention is ≥2.

[0011] According to a specific embodiment of the present invention, the nucleotide sequence of the polynucleotide of the present invention is as follows:

[0012] A1-L1-A2-L2-A3-L3-……-L m -A n

[0013] Among them, A1, A2, A3, ... A n Indicates polyA sequence fragment, A1 to A n-1 Each polyA sequence fragment (i.e., along the 5'→3' direction, the first n-1 polyA sequence fragments, that is, the remaining polyA sequence fragments except the last polyA sequence fragment closest to the 3' end) independently consists of 2-120 nt, preferably 8-80 nt of A, A n (i.e., along the 5'→3' direction, the last polyA sequence fragment, that is, the polyA sequence fragment closest to the 3' end) is composed of 1-120 nt, preferably 1-80 nt of A;

[0014] L1, L2, L3, ... L m represents a linker sequence fragment, each linker sequence fragment independently consists of 1-24 nt, preferably 1-20 nt, of C and / or G;

[0015] n represents the number of polyA sequence fragments, n≥2; m represents the number of linker sequence fragments, m≥2; and n=m or n=m+1.

[0016] According to a specific embodiment of the present invention, the total length of the polynucleotide of the present invention is 42-1000 nt, preferably 62-500 nt, and more preferably 101-248 nt.

[0017] In some specific embodiments of the present invention, the number of linkers of the polynucleotide of the present invention is ≥ 2, and at least one of the 20 nucleotides at the 3' end is C. That is, at least one linker among the 20 nucleotides at the 3' end is C. The remaining linkers may be G or C.

[0018] In some specific embodiments of the present invention, in the polynucleotide of the present invention, the linker in the 20 nucleotides at the 3' terminal end is composed of at least 1 C, and the remaining linkers are all 1 G.

[0019] In some specific embodiments of the present invention, the second position from the 3' end of the polynucleotide of the present invention is C.

[0020] According to a specific embodiment of the present invention, the polynucleotide of the present invention comprises 2-20 segments (i.e., the aforementioned n is 2-20) of polyA sequence fragments, preferably comprises 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 polyA sequence fragments.

[0021] According to a specific embodiment of the present invention, the total number of A in all polyA sequence fragments in the polynucleotide of the present invention is 30-800, preferably 30-300, more preferably 60-240, and more preferably 100-200. According to a specific embodiment of the present invention, except for the last polyA sequence fragment (i.e., A at the 3' end of the polynucleotide of the present invention), n ) are each independently composed of 2-120, such as 2, 5, 8, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110 or 120 consecutive A sequences, A n Consists of 1-120, for example 1, 2, 3, 5, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110 or 120 consecutive A's.

[0022] According to a specific embodiment of the present invention, in the polynucleotide of the present invention, the total number of bases of all Linker sequence fragments is 1-200, preferably 2-100, further preferably 2-48, and more preferably 2-24.

[0023] According to a specific embodiment of the present invention, the polynucleotide of the present invention, wherein each Linker sequence fragment is independently selected from: 1-18 preferably 1-12 Gs; 1-18 preferably 1-12 Cs; a nucleotide sequence composed of 2-24nt C / G and wherein the G content is at least 20%-33% of the total bases in the Linker sequence fragment.

[0024] According to a specific embodiment of the present invention, the polynucleotide of the present invention includes one or more polynucleotides whose sequences are any one of the sequences shown in 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, and SEQ ID NO.20.

[0025] On the other hand, the present invention also provides a vector, which comprises the multi-segmented polyA polynucleotide of the present invention. The multi-segmented polyA polynucleotide of the present invention can be inserted into a plasmid vector by any feasible method in the art, so as to construct the vector of the present invention into which the multi-segmented polyA polynucleotide is inserted. During the specific construction, an enzyme cleavage site can be added to the end of the multi-segmented polyA polynucleotide as needed.

[0026] On the other hand, the present invention also provides a host cell, which comprises the multiple-segmented polyA polynucleotide of the present invention or the vector of the present invention (a vector into which the multiple-segmented polyA polynucleotide is inserted).

[0027] On the other hand, the present invention also 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 has a poly A tail formed by transcription of the multi-segmented polyA polynucleotide of the present invention. 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, and can reduce the loss of polyA sequences during plasmid fermentation, thereby ensuring the efficacy of mRNA vaccines or drugs.

[0028] Therefore, the present invention also provides an mRNA, which comprises 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.

[0029] According to a specific embodiment of the present invention, the target mRNA is used to produce mRNA vaccines or drugs.

[0030] Specific experiments of the present invention show that the multi-segmented polyA polynucleotide provided by the present invention can improve mRNA stability and protein expression, and compared with the traditional unit poly A sequence, it can greatly reduce its recombination rate in Escherichia coli fermentation to make it easier to optimize the amplification process, and can be better applied to the plasmid fermentation involved in the preparation of transcription templates in the production of mRNA vaccines or drugs. It can reduce or avoid the loss of polyA sequences during plasmid fermentation, thereby ensuring the efficacy of mRNA vaccines or drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 Schematic diagram of the design principle of the multi-segmented polyA polynucleotide of the present invention.

[0032] Figure 2 The plasmid map of the vector plasmid pCZ and recombinant polyA used in the specific embodiment of the present invention.

[0033] Figure 3 The stability test results of the fragmented polyA polynucleotide after fermentation in Escherichia coli are shown.

[0034] Figure 4 The results of 24h expression of polynucleotides encoding GFP with different poly A fragments in cells are shown. The numbers in the figure are the SEQ ID NO. numbers of the sequences used in each experimental group.

[0035] Figure 5 The flow cytometry results of each group of cells are displayed, and the quantified fluorescence intensity is visualized. The numbers on the horizontal axis in the figure are the SEQ ID NO. numbers of the sequences used in each experimental group.

[0036] Figure 6 The stability test of mRNA encoding GFP by different polyA polynucleotides in cells is shown. The numbers on the horizontal axis in the figure are the SEQ ID NO. numbers of the sequences used in each experimental group. DETAILED DESCRIPTION

[0037] In order to have a clearer understanding of the technical features, purposes and beneficial effects of the present invention, the technical solution of the present invention is now described in detail below, but it should not be understood as limiting the scope of the present invention. All the raw reagents and materials used in the following experiments can be obtained commercially, and the experimental methods without specific conditions are conventional methods and conventional conditions well known in the art, or according to the conditions recommended by the instrument manufacturer.

[0038] Unless specifically defined otherwise, all technical and scientific terms used herein have the same meanings as commonly understood by one of ordinary skill in the relevant art.

[0039] Example 1

[0040] The present invention provides a multi-segment polyA polynucleotide, the structure design of which is shown in Figure 1 As shown, the main design principles include:

[0041] S=A1+L1+A2+L2+A3+L3+……+L m +A n

[0042] Where A represents one or more consecutive adenosines, and the base number ranges from 1 to 80 nt;

[0043] L represents a linker composed of C or G, and the base number of L ranges from 1 to 20 nt;

[0044] m represents the number of times the L module appears in the sequence, and the range of m is ≥2;

[0045] n represents the number of times module A appears in the sequence, the range of n is ≥2, and n=m or n=m+1;

[0046] S represents the sum of sequence lengths, and the range of S is 42-1000 nt.

[0047] Specifically, the present invention provides a segmented polyA polynucleotide as shown in SEQ ID No. 1 to SEQ ID No. 20 in Table 1.

[0048] Table 1

[0049]

[0050]

[0051] The above sequence was subjected to gene synthesis and constructed into the EcoRV I site on the universal vector pUC-57kana. During synthesis, the following additional sequences were added to the two flanks for subsequent construction. The 5' flanking sequence added was: 5'-CTTTGAATAAAGCCTGAGTAGGAAG-3' (SEQ ID No.23), and the 3' flanking sequence added was: 5'-AGAAGAGCTGAGGGTC TAGAACTAGTGTCG-3' (SEQ ID No.24). After the gene synthesis was completed and verified by sequencing, the target sequence was constructed into the plasmid encoding d2eGFP to form a coherent sequence T7promoter-5'UTR-CDS (d2eGFP)-3'UTR-polyA (multi-polyA design). The construction operation is as follows:

[0052] (1) Design and construction of relevant primers

[0053] F1 (SEQ ID No. 23): 5'-CTTTGAATAAAGCCTGAGTAGGAAG-3';

[0054] R1 (SEQ ID No. 25): 5'-CGACACTAGTTCTAGACCCTCA-3';

[0055] F2 (SEQ ID No. 26): 5'-TGAGGGTCTAGAACTAGTGTCG-3';

[0056] R2 (SEQ ID No. 27): 5'-CTTCCTACTCAGGCTTTATTCAAAG-3'

[0057] (2) Obtaining the target fragment and linearizing the vector

[0058] The acquisition of the target fragment and the linearization of the vector are mainly achieved through PCR. The PCR system is as follows (taking 20 μl as an example):

[0059]

[0060] When obtaining the target fragment, primer F is the primer F1, primer R is the primer R1, and Template is the synthetic gene; when linearizing the vector, primer F is the primer F2, primer R is the primer R2, and Template is a vector plasmid with a coding frame of d2eGFP but without polyA, named pCZ, and the plasmid map is as follows: Figure 2 After the PCR system is prepared, it is placed in a PCR instrument for PCR reaction. The reaction system is: pre-denaturation at 98°C for 3 min; denaturation at 98°C for 30 s, annealing at 56°C for 15 s, extension at 72°C for 45 s, 30 cycles; and final extension at 72°C for 5 min.

[0061] (3) Digestion of reaction system template

[0062] The plasmid template in the reaction system has a great interference on the subsequent transformation, so the above reaction product needs to be digested with DPNⅠ. The plasmid in E. coli is methylated, and DPNⅠ can specifically digest methylated DNA, so the plasmid template in the PCR reaction system can be removed without affecting the amount of PCR product. The specific operation is to configure the digestion system: 17μl of PCR reaction product, 2μl of buffer, and 1μl of DPNⅠ. After the configuration is completed, place it in the PCR instrument and set it to 37℃ constant temperature reaction for 30min.

[0063] (4) Transformation and sequencing

[0064] The above reaction products including the target fragment and the linearized vector are mixed, and then treated with T5 exonuclease to achieve the purpose of in vitro recombination and then transferred into E. coli for screening and replication. The specific operation is to configure the transformation system: 2μl of target fragment, 1μl of linearized vector, 0.1μl of T5 exonuclease, 0.5μl of buffer, and 0.4μl of H2O. After the system is prepared, incubate it on an ice-water mixture for 5 minutes, add 50 μl of competent E. coli, continue to incubate it on an ice-water mixture for 30 minutes, then perform heat shock, place the mixture in a 42°C water bath for 45 seconds, take it out and return it to the ice-water mixture for incubation for 2 minutes, add 950 μl of liquid LB medium, place it in a 30°C shaker for incubation for 1 hour, take it out and centrifuge it at 6000g for 1 minute, remove 800 μl of the supernatant, resuspend the bacteria in the remaining liquid medium and spread it on a kana-resistant solid plate, turn it upside down in a 30°C incubator for incubation for 20 hours, and then pick 5 homogeneous monoclonal clones for sequencing verification after the normal-sized monoclonal clones grow.

[0065] Example 2: DNA sequence stability verification of multi-segmented polyA polynucleotides

[0066] (1) Transformation of plasmid containing target gene

[0067] Each of the above-mentioned partial synthetic genes SEQ ID No.1-SEQ ID No.22 was re-transformed into the competent E. coli cells. The specific operation steps were as follows: the synthetic gene was diluted with enzyme-free water to 1 ng / μl, the competent E. coli cells stbl2 were taken out of the -80°C refrigerator and placed on an ice-water mixture for standing. After 5 minutes, the competent cells were completely thawed, a sterile 1.5 ml EP tube was taken and marked accordingly, and 50 μl of competent cells were added to each tube, and then 1 μl of plasmid was added in sequence according to the mark on the tube, and the competent cells and plasmid were mixed by gentle pipetting 5 times. 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 efficiency of the competent cells. After the competent cells and plasmids are fully mixed, they are placed on an ice-water mixture for incubation. After 30 minutes, heat shock is performed, that is, they are placed in a 42°C water bath for incubation for 45 seconds, and then immediately taken out and placed on an ice-water mixture for 2 minutes. Then, 950 μl of LB liquid culture medium is added to each tube in a sterile operating table, and the EP tube is placed in a 30°C shaking incubator for incubation for 1 hour. After the incubation is completed, it is taken out from the shaking incubator, and 100 μl is taken from each tube with a pipette for coating. The solid plate resistance selects kana resistance. After evenly coating, turn it upside down in a 30°C incubator for incubation for 20 hours. The acceptable standard is that a single colony grows on the plate and the number is about 100-300, and the next step of identification can be carried out.

[0068] (2) Verification of the fermentation stability of polyA DNA sequences

[0069] Take out the PCR tube rack, arrange the corresponding number of eight-tube strips, pour the LB medium into the gun slot, then use the gun to add 120μl LB medium to the eight-tube strips, use tweezers to pick up the single colony on the plate with the sterilized small white gun tip and put it into the eight-tube strips, gently shake the PCR tube rack to make the bacteria on the gun tip fully mixed in the medium, take out the small white gun tip, cover the eight-tube strips at the same time and mark the tube cover with an alcohol-resistant marker, place it in a 30℃ shaker to culture and mix for 2h, and write relevant records in the notebook (date, number, etc.). After taking it out of the shaker, spin it in a handheld centrifuge, gently flick off the bubbles with your fingers, spin it again for 15s, and store it at 4℃ for later use.

[0070] Colony PCR reaction:

[0071] Prepare the following PCR reaction system, add the prepared reaction solution to a 96-well plate, and use a spray gun to add 1μl of bacterial solution. In addition, you need to add a set of reactions with the original gene plasmid as a template as a positive control, and a reaction with water as a template as a negative control. Amplify according to the PCR program:

[0072]

[0073] In the present invention, the initial concentrations of the 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 the primer F is as follows: 5'-gtaaaacgacggccagt-3' (SEQ ID No. 28); the sequence of the primer R is as follows: 5'-caggaaacagctatgac-3' (SEQ ID No. 29). In the present invention, the amplification procedure of the PCR is preferably as follows: pre-denaturation at 98°C for 10 min; denaturation at 98°C for 30 s, annealing at 60°C for 15 s, extension at 72°C for 30 s, 30 cycles; final extension at 72°C for 5 min.

[0074] Agarose gel electrophoresis:

[0075] 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 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 diagram. 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 at a volume ratio of 1:1000. After amplification, perform another verification to obtain the final recombination rate after amplification. The specific operation process is as follows:

[0076] 100 μl of the bacterial suspension corresponding to the non-recombinant strain obtained in the above test was selected and pipetted into a triangular flask containing 100 ml of sterilized liquid LB medium (with kanamycin added) and placed in a 30°C metal shaking incubator for shaking culture for about 30 hours until the bacteria in the bottle were completely turbid. 1 μl of the bacterial suspension was taken out and diluted to 1 ml of liquid LB medium with kanamycin for full mixing, and then 2 μl of the mixed bacterial suspension was taken and mixed in a new tube of 1 ml of liquid LB medium with kanamycin, and three gradient volumes of 50 μl, 100 μl, and 200 μl were respectively applied on a kana-resistant solid plate, and then inverted in a 30°C incubator for static culture for 20 hours, and a plate with obvious single colonies and a number of about 100-300 was selected for secondary identification.

[0077] Take out the PCR tube rack, arrange the corresponding number of eight-tube strips, pour the LB medium into the gun slot, then use the gun to add 120μl LB medium to the eight-tube strips, use tweezers to pick up the single colony on the plate with the sterilized small white gun tip and put it into the eight-tube strips, gently shake the PCR tube rack to make the bacteria on the gun tip fully mixed in the medium, take out the small white gun tip, cover the eight-tube strips at the same time and mark the tube cover with an alcohol-resistant marker, place it in a 30℃ shaker to culture and mix for 2h, and write relevant records in the notebook (date, number, etc.). After taking it out of the shaker, spin it in a handheld centrifuge, gently flick off the bubbles with your fingers, spin it again for 15s, and store it at 4℃ for later use.

[0078] Colony PCR reaction:

[0079] Prepare the following PCR reaction system, add the prepared reaction solution to a 96-well plate, and use a spray gun to add 1μl of bacterial solution. In addition, you need to add a set of reactions with the original gene plasmid as a template as a positive control, and a reaction with water as a template as a negative control. Amplify according to the PCR program:

[0080]

[0081] In the present invention, the initial concentrations of the 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 the primer F is as follows: 5'-gtaaaacgacggccagt-3' (SEQ ID No. 28); the sequence of the primer R is as follows: 5'-caggaaacagctatgac-3' (SEQ ID No. 29). In the present invention, the amplification procedure of the PCR is preferably as follows: pre-denaturation at 98°C for 10 min; denaturation at 98°C for 30 s, annealing at 60°C for 15 s, extension at 72°C for 30 s, 30 cycles; final extension at 72°C for 5 min.

[0082] Agarose gel electrophoresis:

[0083] First, prepare 3% agarose gel (weigh 3g agarose and add it to 100ml TAE solution), add 0.5μl bromophenol blue to the eight-tube tube that completes the PCR reaction, shake and mix, then spot the sample, take pictures of the electrophoresed agarose gel and save it. Based on the results of agarose gel electrophoresis, judge and record the recombination rate after the final sequence amplification fermentation according to the above-mentioned method and principle for analyzing the recombination rate, take part of the bacterial solution for plasmid extraction and sequencing verification, and extract the plasmid using the OMEGA PlasmidMini Kit I kit. The experimental operation process can refer to the instructions of the kit.

[0084] The stability test results of the multi-segmented polyA polynucleotide after fermentation in Escherichia coli are shown in Figure 3 . The results of plasmid sequencing were consistent with those of gel analysis.

[0085] Example 3 Synthesis of mRNA with multiple polyA polynucleotide fragments

[0086] (1) Linearization of plasmid template

[0087] The plasmid constructed successfully above was linearized, and the plasmid was digested with the ⅡS restriction endonuclease BspQⅠ, which can disconnect the plasmid from the end of polyA to prevent the inability to terminate transcription during the transcription process and cause the transcription of non-designed sequences. The enzyme digestion system is recombinant plasmid (8μg), 10×Buffer (2μl), BspQⅠ (9.6U, 0.96μl), and water is added to 20μl. After the enzyme digestion system is prepared, it is placed in a 50℃ metal bath for reaction for 12h, and then detected by agarose gel electrophoresis. The undigested plasmid is used as a control. After enzyme digestion, the single size of the band meets the design expectation and is significantly larger than the undigested supercoiled plasmid. The identified enzyme digestion product is purified using the purification kit omega Cycle Pure Kit. The purification operation can refer to the kit manual.

[0088] (2) Preparation of mRNA by in vitro transcription

[0089] The purified linearized plasmid was used as a transcription template to prepare mRNA with different polyA designs by in vitro transcription. The in vitro transcription was carried out at a constant temperature of 37°C. The following system was prepared in sequence, with the order of adding from top to bottom:

[0090]

[0091] (3) Digestion and purification of DNA template in in vitro transcription reaction system

[0092] After the mRNA synthesis reaction is completed, add 10 μl of DNAseⅠ to each tube, slowly pipette 10 times with the tip of the pipette to ensure full mixing, and place it in a 37℃ metal bath for 45 minutes. After the reaction is completed, use the purification kit MEGAclear TM Kit for purification. The purification operation can refer to the instructions of the kit. After purification, the concentration was measured using NanoDropone. The acceptance standard was a concentration greater than or equal to 300ng / μl. According to the measured concentration, 2μl of the purified product was diluted to 100ng / μl, and then mixed with an equal volume of 2× loading buffer. After heating at 75℃ for 5min, centrifuge for 3s on a handheld centrifuge, and 4μl was taken for agarose gel electrophoresis. The acceptance standard was that the band size was clear, single and correct, with no obvious mixed bands or dragging.

[0093] Example 4: Cell experiment detection

[0094] Cell transfection

[0095] The host cells used for transfection are 293T. The cells are inoculated into a 24-well plate in advance. 24 hours after inoculation, the cell status in the 24-well plate is observed, and the confluence is about 90%. In a biosafety cabinet, prepare the required volume of 90% DMEM + 10% FBS medium. Discard the medium in the well plate 30 minutes before transfection, and add 0.5 ml of fresh medium (90% DMEM + 10% FBS) to each well.

[0096] Prepare the transfection system: take 25μl of opti-MEM, add 1μg of mRNA sample or control GFP-120A, gently blow and mix with a pipette tip, then take 25μl of opti-MEM and 1.5μl of transfection reagent LipofectamineTM3000 and mix them evenly, take 25μl of the diluted mRNA and diluted transfection reagent in equal volumes and mix them thoroughly, incubate at appropriate temperature for 15min, then drop the mixture into the pre-prepared well plate containing host cells, and culture in a carbon dioxide incubator for 24h or 48h before conducting corresponding tests.

[0097] Inverted fluorescence microscopy

[0098] The reporter gene encoded by the transfected mRNA is d2eGFP (with a half-life of only 2h, the expressed GFP will not accumulate over time, and the observed fluorescence is more accurate), so the expression of mRNA in cells can be determined by microscopic examination under an inverted fluorescence microscope. The specific operation is to culture the well plate cells transfected with the target mRNA for the target time, take them out of the carbon dioxide incubator, place them under the objective lens of the inverted fluorescence microscope, observe through the eyepiece, select the appropriate magnification and the appropriate field of view, adjust the focus knob until the field of view is clear, turn on the computer connected to the inverted fluorescence microscope and the control software, take a picture of the field of view corresponding to the name of the mRNA sample in the observed well and name it accordingly.

[0099] The results of 24h expression of GFP encoded by polyA with different fragments in cells are shown in Figure 4 .

[0100] Each group of cells was subjected to flow cytometry, and the quantified fluorescence intensity was visualized. The results are shown in Figure 5 shown.

[0101] RT-qPCR detection of mRNA stability

[0102] The total RNA in the cells cultured at different times was extracted using the Total RNA Extraction Kit. The operation steps can refer to the instructions of the kit. The extracted total RNA was reverse transcribed. The reverse transcription can be performed using a reverse transcription kit (TAKARA / RR037A). The specific operation steps are to take out PCR tubes corresponding to the number of samples, write the corresponding labels, and prepare the reverse transcription reaction systems in Table 2 respectively:

[0103] Table 2

[0104] Components Usage 5*PrimeScript Buffer 2μl PrimeScript RT Enzyme Mix I 0.5μl Oligo dT Primer 0.5μl Primer-F 0.5μl mRNA template 500ng mRNA <![CDATA[RNase Free dH2O]]> Make up to 10 μl

[0105] Oligo dT Primer is the primer that comes with the kit, and Primer-F is a specific sequence synthetic primer with the sequence information of 5'-CAAGGGCGAGGAGCTG-3' (SEQ ID No. 30). mRNA template is the different total RNA extracted above. After preparing the above reaction system, mix well, and then perform reverse transcription according to the procedure in Table 3:

[0106] Table 3

[0107] step Temperature(℃) time 1 37 15min 2 85 5s 3 4 save

[0108] After reverse transcription, remove all the PCR tubes from the PCR instrument, add 140 μl of enzyme-free water and mix gently, and then proceed to the next step or freeze in a -20°C refrigerator for future use. TM Fast Advanced Master Mix (2X), the preparation system is as shown in Table 4:

[0109] Table 4

[0110] Components Usage Nuclease-Free Water 8.8μl Upstream primer (10 μM) 0.4μl Downstream primer (10 μM) 0.4μl <![CDATA[TaqMan TM Fast Advanced Master Mix(2X)]]> 10μl probe 0.4μL

[0111] The sequence of the upstream primer is 5'-CTCGTGACCACCCTGAC-3' (SEQ ID No. 31), the sequence of the downstream primer is 5'-GGTCTTGTAGTTGCCGTCG-3' (SEQ ID No. 32), and the probe sequence is 5'-CGACCACATGAAGCAGCACGACTTC-3' (SEQ ID No. 33); after the above reaction system is prepared, it is placed in a qPCR instrument (ABI 12K) for reaction, and the reaction program is shown in Table 5:

[0112] Table 5

[0113]

[0114] After the reaction is completed, data analysis is performed. Before the analysis, a control sample of known concentration is used as a standard, and different concentration gradient dilutions are performed to establish a standard curve. In this embodiment, the GFP-120A plasmid is used as a standard for concentration gradient dilution, and the gradient dilution is 0.04ng / μL~0.008ng / μL~0.0016ng / μL~0.00032ng / μL~0.000064ng / μL~0.0000128ng / μL~0.0000064ng / μL~0.00000256ng / μL~0.000000512ng / μL~0.0000001024ng / μL for the above reaction detection, and a standard curve relationship between the CT value and the DNA concentration is established. With this standard curve, the content of the target cDNA in different samples can be detected, calculated and analyzed, that is, the content of the original mRNA can be compared. In this way, the target mRNA content of the above samples and controls in cells cultured for 24h and 48h was obtained, and the content of each sample at 24h was normalized to that at 48h to obtain the difference in the content of each sample mRNA at 48h relative to that at 24h.

[0115] Stability test of mRNA encoding GFP with different polyA fragments in cells Figure 6 .

[0116] It can be seen from the above embodiments that the multi-segmented polyA provided by the present invention can improve the stability of mRNA with the sequence design in cells, and does not affect the normal translation of mRNA, thereby greatly improving the expression time of mRNA and the protein expression amount per unit time, and can improve the stability of the DNA corresponding to the designed sequence cloned into the plasmid in Escherichia coli fermentation, and has the advantage of being easier to industrially scale up when used for mRNA products.

[0117] 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 principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A polynucleotide comprising two or more polyA sequence fragments, wherein adjacent polyA sequence fragments are connected by a linker, wherein: Each polyA sequence segment consists of one or more consecutive A's, and each linker consists of C and / or G.

2. The polynucleotide according to claim 1, wherein the number of linkers is ≥ 2; Preferably, the nucleotide sequence of the polynucleotide is as follows: <h2 style=";text-align:left;direction:ltr">A1-L1-A2-L2-A3-L3-……-L<h2 style=";text-align:left;direction:ltr"> m <h2 style=";text-align:left;direction:ltr"> -A<h2 style=";text-align:left;direction:ltr"> n in, A1、A2、A3、……A n Indicates polyA sequence fragment, A1 to A n-1 Each polyA sequence fragment is independently composed of 2-120 nt, preferably 8-80 nt of A. n It is composed of 1-120 nt, preferably 1-80 nt of A; L1, L2, L3, ... L m represents a linker sequence fragment, each linker sequence fragment independently consists of 1-24 nt, preferably 1-20 nt, of C and / or G; n represents the number of polyA sequence fragments, n≥2; m represents the number of linker sequence fragments, m≥2; and n=m or n=m+1.

3. The polynucleotide according to claim 1 or 2, which has a total length of 42-1000 nt, preferably 62-500 nt, more preferably 101-248 nt; Preferably, at least one of the 20 nucleotides at the 3' terminal of the polynucleotide is C.

4. The polynucleotide according to any one of claims 1 to 3, comprising 2-20 polyA sequence segments, preferably comprising 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 polyA sequence segments; Preferably, the total number of A in all polyA sequence fragments in the polynucleotide is 30-800, preferably 30-300, more preferably 60-240, and more preferably 100-200.

5. The polynucleotide according to any one of claims 1 to 4, wherein the total number of bases in all linker sequence fragments is 1-200, preferably 2-100, more preferably 2-48, and more preferably 2-24.

6. The polynucleotide according to any one of claims 1 to 5, wherein Each Linker sequence fragment is independently selected from: 1-18 preferably 1-12 G; 1-18 preferably 1-12 C; A nucleotide sequence consisting of 2-24 nt C / G, wherein the G content is at least 20%-33% of the total bases in the Linker sequence fragment.

7. The polynucleotide according to claim 1, comprising one or more polynucleotides having a sequence of any one of the sequences shown in 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, and SEQ ID NO.

20. A vector comprising the polynucleotide according to any one of claims 1 to 7.

9. A host cell comprising the polynucleotide according to any one of claims 1 to 7 or the vector according to claim 8.

10. Use of the polynucleotide according to any one of claims 1 to 7, the vector according to claim 8, and the host cell according to claim 9 in in vitro transcription to produce target mRNA; preferably, the target mRNA is used to produce mRNA vaccines or drugs.

11. An mRNA comprising a poly A tail formed by in vitro transcription of the polynucleotide according to any one of claims 1 to 7.

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