Multi-interval poly (A) sequence and application thereof

Abstract

The invention relates to a multi-interval type poly (A) sequence, which is shown as SEQ ID No.14 or SEQ ID No.15. The invention also relates to a preparation method of the multi-interval type poly (A) sequence. The plasmid constructed by adopting the sequence is high in translation efficiency and strong in stability, stable amplification can be carried out without using special strains and conditions, the plasmid yield cannot be reduced, and meanwhile, the protein expression quantity of the plasmid is obviously superior to that of a poly (A) sequence in the prior art.

Description

Technical Field

[0001] The present invention relates to a multi-spaced poly(A) sequence and its application, belonging to the field of biotechnology. Background Art

[0002] For an mRNA molecule to be efficiently translated, it needs to have a 5'-cap structure and a 3'-poly(A) sequence. The 3'-poly(A) sequence can be added after in vitro transcription using poly(A) polymerase, or a poly(A) sequence can be directly added to the template for synchronous poly(A) tail addition during IVT; since the former process steps are relatively complex, the resulting poly(A) tail length distribution is very wide and there are obvious differences between different batches, so the industrial community adopts the latter, that is, directly adding a poly(A) sequence to the template for synchronous poly(A) tail addition during IVT. The IVT template is generally generated by plasmid linearization, and plasmids containing poly(A) often have the problem that poly(A) is very unstable in Escherichia coli and leads to deletion. For a poly(A) sequence with a length of 110 bp, a series of measures need to be taken to ensure that poly(A) is not deleted, while for poly(A) sequences longer than 120 bp, deletion is difficult to avoid. Early studies have shown that before the poly(A) length reaches 120 bp, the translation efficiency increases with the increase of the poly(A) length. To avoid the loss of poly(A), the commonly adopted solution is to add other bases. For example, the solution of BioNTech company is 30A + 10 bp linker + 70A, that is, inserting a 10 bp linker between the 30th A and the 31st A of the total 100 bp poly(A) (the same applies hereinafter); another example is that a literature (DOI: 10.1261 / rna.069286.118) reports and discloses solutions of 60A + linker + 60A, 40A + linker + 40A + linker + 40A.

[0003] However, the existing solutions have not simultaneously solved the problems of stability and translation efficiency. For example, the translation efficiency of the above-mentioned solution of BioNTech company is still lower than that of a 120 bp poly(A); moreover, these existing solutions still cannot maintain length stability for a long time under conventional culture conditions. Therefore, it is urgent to develop a poly(A) sequence with high translation efficiency and strong stability. Summary of the Invention

[0004] The main object of the present invention is: to overcome the problems existing in the prior art, and propose a multi-spaced poly(A) sequence, which can make the translation efficiency of the protein expression plasmid high and the stability strong; at the same time, the application of this sequence is proposed.

[0005] The technical solution for the present invention to solve its technical problems is as follows: Multi - spacer poly(A) sequence, with the sequence SEQ ID No.14: A 30-35 T 2-4 A 30-35 T 2-4 A 30-35 T 2-4 A 30-35 , or SEQ ID No.15: A 30-35 T 2-4 A 30-35 T 2-4 A 30-35 T 2-4 A 30-35 T 2-4 A 30-35 。

[0006] Preferably, the sequence of the multi - spacer poly(A) sequence is one of SEQ ID No.1, SEQ ID No.10, SEQ ID No.12, SEQ ID No.13.

[0007] The above - mentioned poly(A) sequence can make the translation efficiency of the protein expression plasmid high and the stability strong.

[0008] The present invention also proposes: A plasmid containing the multi - spacer poly(A) sequence described above.

[0009] Preferably, the plasmid is a protein expression plasmid.

[0010] Preferably, the backbone sequence of the plasmid is SEQ ID No.16, wherein the insertion site of the poly(A) sequence is between the 2784th base and the 2785th base of the backbone sequence.

[0011] The above - mentioned plasmid has high translation efficiency and strong stability, can be stably amplified without using special strains and conditions, and will not cause a decrease in plasmid yield.

[0012] The present invention also proposes: The application of the multi - spacer poly(A) sequence described above in constructing plasmids.

[0013] The application of the plasmid described above as a template plasmid DNA for expressing a target protein.

[0014] A method for preparing a target protein using the plasmid described above. A host cell containing the plasmid described above.

[0015] A method for preparing a target protein using the host cell described above.

[0016] Compared with the prior art, the multi-spacer poly(A) sequence of the present invention can significantly improve the translation efficiency of protein expression plasmids and endow them with good plasmid stability. The plasmid using the multi-spacer poly(A) sequence of the present invention does not require the use of special strains and conditions, and can be stably amplified even under standard molecular cloning conditions in DH5α or Turbo, without causing a decrease in plasmid yield. At the same time, its protein expression level is significantly better than that of the poly(A) sequence of the prior art. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 It is the map of the plasmid used in Example 1 of the present invention. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0018] The present invention will be further described in detail below in conjunction with embodiments. However, the present invention is not limited to the given examples. Embodiment

[0019] I. The basic process of this embodiment is as follows: According to the literature (DOI: 10.1038 / nsmb.3509), a 30bp poly(A) can be recognized by PABPC1 and effectively initiate translation. However, the research team of the inventors found in actual experiments that although the 30bp poly(A) sequence can translate to produce proteins, its translation efficiency is far lower than that of the 110bp poly(A) sequence. The 60bp+60bp or 30bp+70bp poly(A) sequences reported in the literature are not stable enough during passage, and still have a high deletion ratio under continuous passage at 37°C, and their translation efficiency has no advantage compared with the pure A poly(A) sequence. Considering these problems, the research team of the inventors further found through research that by setting multiple 30bp poly(A) sequences, the translation efficiency can be improved, and it can be significantly higher than that of the 110 consecutive A poly(A) sequence. At the same time, even a small number of base insertions can increase the stability of poly(A). On this basis, in this embodiment, a poly(A) sequence with high enough translation efficiency and stable passage in the plasmid is further obtained through systematic screening.

[0020] This embodiment constructs a sequence to replace the conventional A 80-120 sequence (i.e., poly(A) sequence), and compares it with the sequence of the BioNTech company's solution mentioned in the background art and the sequence with the best effect in the literature (DOI: 10.1038 / nsmb.3509) (other sequences with poor effects in the literature are not involved in the comparison).

[0021] The sequences constructed in this embodiment are SEQ ID No.1-3, specifically as follows: SEQ ID No.1: A 30 TTA 30 TTA 30 TTA 30 , denoted as SEQ1.

[0022] SEQ ID No.2: A 30 CCA 30 CCA 30 CCA 30 , denoted as SEQ2.

[0023] SEQ ID No.3: A 30 GGA 30 GGA 30 GGA 30 , denoted as SEQ3.

[0024] The comparison sequences are SEQ ID No.4 - 9, specifically as follows: SEQ ID No.4: A 30 GCATATGACTA 70 , denoted as SEQ4.

[0025] SEQ ID No.5: A 110 , denoted as SEQ5.

[0026] SEQ ID No.6: A 60 ATGCATA 60 , denoted as SEQ6.

[0027] SEQ ID No.7: A 60 TA 60 , denoted as SEQ7.

[0028] SEQ ID No.8: A 60 CA 60 , denoted as SEQ8.

[0029] SEQ ID No.9: A 60 GA 60 , denoted as SEQ9.

[0030] According to the results of the comparative experiments, it was found that SEQ1 and SEQ2 had the best expression effects, but SEQ2 had slightly worse stability. Therefore, in this example, the approximate sequences SEQ ID No.10 - 13 of SEQ1 were further compared, specifically as follows: SEQ ID No.10: A 30 TTTTA 30 TTTTA 30 TTTTA 30 , denoted as SEQ10.

[0031] SEQ ID No.11: A 30 TTA 30 TTA 30 , denoted as SEQ11.

[0032] SEQ ID No.12: A 30 TTA 30 TTA 30 TTA 30 TTA 30 , denoted as SEQ12.

[0033] SEQ ID No.13: A 35 TTA 35 TTA 35 TTA 35 , denoted as SEQ13.

[0034] It was found by comparison that, except for the relatively low expression level of SEQ11, the expression levels of other sequences were comparable.

[0035] Based on the above results, it can be known that: SEQ ID No.14 (denoted as SEQ14): A 30-35 T 2-4 A 30-35 T 2-4 A 30-35 T 2-4 A 30-35 , and SEQ ID No.15 (denoted as SEQ15): A 30-35 T 2-4 A 30-35 T 2-4 A 30-35 T 2-4 A 30-35 T 2-4 A 30-35 , can be used as a poly(A) sequence.

[0036] II. The specific experimental content and results of this example are as follows: In this example, the GFP-Fluc IVT template was used as the plasmid for poly(A) testing, and its map is as Figure 1 shown. The plasmid backbone sequence is SEQ ID No.16 (denoted as SEQ16), and among them, the insertion site of the poly(A) sequence is between the 2784th and 2785th bases of the backbone sequence.

[0037] (1) Comparison of the translation efficiency of different forms of poly(A) sequences The applicant synthesized the following plasmid vectors by himself: UTR2-GFP-Fluc-PA4T: The backbone is SEQ16 and the inserted sequence is SEQ1.

[0038] UTR2-GFP-Fluc-PA4C: The backbone is SEQ16 and the inserted sequence is SEQ2.

[0039] UTR2-GFP-Fluc-PA4G: The backbone is SEQ16 and the inserted sequence is SEQ3.

[0040] UTR2-GFP-Fluc-PA3070: The backbone is SEQ16 and the inserted sequence is SEQ4.

[0041] UTR2-GFP-Fluc-PA110: The backbone is SEQ16 and the inserted sequence is SEQ5.

[0042] UTR2-GFP-Fluc-PA60X2: The backbone is SEQ16 and the inserted sequence is SEQ6.

[0043] UTR2-GFP-Fluc-PA60T2: The backbone is SEQ16 and the inserted sequence is SEQ7.

[0044] UTR2-GFP-Fluc-PA60C2: The backbone is SEQ16 and the inserted sequence is SEQ8.

[0045] UTR2-GFP-Fluc-PA60G2: The backbone is SEQ16 and the inserted sequence is SEQ9.

[0046] Linearize using XbaI (NEB R0145) and operate according to the supplier's instructions.

[0047] The digested product was directly recovered using the Yeasen MolPure® PCR Product Purification Kit (19106ES70) and quantified using a NanoDrop micro-spectrophotometer.

[0048] Use the T7 in vitro transcription kit (AGCN), catalog number 10110N, from Jiangsu Shenji Biotechnology Co., Ltd.; the GAG CAP used is "m7G(5')ppp(5')(2'OMeA)pG 100mM Ammonium Solution", catalog number CAP3011. Carry out the in vitro transcription reaction system as shown in Table 1 at 37°C for 4 hours.

[0049] Table 1. In vitro transcription reaction system

[0050] After the reaction, DNase I digestion was performed to remove the template, and purification was carried out using Novoprotein RNA purification magnetic beads (N412) to obtain mRNA.

[0051] HEK293T cells were transfected with Thermo Lipofectamine™ MessengerMAX™ transfection reagent (LMRNA008). 200 ng of mRNA was transfected into each well of a 96-well plate, and three parallel replicates were performed for each type of mRNA. No mRNA was added to the negative control (NC).

[0052] After culturing for 24 hours, inspection was carried out using a firefly luciferase reporter gene detection kit (Beyotime RG006). Each well was lysed with 100 μL of reporter gene cell lysis buffer, and the lysate was diluted 10-fold with ultrapure water. 5 μL was taken for detection according to the supplier's instructions. The detection results are shown in Table 2.

[0053] Table 2. Detection Results

[0054] From the above results, it can be seen that the translation efficiency of SEQ1 and SEQ2 is better than that of other poly(A) sequences. Although SEQ1 is slightly lower than SEQ2, there is no significant difference between the two.

[0055] (2) Plasmid Stability Test All plasmids used in (1) were transfected into NEB Stable competent cells (NEB C3040) respectively, and cultured overnight on a kanamycin LB plate at 37°C. 40 clones were picked respectively and cultured overnight in a kanamycin LB liquid medium at 37°C. Sequencing was performed using primer SEQ ID No.17 (denoted as SEQ17): GATGTGCTGCAAGGCGATTA. The number of clones with a complete poly(A) sequence is shown in Table 3 below.

[0056] Table 3. Number of Clones with a Complete poly(A) Sequence

[0057] From the above results, it can be seen that the stability of UTR2-GFP-Fluc-PA4T is significantly higher than that of other plasmids, that is, the plasmid using SEQ1 has better stability.

[0058] (3) Plasmid Stability Test in Different Strains The competent strains used include: DH10B-T1 (12331013), DH5α-T1 (12297016), and Stbl3 (C737303) from Thermo, Turbo (C2984) from NEB, JM109 (200235), and XL1-Blue (200249) from Agilent.

[0059] UTR2-GFP-Fluc-PA4T was used to transform each competent strain, and the cells were cultured overnight on a kanamycin LB plate at 37°C. 40 clones were picked respectively, cultured overnight in a kanamycin LB liquid medium at 37°C with shaking, and sequenced using primer SEQ17. The number of clones with a complete poly(A) sequence is shown in Table 4 below.

[0060] Table 4. Number of clones with a complete poly(A) sequence

[0061] From the above results, it can be seen that when using UTR2-GFP-Fluc-PA4T, there is little difference between different strains, and all can maintain good integrity.

[0062] (4)Comparison of translation efficiency of other forms of T insertion sequences (SEQ1 approximate sequences) The steps of synthesizing mRNA and luciferase detection were the same as in (1). Using plasmid backbone SEQ16, sequences SEQ1, SEQ10, SEQ11, SEQ12, and SEQ13 were inserted respectively. The detection results are shown in Table 5.

[0063] Table 5. Detection results

[0064] From the above results, it can be seen that except for the low expression level of SEQ11, the expression levels of other sequences are comparable.

[0065] Based on the above embodiments: Plasmids with the spacer poly(A) sequence of the present invention do not require the use of special strains and conditions. Even under standard molecular cloning conditions in DH5α or Turbo, they can be stably amplified without causing a decrease in plasmid yield. At the same time, their protein expression level is significantly better than that of the poly(A) sequence with a length of 110 bp.

[0066] In addition to the above embodiments, the present invention may have other embodiments. All technical solutions formed by equivalent replacement or equivalent transformation fall within the protection scope required by the present invention.

Claims

1. A multi-spacer poly(A) sequence, characterized in that, The sequence is SEQ ID No.14: A 30-35 T 2-4 A 30-35 T 2-4 A 30- 35 T 2-4 A 30-35 , or SEQ ID No.15: A 30-35 T 2-4 A 30-35 T 2-4 A 30-35 T 2-4 A 30-35 T 2-4 A 30-35 。 2. The multi-spaced poly(A) sequence according to claim 1, characterized in that, The sequence is one of SEQ ID No.1, SEQ ID No.10, SEQ ID No.12, and SEQ ID No.

13.

3. A plasmid containing the multi-spacer poly(A) sequence described in claim 1 or 2.

4. The plasmid according to claim 3, characterized in that, The plasmid is a protein expression plasmid.

5. The plasmid according to claim 3, characterized in that, The backbone sequence of the plasmid is SEQ ID No.16, and the insertion site of the poly(A) sequence is between the 2784th base and the 2785th base of the backbone sequence.

6. Use of the multi-spacer poly(A) sequence described in claim 1 or 2 for constructing a plasmid.

7. Use of the plasmid described in any one of claims 3 to 5 as a template plasmid DNA for expressing a target protein.

8. A method for preparing a target protein using the plasmid described in any one of claims 3 to 5.

9. A host cell containing the plasmid described in any one of claims 3 to 5.

10. A method for preparing a target protein using the host cell described in claim 9.

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