Multi-spaced poly(A) sequences and their applications

Abstract

The present invention relates to a multi-spaced poly(A) sequence, the sequence of which is SEQ ID No. 14 or SEQ ID No. 15. Plasmids constructed using this sequence have high translation efficiency and strong stability, can be stably amplified without the use of special strains and conditions, and do not result in reduced plasmid yield. At the same time, the protein expression level is significantly better than that of poly(A) sequences in the prior art.

Description

Technical Field

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

[0002] For efficient translation, an mRNA molecule requires 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 by adding the poly(A) sequence directly to the template for simultaneous IVT tailing. Because the former involves a relatively complex process and the resulting poly(A) tail has a wide distribution in length and significant batch-to-batch variability, the industry generally adopts the latter approach, which involves adding the poly(A) sequence directly to the template for simultaneous IVT tailing. IVT templates are typically generated using plasmid linearization. However, plasmids containing poly(A) sequences often suffer from poly(A) instability in E. coli, leading to deletions. For a poly(A) sequence of 110 bp, a series of measures are required to prevent poly(A) deletions, while deletions are unavoidable for poly(A) sequences greater than 120 bp. Early studies have shown that translation efficiency increases with poly(A) length before reaching 120 bp. To avoid the loss of poly(A), the commonly used scheme is to add other bases. For example, BioNTech's scheme is 30A+10bp linker+70A, that is, a 10bp linker is inserted between the 30th A and the 31st A of the poly(A) with a total length of 100bp (and so on below); for example, a document (DOI:10.1261 / rna.069286.118) reported the schemes of 60A+linker+60A and 40A+linker+40A+linker+40A.

[0003] However, existing solutions have not simultaneously addressed the issues of stability and translation efficiency. For example, the aforementioned BioNTech solution still has a lower translation efficiency than a 120-bp poly(A). Furthermore, these existing solutions still cannot maintain a stable length over long periods of time under conventional culture conditions. Therefore, the development of a poly(A) sequence with both high translation efficiency and strong stability is urgently needed. Summary of the Invention

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

[0005] The technical solution of the present invention to solve the technical problem is as follows:

[0006] A multi-spaced poly(A) sequence having 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 .

[0007] Preferably, the sequence of the multi-spaced poly(A) sequence is one of SEQ ID No.1, SEQ ID No.10, SEQ ID No.12, and SEQ ID No.13.

[0008] The poly(A) sequence can make the translation efficiency of the protein expression plasmid high and the stability strong.

[0009] The present invention also proposes:

[0010] A plasmid containing the multi-spacer poly(A) sequence described above.

[0011] Preferably, the plasmid is a protein expression plasmid.

[0012] Preferably, the backbone sequence of the plasmid is SEQ ID No. 16, wherein the poly(A) sequence insertion site is between bases 2784 and 2785 of the backbone sequence.

[0013] The above-mentioned plasmid has high translation efficiency and strong stability, and can be stably amplified without the use of special strains and conditions, and will not lead to a decrease in plasmid yield.

[0014] The present invention also proposes:

[0015] The aforementioned multi-spacer poly(A) sequence is used to construct a plasmid.

[0016] The plasmid described above is used as a template plasmid DNA for expressing the target protein.

[0017] A method for preparing a target protein using the plasmid described above. A host cell containing the plasmid described above.

[0018] The method for preparing the target protein is carried out using the host cells described above.

[0019] Compared to existing technologies, the multi-spaced poly(A) sequence of the present invention significantly improves the translation efficiency of protein expression plasmids and provides excellent plasmid stability. Plasmids employing the multi-spaced poly(A) sequence of the present invention do not require specialized strains or conditions and can be stably amplified using standard molecular cloning conditions in DH5α or Turbo cells without reducing plasmid yield. Furthermore, the protein expression levels achieved are significantly superior to those achieved with existing poly(A) sequences. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 This is a map of the plasmid used in Example 1 of the present invention. DETAILED DESCRIPTION

[0021] The present invention will be described in further detail below with reference to the following examples, but the present invention is not limited to the examples given. Example

[0022] 1. The basic process of this embodiment is as follows:

[0023] According to the literature (DOI: 10.1038 / nsmb.3509), a 30bp poly(A) sequence is recognized by PABPC1 and effectively initiates translation. However, during actual experiments, the inventors' research team found that while a 30bp poly(A) sequence could be translated to produce protein, its translation efficiency was far lower than that of a 110bp poly(A) sequence. Furthermore, the 60bp+60bp or 30bp+70bp poly(A) sequences reported in the literature were not stable enough during cell culture, and even after continuous cell culture at 37°C, they still had a high deletion rate and no advantage in translation efficiency over a pure A poly(A) sequence. Considering these issues, the inventors' research team further discovered that by incorporating multiple 30bp poly(A) sequences, translation efficiency could be significantly improved compared to a poly(A) sequence consisting of 110 consecutive A bases. Furthermore, even small base insertions could enhance the stability of the poly(A) sequence. On this basis, this example further systematically screened poly(A) sequences with high translation efficiency and the ability to be stably propagated in plasmids.

[0024] This example constructs a sequence to replace the conventional A 80-120 The sequence (i.e., poly(A) sequence) was compared with the sequence of the BioNTech solution mentioned in the background technology and the best sequence in the literature (DOI: 10.1038 / nsmb.3509) (other sequences with poor effects in the literature were not included in the comparison).

[0025] The sequences constructed in this example are SEQ ID No. 1-3, which are as follows:

[0026] SEQ ID No.1: A 30 TTA 30 TTA 30 TTA 30 , denoted as SEQ1.

[0027] SEQ ID No. 2: A 30 CCA 30 CCA 30 CCA 30 , denoted as SEQ2.

[0028] SEQ ID No.3:A 30 GGA 30 GGA 30 GGA 30 , denoted as SEQ3.

[0029] The comparison sequences are SEQ ID No. 4-9, which are as follows:

[0030] SEQ ID No.4:A 30 GCATATGACTA 70 , denoted as SEQ4.

[0031] SEQ ID No.5:A 110 , denoted as SEQ5.

[0032] SEQ ID No.6:A 60 ATGCATA 60 , denoted as SEQ6.

[0033] SEQ ID No.7:A 60 TA 60 , denoted as SEQ7.

[0034] SEQ ID No.8:A 60 CA 60 , denoted as SEQ8.

[0035] SEQ ID No.9:A 60 GA 60 , denoted as SEQ9.

[0036] According to the results of the comparative experiments, SEQ1 and SEQ2 have the best expression effects, but SEQ2 has slightly lower stability. Therefore, this example further compares the similar sequences of SEQ1, SEQ ID No. 10-13, as follows:

[0037] SEQ ID No.10: A30 TTTTA 30 TTTTA 30 TTTTA 30 , denoted as SEQ10.

[0038] SEQ ID No.11: A 30 TTA 30 TTA 30 , denoted as SEQ11.

[0039] SEQ ID No.12: A 30 TTA 30 TTA 30 TTA 30 TTA 30 , denoted as SEQ12.

[0040] SEQ ID No.13:A 35 TTA 35 TTA 35 TTA 35 , denoted as SEQ13.

[0041] Comparison showed that except for SEQ11, which had a relatively low expression level, the expression levels of other sequences were comparable.

[0042] Based on the above results, we can know that:

[0043] 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.

[0044] 2. The specific experimental contents and results of this embodiment are as follows:

[0045] This example uses GFP-Fluc IVT template as the plasmid for poly(A) testing, and its map is shown in FIG. Figure 1The plasmid backbone sequence is SEQ ID No. 16 (denoted as SEQ16), wherein the poly(A) sequence insertion site is between bases 2784 and 2785 of the backbone sequence.

[0046] (1) Comparison of translation efficiency of different poly(A) sequences

[0047] The following plasmid vectors were synthesized by the applicant:

[0048] UTR2-GFP-Fluc-PA4T: the backbone is SEQ16, and the inserted sequence is SEQ1.

[0049] UTR2-GFP-Fluc-PA4C: the backbone is SEQ16, and the inserted sequence is SEQ2.

[0050] UTR2-GFP-Fluc-PA4G: the backbone is SEQ16, and the inserted sequence is SEQ3.

[0051] UTR2-GFP-Fluc-PA3070: the backbone is SEQ16, and the inserted sequence is SEQ4.

[0052] UTR2-GFP-Fluc-PA110: the backbone is SEQ16, and the inserted sequence is SEQ5.

[0053] UTR2-GFP-Fluc-PA60X2: the backbone is SEQ16, and the inserted sequence is SEQ6.

[0054] UTR2-GFP-Fluc-PA60T2: the backbone is SEQ16, and the inserted sequence is SEQ7.

[0055] UTR2-GFP-Fluc-PA60C2: the backbone is SEQ16, and the inserted sequence is SEQ8.

[0056] UTR2-GFP-Fluc-PA60G2: the backbone is SEQ16, and the inserted sequence is SEQ9.

[0057] Linearization was performed using XbaI (NEB R0145) according to the supplier's instructions.

[0058] The digested products were directly recovered using the MolPure® PCR Product Purification Kit (19106ES70) and quantified using a NanoDrop micro-spectrophotometer.

[0059] The T7 in vitro transcription kit (AGCN, Jiangsu Shenji Biotechnology Co., Ltd.) was used, catalog number 10110N. The GAG ​​CAP used was "m7G(5')ppp(5')(2'OMeA)pG 100mM Ammonium Solution," catalog number CAP3011. The in vitro transcription reaction system shown in Table 1 was incubated at 37°C for 4 hours.

[0060] Table 1. In vitro transcription reaction system

[0061]

[0062] After the reaction, the template was removed by DNase I digestion, and mRNA was purified using Novozymes RNA purification magnetic beads (N412) to obtain mRNA.

[0063] HEK293T cells were transfected using Thermo Scientific Lipofectamine™ MessengerMAX™ transfection reagent (LMRNA008) with 200 ng of mRNA per well of a 96-well plate. Each mRNA was transfected in triplicate. A negative control (NC) was performed without mRNA.

[0064] After 24 hours of culture, cells were assayed using the Firefly Luciferase Reporter Gene Assay Kit (Beyotime RG006). Each well was lysed with 100 μL of reporter gene cell lysis buffer. The lysate was diluted 10-fold with ultrapure water, and 5 μL was assayed according to the supplier's instructions. The results are shown in Table 2.

[0065] Table 2. Test results

[0066]

[0067] 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.

[0068] (2) Plasmid stability test

[0069] All plasmids used in (1) were used to transfect NEB Stable competent cells (NEB C3040), cultured on kanamycin LB plates at 37°C overnight, and 40 clones were selected and cultured in kanamycin LB liquid medium at 37°C overnight.

[0070] Sequencing was performed using primer SEQ ID No. 17 (denoted as SEQ17): GATGTGCTGCAAGGCGATTA. The number of clones with complete poly(A) sequences is shown in Table 3 below.

[0071] Table 3. Number of clones with complete poly(A) sequences

[0072]

[0073] 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.

[0074] (3) Plasmid stability test of different strains

[0075] The competent strains used included: DH10B-T1 (12331013), DH5α-T1 (12297016) and Stbl3 (C737303) from Thermo, Turbo (C2984) from NEB, JM109 (200235) and XL1-Blue (200249) from Agilent.

[0076] UTR2-GFP-Fluc-PA4T was used to transform each competent strain, and the cells were cultured on kanamycin LB plates overnight at 37°C. 40 clones were selected and incubated in kanamycin LB liquid medium at 37°C overnight. Sequencing was performed using primer SEQ17. The number of clones with complete poly(A) sequences is shown in Table 4 below.

[0077] Table 4. Number of clones with complete poly(A) sequences

[0078]

[0079] From the above results, it can be seen that when using UTR2-GFP-Fluc-PA4T, there is little difference between different strains and they can all maintain good integrity.

[0080] (4) Comparison of translation efficiency of other forms of T insertion sequences (sequences similar to SEQ1)

[0081] The steps for synthesizing mRNA and luciferase detection were the same as (1), using the plasmid backbone SEQ16 and inserting the sequences SEQ1, SEQ10, SEQ11, SEQ12, and SEQ13, respectively. The detection results are shown in Table 5.

[0082] Table 5. Test results

[0083]

[0084] 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.

[0085] Based on the above embodiments:

[0086] The plasmid using the spacer poly(A) sequence of the present invention does not require the use of special strains and conditions. Even in DH5α or Turbo, it can be stably amplified using standard molecular cloning conditions without causing a decrease in plasmid yield. At the same time, its protein expression amount is significantly better than that of a poly(A) sequence of 110 bp in length.

[0087] In addition to the above embodiments, the present invention may also have other implementations. Any technical solution formed by equivalent replacement or equivalent transformation falls within the scope of protection required by the present invention.

Claims

1. A multi-spacer poly(A) gene segment characterized by: The structure of the gene fragment is A 30-35 T 2-4 A 30-35 T 2- 4A 30-35 T 2-4 A 30-35 , or 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 ; The sequence of the gene fragment is one of SEQ ID No.1, SEQ ID No.10, SEQ ID No.12, and SEQ ID No.

13.

2. A plasmid containing the multi-spaced poly(A) gene segment according to claim 1.

3. The plasmid according to claim 2, characterized in that The plasmid is a protein expression plasmid.

4. The plasmid according to claim 2, wherein The backbone sequence of the plasmid is SEQ ID No. 16, wherein the multi-spaced poly (A) gene fragment insertion site is between the 2784th and 2785th bases of the backbone sequence.

5. Use of the multi-spaced poly (A) gene segment according to claim 1 for constructing a plasmid.

6. Use of the plasmid according to any one of claims 2 to 4 as a template plasmid DNA for expressing a target protein.

7. A method for preparing a target protein using the plasmid according to any one of claims 2 to 4.

8. A host cell containing the plasmid according to any one of claims 2 to 4.

9. A method for preparing a target protein using the host cell according to claim 8.

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