A mutant T7 RNA polymerase with reduced transcription byproducts, its preparation method and applications

Abstract

The present application relates to the technical field of biology, and discloses a mutant T7 RNA polymerase with reduced by-products in transcription process, and a preparation method and application thereof. The mutant T7 RNA polymerase of the present application is a protein as described in A1) or A2): A1) the mutant T7 RNA polymerase comprises amino acid substitution sites A124E / A258D, A124E / M190D or A124E / S192P relative to a wild-type T7 RNA polymerase; the amino acid sequence of the wild-type T7 RNA polymerase is shown as SEQ ID NO:1; A2) a fusion protein obtained by connecting a tag to the N terminal or / and C terminal of the amino acid sequence described in A1). Relative to the wild-type T7 RNA polymerase, the mutant T7 RNA polymerase of the present application has reduced by-products such as dsRNA in the transcription process.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and in particular to a mutant T7 RNA polymerase that reduces byproducts during transcription, its preparation method, and its applications. Background Technology

[0002] T7 RNA polymerase is a potent transcriptase widely used in gene expression research, virology, vaccine development, and other fields. It can efficiently synthesize RNA from DNA templates. Its high specificity and efficiency have led to its widespread application in in vitro transcription systems.

[0003] However, T7 RNA polymerase has certain problems in its use, especially in large-scale production, where the generation of byproducts has become a pressing issue. One typical byproduct is double-stranded RNA (dsRNA). dsRNA can interfere with downstream experimental results and even be toxic to cells, affecting the accuracy of gene function studies or RNA interference experiments. Related technologies have attempted to reduce dsRNA production by optimizing reaction conditions and introducing inhibitors. However, these methods are generally limited in efficiency and effectiveness, and lead to increased production costs and more complex reaction systems. Therefore, developing a novel T7 RNA polymerase that can reduce dsRNA production is crucial. Summary of the Invention

[0004] This invention aims to solve one of the technical problems existing in the prior art. To this end, this invention proposes a mutant T7 RNA polymerase with low byproduct yield.

[0005] The present invention also proposes a biomaterial related to the above-mentioned mutant T7 RNA polymerase.

[0006] The present invention also proposes a method for preparing the above-mentioned mutant T7 RNA polymerase.

[0007] The present invention also proposes a method for using the above-mentioned mutant T7 RNA polymerase.

[0008] This invention also proposes an application of the above-mentioned mutant T7 RNA polymerase or biological material in the preparation of nucleic acid transcription products.

[0009] The present invention also proposes an application of the above-mentioned mutant T7 RNA polymerase in the in vitro transcription and synthesis of RNA.

[0010] The present invention also proposes a kit for nucleic acid transcription.

[0011] In a first aspect, the present invention provides a mutant T7 RNA polymerase, which is the protein described in A1) or A2): A1) Compared to the wild-type T7 RNA polymerase, the mutant T7 RNA polymerase contains amino acid substitution sites A124E / A258D, A124E / M190D, or A124E / S192P; the amino acid sequence of the wild-type T7 RNA polymerase is shown in SEQ ID NO: 1; A2) A fusion protein obtained by attaching a tag to the N-terminus and / or C-terminus of the amino acid sequence described in A1).

[0012] The mutant T7 RNA polymerase according to embodiments of the present invention has at least the following beneficial effects: The mutant T7 RNA polymerase of this invention can reduce the generation of byproducts such as double-stranded RNA (dsRNA), making it suitable for the development of in vitro diagnostic reagents and synthetic biology applications, especially for applications that efficiently synthesize full-length RNA and reduce immunogenic byproducts.

[0013] In some embodiments of the present invention, when the mutant T7 RNA polymerase contains the amino acid substitution site A124E / A258D, the mutant T7 RNA polymerase further contains at least one of the following amino acid substitution sites: H161E, S192P, V174K, K163W, M46L, N165P, Q169D, M219L, and K363P.

[0014] In some embodiments of the present invention, the amino acid substitution sites of the mutant T7 RNA polymerase are selected from the following combinations of mutations: A124E / A258D, A124E / A258D / H161E, A124E / A258D / S192P, A124E / A258D / V174K, A124E / A258D / K163W, A124E / A258D / M4 6L, A124E / A258D / N165P, A124E / A258D / Q169D, A124E / A258D / M219L, A124E / A258D / K363P, A124E / M190D, A124E / S192P.

[0015] In some embodiments of the present invention, the tag includes at least one of the tags that facilitate the dissolution, purification, and detection of mutant T7 RNA polymerase. For example, tags facilitating the dissolution of mutant T7 RNA polymerase include, but are not limited to, nus tags or maltose-binding protein tags; tags facilitating the purification of mutant T7 RNA polymerase include, but are not limited to, strep tags, His tags, GST tags, pelB signal tags, or ompA signal tags; tags facilitating the detection of mutant T7 RNA polymerase include, but are not limited to, horseradish peroxidase (HRP) tags, β-galactosidase tags, luciferase tags, green fluorescent protein (GFP) tags, HcRed tags, DsRed tags, or cyan fluorescent protein (CFP) tags. Specifically, the tag may be a His tag. It is understood that the mutant T7 RNA polymerase of the present invention may contain one or more tags; multiple tags may include combinations of multiple identical tags, or combinations of multiple different tags.

[0016] A second aspect of the invention provides biological material related to the mutant T7 RNA polymerase described in the first aspect, said biological material being any one of B1) to B4): B1) A nucleic acid molecule encoding the mutant T7 RNA polymerase described in the first aspect embodiment; B2), an expression cassette containing the nucleic acid molecule described in B1); B3), a recombinant vector containing the nucleic acid molecule described in B1) or the expression cassette described in B2); B4) Recombinant biological cells containing the nucleic acid molecule described in B1), the expression cassette described in B2), or the recombinant vector described in B3).

[0017] In some embodiments of the present invention, the nucleic acid sequence of the nucleic acid molecule is shown as any one of B1-1) to B1-14): B1-1) The nucleic acid molecule obtained by mutating nucleotides 397-399 of the nucleic acid sequence shown in SEQ ID NO: 2 from “GCG” to “GAA” and nucleotides 799-801 from “GCG” to “GAT” is nucleotide 28-2676. B1-2) The nucleic acid molecule obtained by mutating nucleotides 397-399 of the nucleic acid sequence shown in SEQ ID NO: 2 from “GCG” to “GAA”, nucleotides 508-510 from “CAT” to “GAA”, and nucleotides 799-801 from “GCG” to “GAT”, is nucleotide 28-2676. B1-3) The nucleic acid molecule obtained by mutating nucleotides 397-399 of the nucleic acid sequence shown in SEQ ID NO: 2 from “GCG” to “GAA”, nucleotides 601-603 from “AGC” to “CCG”, and nucleotides 799-801 from “GCG” to “GAT”, is the 28th-2676th nucleotide; B1-4) The nucleic acid molecule obtained by mutating nucleotides 397-399 of the nucleic acid sequence shown in SEQ ID NO: 2 from “GCG” to “GAA”, nucleotides 547-549 from “GTG” to “AAA”, and nucleotides 799-801 from “GCG” to “GAT” is nucleotide 28-2676. B1-5) The nucleic acid molecule obtained by mutating nucleotides 397-399 of the nucleic acid sequence shown in SEQ ID NO: 2 from “GCG” to “GAA”, nucleotides 514-516 from “AAA” to “TGG”, and nucleotides 799-801 from “GCG” to “GAT” is nucleotide 28-2676. B1-6) The nucleic acid molecule obtained by mutating nucleotides 163-165 of the nucleic acid sequence shown in SEQ ID NO: 2 from “ATG” to “CTG”, nucleotides 397-399 from “GCG” to “GAA”, and nucleotides 799-801 from “GCG” to “GAT”, consists of nucleotides 28-2676. B1-7) The nucleic acid molecule obtained by mutating nucleotides 397-399 of the nucleic acid sequence shown in SEQ ID NO: 2 from “GCG” to “GAA”, nucleotides 520-522 from “AAC” to “CCG”, and nucleotides 799-801 from “GCG” to “GAT” is nucleotide 28-2676. B1-8) The nucleic acid molecule obtained by mutating nucleotides 397-399 of the nucleic acid sequence shown in SEQ ID NO: 2 from “GCG” to “GAA”, nucleotides 532-534 from “CAG” to “GAT”, and nucleotides 799-801 from “GCG” to “GAT” is nucleotide 28-2676. B1-9) The nucleic acid molecule obtained by mutating nucleotides 397-399 of the nucleic acid sequence shown in SEQ ID NO: 2 from “GCG” to “GAA”, nucleotides 682-684 from “ATG” to “CTG”, and nucleotides 799-801 from “GCG” to “GAT” is nucleotide 28-2676. B1-10) The nucleic acid molecule obtained by mutating nucleotides 397-399 of the nucleic acid sequence shown in SEQ ID NO: 2 from “GCG” to “GAA”, nucleotides 799-801 from “GCG” to “GAT”, and nucleotides 1114-1116 from “AAA” to “CCG”, consists of nucleotides 28-2676. B1-11) The nucleic acid molecule obtained by mutating nucleotides 397-399 of the nucleic acid sequence shown in SEQ ID NO: 2 from “GCG” to “GAA” and nucleotides 595-597 from “ATG” to “GAT” is nucleotide 28-2676. B1-12) The nucleic acid molecule obtained by mutating nucleotides 397-399 of the nucleic acid sequence shown in SEQ ID NO: 2 from “GCG” to “GAA” and nucleotides 601-603 from “AGC” to “CCG” is nucleotide 28-2676. The nucleic acid sequence shown in B1-13) has more than 70% identity with any of B1-1) to B1-12) and encodes the mutant T7 RNA polymerase; B1-14) hybridizes under stringent conditions with any one of the specified nucleic acid sequences from B1-1) to B14) and encodes the mutant T7 RNA polymerase.

[0018] In some embodiments of the present invention, the 70% or more of identity can be 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity.

[0019] In some embodiments of the present invention, the expression cassette refers to DNA capable of expressing the mutant T7 RNA polymerase in host cells. This DNA may include not only a promoter to initiate transcription of the mutant T7 RNA polymerase gene, but also a terminator to terminate transcription of the mutant T7 RNA polymerase gene. Furthermore, the expression cassette may also include an enhancer sequence.

[0020] In some embodiments of the present invention, the vector may be a plasmid, a granule, a bacteriophage, or a viral vector. For example, it may be a PET-28a vector.

[0021] In some embodiments of the present invention, the recombinant vector may be a recombinant vector obtained by inserting a DNA molecule encoding the mutant T7 RNA polymerase into the multiple cloning site of the vector.

[0022] In some embodiments of the present invention, the biological cells include prokaryotic cells and eukaryotic cells. The prokaryotic cells include bacteria or algae. The eukaryotic cells include fungi, mammalian cells, or insect cells. The bacteria may be *Escherichia coli*, such as... E. coli BL21. The recombinant organism does not contain reproductive material.

[0023] In some embodiments of the present invention, the recombinant biological cell is a recombinant biological cell obtained by introducing the nucleic acid molecule described in B1), the expression cassette described in B2), or the recombinant vector described in B3) into a biological cell. Specifically, it can be introduced into a biological cell... E. coli Recombinant Escherichia coli obtained by introducing a recombinant vector into BL21.

[0024] A third aspect of the present invention provides a method for preparing the mutant T7 RNA polymerase according to any one of the first aspects, comprising: The coding gene of the mutant T7 RNA polymerase described in the first aspect embodiment is introduced into a biological cell to express the coding gene, thereby obtaining the mutant T7 RNA polymerase.

[0025] In some embodiments of the present invention, the biological cells include prokaryotic cells and eukaryotic cells.

[0026] In some embodiments of the present invention, the prokaryotic cells include bacteria or algae. The bacteria may be *Escherichia coli* (e.g., *Escherichia coli*). E. coli BL21).

[0027] In some embodiments of the present invention, the eukaryotic cells include fungi (such as yeast), mammalian cells (such as HEK293 cells), or insect cells.

[0028] A fourth aspect of the present invention provides a method comprising the following steps: The nucleic acid template was contacted with the mutant T7 RNA polymerase described in the first aspect embodiment for in vitro transcription.

[0029] A fifth aspect of the invention provides the use of any one of C1) to C2) in the preparation of nucleic acid transcription products; C1) The mutant T7 RNA polymerase described in the first aspect embodiment; C2), the biomaterials described in the second aspect embodiment.

[0030] A sixth aspect of the present invention provides the application of the mutant T7 RNA polymerase described in the first aspect embodiment in the in vitro transcription and synthesis of RNA.

[0031] A seventh aspect of the present invention provides a kit comprising the mutant T7 RNA polymerase described in the first aspect embodiment.

[0032] In some embodiments of the present invention, the kit further includes at least one of a reaction premix and an enzyme preservation solution.

[0033] In some embodiments of the present invention, the reaction premix includes RNase inhibitors, spermidine, Tris-HCl, DTT, NTPs, and Mg. 2+ At least one of them.

[0034] In some embodiments of the present invention, the enzyme preservation solution contains at least one of KPB, NaCl, DTT, EDTA, and glycerol.

[0035] It is understood that the reaction premix and enzyme preservation solution should preferably not affect the activity of the mutant T7 RNA polymerase.

[0036] Other features and advantages of the present invention will be set forth in the following description. Attached Figure Description

[0037] Figure 1 To compare the transcriptional performance of wild-type T7 RNA polymerase and mutant T7 RNA polymerase M1-M12 in Example 1 at different temperatures; where 1: 37℃, 2: 42℃, 3: 45℃; Figure 2 To detect the standard curve constructed in Example 2; Figure 3 The results were analyzed to determine the percentage of dsRNA, a transcriptional byproduct of mutant T7 RNA polymerase M1-M12, in Example 2. Detailed Implementation

[0038] The following will describe the concept and technical effects of the present invention clearly and completely with reference to embodiments, so as to fully understand the purpose, features and effects of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are all within the scope of protection of the present invention.

[0039] In the description of this invention, the term "amino acid" refers to the basic unit that constitutes a protein, giving the protein a specific molecular structure and morphology, and endowing its molecules with biochemical activity. For example, the "amino acid" used in this invention includes the following 20 natural amino acids: alanine (Ala or A), glycine (Gly or G), isoleucine (Ile or I), asparagine (Asn or N), arginine (Arg or R), lysine (Lys or K), lysine (Lys or K), cysteine ​​(Cys or C), aspartic acid (Asp or D), glutamic acid (Glu or E), glutamine (Gln or Q), histidine (His or H), leucine (Leu or L), methionine (Met or M), phenylalanine (Phe or F), proline (Pro or P), serine (Ser or S), threonine (Thr or T), tryptophan (Trp or W), valine (Val or V), and tyrosine (Tyr or Y). The "*" in the amino acid sequence is a stop codon.

[0040] In the description of this invention, each amino acid substitution is represented by a triplet: letter-number-letter; wherein the number indicates the position of the mutated amino acid, the letter before the number corresponds to the amino acid involved in the mutation, and the letter after the number indicates the amino acid used to replace the amino acid before the number.

[0041] The following detailed description is provided with reference to specific embodiments. Unless otherwise specified, conditions in the following embodiments are performed according to conventional conditions (e.g., conditions described in literature or books) or conditions recommended by the manufacturer. Reagents or instruments used in the embodiments, unless otherwise specified by the manufacturer, are all commercially available conventional products.

[0042] Example 1: Obtaining mutant T7 RNA polymerase The amino acid sequence of wild-type T7 RNA polymerase is shown in SEQ ID NO: 1. Compared to wild-type T7 RNA polymerase, the mutation sites of mutant T7 RNA polymerase M1 are A124E and A258D; the mutation sites of mutant T7 RNA polymerase M2 are A124E, A258D, and H161E; the mutation sites of mutant T7 RNA polymerase M3 are A124E, A258D, and S192P; the mutation sites of mutant T7 RNA polymerase M4 are A124E, A258D, and V174K; the mutation sites of mutant T7 RNA polymerase M5 are A124E, A258D, and K163W; the mutation sites of mutant T7 RNA polymerase M6 are A124E, A258D, and M46L; and the mutation sites of mutant T7 RNA polymerase M7 are A124E, A258D, and N165P. The mutation sites for T7 RNA polymerase M8 are A124E, A258D, and Q169D; the mutation sites for T7 RNA polymerase M9 are A124E, A258D, and M219L; the mutation sites for T7 RNA polymerase M10 are A124E, A258D, and K363P; the mutation sites for T7 RNA polymerase M11 are A124E and M190D; and the mutation sites for T7 RNA polymerase M12 are A124E and S192P.

[0043] MNTINIAKNDFSDIELAAIPFNTLADHYGERLAREQLALEHESYEMGEARFRKMFERQLKAGEVADNAAAKPLITTLLPKMIARINDWFEEVKAKRGKRPTAFQFLQEIKPEAVAYITIKTTLACLTSADNTTVQAVASAIGRAIEDEARFGRIRDLEAKHFKKNVEEQLNKRVGHVYKKAFMQVVEADMLSKGLLGGEAWSSWHKEDSIHVGVRCIEMLIESTGMVSLHRQNAGVVGQDSETIELAPEYAEAIATRAGALAGISPMFQPCVVPPKPWTGITGGGYWANGRRPLALVRTHSKKALMRYEDVYMPEVYKAINIAQNTAWKINKKVLAVANVITKWKHCPVEDIPAIEREELPMKPEDIDMNPEALTAWKRAAAAVYRKDKARKSRRISLEFMLEQANKFANHKAIWFPYNMDWRGRVYAVSMFNPQGNDMTKGLLTLAKGKPIGKEGYYWLKIHGANCAGVDKVPFPERIKFIEENHENIMACAKSPLENTWWAEQDSPFCFLAFCFEYAGVQHHGLSYNCSLPLAFDGSCSGIQHFSAMLRDEVGGRAVNLLPSETVQDIYGIVAKKVNEILQADAINGTDNEVVTVTDENTGEISEKVKLGTKALAGQWLAYGVTRSVTKRSVMTLAYGSKEFGFRQQVLEDTIQPAIDSGKGLMFTQPNQAAGYMAKLIWESVSVTVVAAVEAMNWLKSAAKLLAAEVKDKKTGEILRKRCAVHWVTPDGFPVWQEYKKPIQTRLNLMFLGQFRLQPTINTNKDSEIDAHKQESGIAPNFVHSQDGSHLRKTVVWAHEKYGIESFALIHDSFGTIPADAANLFKAVRETMVDTYESCDVLADFYDQFADQLHESQLDKMPALPAKGNLNLRDILESDFAFA (SEQ ID NO: 1).

[0044] 1. Construction of expression vector: The codons of the wild-type T7 RNA polymerase encoding gene were optimized, and a nucleotide sequence encoding a 6×histidine tag (HHHHHH) and a linker peptide (GS) was designed at the 5' end of the coding sequence. The optimized nucleotide sequence is shown in SEQ ID NO: 2. Using PCR point mutagenesis, primers were designed based on the mutation site of the mutant T7 RNA polymerase to perform point mutations, obtaining the nucleic acid molecule encoding the mutant T7 RNA polymerase.

[0045] The specific mutations in the nucleic acid molecules of each mutant T7 RNA polymerase compared to the wild-type T7 RNA polymerase are as follows: Mutant T7 RNA polymerase M1 (A124E / A258D): Nucleotides 397-399 are mutated from “GCG” to “GAA”, and nucleotides 799-801 are mutated from “GCG” to “GAT”. Mutant T7 RNA polymerase M2 (A124E / A258D / H161E): nucleotides 397-399 are mutated from “GCG” to “GAA”, nucleotides 508-510 are mutated from “CAT” to “GAA”, and nucleotides 799-801 are mutated from “GCG” to “GAT”. Mutant T7 RNA polymerase M3 (A124E / A258D / S192P): Nucleotides 397-399 are mutated from “GCG” to “GAA”, nucleotides 601-603 are mutated from “AGC” to “CCG”, and nucleotides 799-801 are mutated from “GCG” to “GAT”. Mutant T7 RNA polymerase M4 (A124E / A258D / V174K): nucleotides 397-399 are mutated from “GCG” to “GAA”, nucleotides 547-549 are mutated from “GTG” to “AAA”, and nucleotides 799-801 are mutated from “GCG” to “GAT”. Mutant T7 RNA polymerase M5 (A124E / A258D / K163W): nucleotides 397-399 are mutated from “GCG” to “GAA”, nucleotides 514-516 are mutated from “AAA” to “TGG”, and nucleotides 799-801 are mutated from “GCG” to “GAT”. Mutant T7 RNA polymerase M6 (A124E / A258D / M46L): Nucleotides 163-165 are mutated from "ATG" to "CTG", nucleotides 397-399 are mutated from "GCG" to "GAA", and nucleotides 799-801 are mutated from "GCG" to "GAT". Mutant T7 RNA polymerase M7 (A124E / A258D / N165P): nucleotides 397-399 are mutated from “GCG” to “GAA”, nucleotides 520-522 are mutated from “AAC” to “CCG”, and nucleotides 799-801 are mutated from “GCG” to “GAT”. Mutant T7 RNA polymerase M8 (A124E / A258D / Q169D): Nucleotides 397-399 are mutated from "GCG" to "GAA", nucleotides 532-534 are mutated from "CAG" to "GAT", and nucleotides 799-801 are mutated from "GCG" to "GAT". Mutant T7 RNA polymerase M9 (A124E / A258D / M219L): nucleotides 397-399 are mutated from “GCG” to “GAA”, nucleotides 682-684 are mutated from “ATG” to “CTG”, and nucleotides 799-801 are mutated from “GCG” to “GAT”. Mutant T7 RNA polymerase M10 (A124E / A258D / K363P): nucleotides 397-399 are mutated from “GCG” to “GAA”, nucleotides 799-801 are mutated from “GCG” to “GAT”, and nucleotides 1114-1116 are mutated from “AAA” to “CCG”. Mutant T7 RNA polymerase M11 (A124E / M190D): Nucleotides 397-399 are mutated from “GCG” to “GAA”, and nucleotides 595-597 are mutated from “ATG” to “GAT”. Mutant T7 RNA polymerase M12 (A124E / S192P): Nucleotides 397-399 are mutated from “GCG” to “GAA”, and nucleotides 601-603 are mutated from “AGC” to “CCG”.

[0046]

[0047] The corresponding DNA molecules were ligated into the PET-28a vector to obtain recombinant expression vectors PET-28a / WT, PET-28a / M1, PET-28a / M2, PET-28a / M3, PET-28a / M4, PET-28a / M5, PET-28a / M6, PET-28a / M7, PET-28a / M8, PET-28a / M9, PET-28a / M10, PET-28a / M11, and PET-28a / M12. Each recombinant expression vector was transformed into E. coli BL21 DE3 Star competent cells, and positive single colonies were screened for sequencing verification, obtaining single colonies with valid verification results.

[0048] 2. Protein expression: The single colony verified in step 1 above was inoculated into 100 mL LB medium containing 50 μg / mL ampicillin and cultured overnight at 37°C with shaking. The seed culture from the overnight culture was then inoculated into 2 L LB medium containing 50 μg / mL ampicillin at a volume ratio of 1:100 and cultured at 37°C with shaking until 0D. 600 The concentration was 0.6-0.8; 10% IPTG was added to a final concentration of 0.4 mmol / L, and the cells were induced by shaking at 37℃ for 6-8 h; the cells were collected by centrifugation and weighed, and the wet weight of the cells was recorded and stored at -80℃.

[0049] 3. Protein purification: (1) High-pressure disruption of induced expression cells: Bacterial cells frozen at -80℃ after induction of expression were collected. Based on the recorded wet weight of the cells, 5 mL of lysis buffer (50 mM Tris-HCl, pH 7.8 at 25℃, 300 mM NaCl, 50 mM Imidazole, 5% Glycerol) was added per gram of cells to resuspend the cells. The cells were then lysed using a high-pressure homogenizer at 650 bar for three cycles. The lysed cells were centrifuged at 12000 rpm for 30 min at 4℃, and the supernatant and precipitate were separated. The supernatant was transferred to a 200 mL sterile beaker. SDS-PAGE electrophoresis was performed on both the supernatant and the precipitate.

[0050] (2) Nickel ion affinity chromatography purification: The selected chromatography column was a Ni-NTA Purose 6 Fast Flow (purchased from Jiaxing Qianchun Biotechnology Co., Ltd.). The binding buffer was buffer A (50 mM Tris-HCl, pH 7.8 at 25℃, 100 mM NaCl, 80 mM Imidazole, 5% Glycerol); the elution buffer was buffer B (50 mM Tris-HCl, pH 7.8 at 25℃, 100 mM NaCl, 500 mM Imidazole, 5% Glycerol). The supernatant obtained in step (1) was loaded into the chromatography column that had been equilibrated with buffer A. After loading, the column was first rinsed with equilibration buffer A, and then eluted with buffer B at a gradient of 0%-100%. The eluted fraction was detected by SDS-PAGE protein electrophoresis, and the eluent containing the target protease was collected according to the detection results.

[0051] (3) Secondary nickel ion affinity chromatography purification: Repeat step (2) to perform SDS-PAGE protein electrophoresis on the eluent. Collect the eluent containing the target protease according to the detection results, and dialyze the eluent into buffer C (50 mM Tris-HCl, pH 7.8 at 25℃, 50 mM NaCl, 1 mM DTT, 0.1 mM EDTA). Collect the dialysate for later use.

[0052] (4) Strong anion exchange chromatography purification: The selected chromatography column was a Q Purose 6 Fast Flow (purchased from Jiaxing Qianchun Biotechnology Co., Ltd.), with binding buffer C and elution buffer D (50 mM Tris-HCl, pH 7.8 at 25℃, 800 mM NaCl, 1 mM DTT, 0.1 mM EDTA). The dialysate obtained in step (3) was added to the chromatography column that had been equilibrated with buffer C. After the addition was complete, the column was first rinsed with buffer C, and then eluted with buffer D at a gradient of 0%-100%. The eluted fraction was detected by SDS-PAGE protein electrophoresis. The eluent was collected according to the detection results and dialyzed into a storage solution (20 mM KPB (pH 7.9), 100 mM NaCl, 1 mM DTT, 0.1 mM EDTA, 50% glycerol) for later use.

[0053] Detection Example 1 Two single-stranded DNA molecules (SEQ ID NO: 3 and SEQ ID NO: 4) were designed and annealed to create a 30 bp double-stranded DNA template containing the T7 promoter. T7 RNA polymerase in vitro transcription systems were prepared according to Table 1 using wild-type T7 RNA polymerase and mutant T7 RNA polymerases M1-M12, respectively, and transcribed at 37℃ / 42℃ / 45℃ for 3 h. The transcribed products were digested with DNase I, and the products were analyzed using 15% urea for Native Page assay.

[0054] DNA single strand 1: 5'-TTCTAATACGACTCACTATAGGGAAACATCTTGTTGAGAGCAGTATACAACTC-3' (SEQ ID NO: 3); DNA single strand 2: 5'-GAGTTGTATACTGCTCTCAACAAGATGTTTCCCTATAGTGAGTCGTATTAGAA-3' (SEQ ID NO: 4).

[0055] Table 1

[0056] The results are as follows Figure 1 As shown.

[0057] Wild-type T7 RNA polymerase and mutant T7 RNA polymerase M1-M12 all exhibit good transcription performance at 37℃, 42℃, and 45℃.

[0058] Detection Example 2 The nucleic acid sequence shown in SEQ ID NO: 5 was used as a template for detecting byproducts. T7 RNA polymerase in vitro transcription systems were prepared according to Table 1 using wild-type T7 RNA polymerase and mutant T7 RNA polymerases M1-M12, and reacted at 37℃ for 2 h. After the reaction, DNase I was added to digest the DNA template, and the mixture was treated at 70℃ for 10 min. The digested transcripts were recovered using an RNA recovery kit (Aikerui Biotechnology, catalog number: AG21033), and the volume of the recovered product was brought to 100 μL with enzyme-free sterile water. The product concentration was recorded. The RNA product obtained from wild-type T7 RNA polymerase was diluted 5000× with enzyme-free sterile water, and the RNA product from the T7 RNA polymerase mutant was diluted 2000× for subsequent ELISA detection. The transcripts were quantified using a Qubit RNA Quantification Kit (Thermo Fisher Scientific). The diluted RNA products were detected using the NovoFastdsRNA ELISA Kit (nearshore protein, catalog number: RD017). A standard curve was first constructed using the standards in the kit. The diluted RNA products and standards were then processed in the same manner according to the kit instructions. The absorbance values ​​at 450 nm were recorded, and the dsRNA content and percentage of the product were calculated (dsRNA content percentage = dsRNA yield / total RNA yield × 100%).

[0059] 5'--3' (SEQ ID NO: 5).

[0060] The constructed standard curve is as follows Figure 2 As shown. The results are as follows. Figure 3 (The ratio of dsRNA produced by transcription to total RNA) and Table 2 are shown.

[0061] Table 2

[0062] Compared to wild-type T7 RNA polymerase, mutant T7 RNA polymerase M1-M12 has a lower dsRNA yield.

[0063] The embodiments of the present invention have been described in detail above. However, the present invention is not limited to the above embodiments. Within the scope of knowledge possessed by those skilled in the art, various changes can be made without departing from the spirit of the present invention. Furthermore, the embodiments of the present invention and the features thereof can be combined with each other unless otherwise specified.

Claims

1. A mutant T7 RNA polymerase, characterized in that, The mutant T7 RNA polymerase is the protein described in A1) or A2): A1) Compared to the wild-type T7 RNA polymerase, the mutant T7 RNA polymerase contains amino acid substitution sites A124E / A258D, A124E / M190D, or A124E / S192P; the amino acid sequence of the wild-type T7 RNA polymerase is shown in SEQ ID NO: 1; A2) A fusion protein obtained by attaching a tag to the N-terminus and / or C-terminus of the amino acid sequence described in A1).

2. The mutant T7 RNA polymerase according to claim 1, characterized in that, When the mutant T7 RNA polymerase contains the amino acid substitution site A124E / A258D, the mutant T7 RNA polymerase further contains at least one of the following amino acid substitution sites: H161E, S192P, V174K, K163W, M46L, N165P, Q169D, M219L, and K363P.

3. Biomaterials related to the mutant T7 RNA polymerase of claim 1 or 2, characterized in that, The biomaterial is any one of B1) to B4): B1) A nucleic acid molecule encoding the mutant T7 RNA polymerase as described in claim 1 or 2; B2), an expression cassette containing the nucleic acid molecule described in B1); B3), a recombinant vector containing the nucleic acid molecule described in B1) or the expression cassette described in B2); B4) Recombinant biological cells containing the nucleic acid molecule described in B1), the expression cassette described in B2), or the recombinant vector described in B3).

4. The biomaterial according to claim 3, characterized in that, The nucleic acid sequence of the nucleic acid molecule is shown in any one of B1-1) to B1-14): B1-1) The nucleic acid molecule obtained by mutating nucleotides 397-399 of the nucleic acid sequence shown in SEQ ID NO: 2 from "GCG" to "GAA" and nucleotides 799-801 from "GCG" to "GAT" is nucleotide 28-2676. B1-2) The nucleic acid molecule obtained by mutating nucleotides 397-399 of the nucleic acid sequence shown in SEQ ID NO: 2 from "GCG" to "GAA", nucleotides 508-510 from "CAT" to "GAA", and nucleotides 799-801 from "GCG" to "GAT" is nucleotide 28-2676. B1-3) The nucleic acid molecule obtained by mutating nucleotides 397-399 of the nucleic acid sequence shown in SEQ ID NO: 2 from "GCG" to "GAA", nucleotides 601-603 from "AGC" to "CCG", and nucleotides 799-801 from "GCG" to "GAT" is nucleotide 28-2676. B1-4) The nucleic acid molecule obtained by mutating nucleotides 397-399 of the nucleic acid sequence shown in SEQ ID NO: 2 from "GCG" to "GAA", nucleotides 547-549 from "GTG" to "AAA", and nucleotides 799-801 from "GCG" to "GAT" is nucleotide 28-2676. B1-5) The nucleic acid molecule obtained by mutating nucleotides 397-399 of the nucleic acid sequence shown in SEQ ID NO: 2 from "GCG" to "GAA", nucleotides 514-516 from "AAA" to "TGG", and nucleotides 799-801 from "GCG" to "GAT" is nucleotide 28-2676. B1-6) The nucleic acid molecule obtained by mutating nucleotides 163-165 from "ATG" to "CTG", nucleotides 397-399 from "GCG" to "GAA", and nucleotides 799-801 from "GCG" to "GAT" after mutating nucleotides 163-165 from "ATG" to "CTG", nucleotides 397-399 from "GCG" to "GAA", and nucleotides 799-801 from "GCG" to "GAT" is nucleotide 28-2676. B1-7) The nucleic acid molecule obtained by mutating nucleotides 397-399 of the nucleic acid sequence shown in SEQ ID NO: 2 from "GCG" to "GAA", nucleotides 520-522 from "AAC" to "CCG", and nucleotides 799-801 from "GCG" to "GAT" is nucleotide 28-2676. B1-8) The nucleic acid molecule obtained by mutating nucleotides 397-399 of the nucleic acid sequence shown in SEQ ID NO: 2 from "GCG" to "GAA", nucleotides 532-534 from "CAG" to "GAT", and nucleotides 799-801 from "GCG" to "GAT" is nucleotide 28-2676. B1-9) The nucleic acid molecule obtained by mutating nucleotides 397-399 of the nucleic acid sequence shown in SEQ ID NO: 2 from "GCG" to "GAA", nucleotides 682-684 from "ATG" to "CTG", and nucleotides 799-801 from "GCG" to "GAT" is nucleotide 28-2676. B1-10) The nucleic acid molecule obtained by mutating nucleotides 397-399 of the nucleic acid sequence shown in SEQ ID NO: 2 from "GCG" to "GAA", nucleotides 799-801 from "GCG" to "GAT", and nucleotides 1114-1116 from "AAA" to "CCG" is nucleotide 28-2676. B1-11) The nucleic acid molecule obtained by mutating nucleotides 397-399 of the nucleic acid sequence shown in SEQ ID NO: 2 from "GCG" to "GAA" and nucleotides 595-597 from "ATG" to "GAT" is nucleotide 28-2676. B1-12) The nucleic acid molecule obtained by mutating nucleotides 397-399 of the nucleic acid sequence shown in SEQ ID NO: 2 from "GCG" to "GAA" and nucleotides 601-603 from "AGC" to "CCG" is nucleotide 28-2676. The nucleic acid sequence shown in B1-13) has more than 70% identity with any of B1-1) to B1-12) and encodes the mutant T7 RNA polymerase; B1-14) hybridizes under stringent conditions with any one of the specified nucleic acid sequences from B1-1) to B14) and encodes the mutant T7 RNA polymerase.

5. A method for preparing the mutant T7 RNA polymerase according to claim 1 or 2, characterized in that, include: The mutant T7 RNA polymerase encoding the mutant T7 RNA polymerase described in claim 1 or 2 is introduced into a biological cell to express the encoding gene, thereby obtaining the mutant T7 RNA polymerase.

6. A method, characterized in that, Includes the following steps: The nucleic acid template is contacted with the mutant T7 RNA polymerase as described in claim 1 or 2 for in vitro transcription.

7. The application of any one of C1) to C2) in the preparation of nucleic acid transcription products; C1), the mutant T7 RNA polymerase as described in claim 1 or 2; C2), the biomaterials described in claim 3 or 4.

8. The use of the mutant T7 RNA polymerase according to claim 1 or 2 in the in vitro transcription and synthesis of RNA.

9. A reagent kit, characterized in that, Includes the mutant T7 RNA polymerase as described in claim 1 or 2.

10. The reagent kit according to claim 9, characterized in that, The kit also includes at least one of the following: reaction premix solution and enzyme preservation solution.

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