Mutant T7 RNA polymerase as well as preparation method and application thereof

Abstract

The invention relates to the technical field of biology, and discloses a mutant T7 RNA polymerase as well as a preparation method and application thereof. The mutant T7 RNA polymerase is the protein of A1) or A2): A1) mutation of amino acid sites is carried out on the basis of an amino acid sequence of wild T7 RNA polymerase, and the amino acid mutation sites comprise M190D and A258D; the amino acid sequence of the wild type T7 RNA polymerase is as shown in SEQ ID NO: 1; a2) is a fusion protein obtained by connecting a label to the N end or / and the C end of the amino acid sequence in A1). Compared with wild type T7 RNA polymerase, the mutant T7 RNA polymerase has the advantage of low yield of by-products.

Description

Technical Field

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

[0002] T7 RNA polymerase is a single-subunit RNA polymerase with a molecular weight of approximately 99 kDa, composed of 883 amino acids. Due to its high promoter specificity and high elongation rate, T7 RNA polymerase is currently the preferred enzyme for in vitro mRNA synthesis and is widely used in the production of various RNA preparations, including vaccines.

[0003] However, T7 RNA polymerase produces a series of double-stranded RNA (dsRNA) byproducts during in vitro transcription. Currently, controlling the content of dsRNA byproducts mainly relies on downstream purification processes, but these methods suffer from high costs, cumbersome procedures, and low yields. Directly modifying T7 RNA polymerase through protein engineering to fundamentally reduce its tendency to produce dsRNA during catalysis represents a more promising and fundamental solution. 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.

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

[0006] The present invention also proposes an enzyme preparation comprising the above-mentioned mutant T7 RNA polymerase.

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

[0008] The present invention also proposes a method for using the above-mentioned mutant T7 RNA polymerase.

[0009] This invention also proposes an application of the above-mentioned mutant T7 RNA polymerase in the preparation of nucleic acid transcription products.

[0010] The present invention also proposes the application of the above-mentioned mutant T7 RNA polymerase or the above-mentioned enzyme preparation in nucleic acid transcription.

[0011] The present invention also proposes a reagent kit.

[0012] In a first aspect, the present invention provides a mutant T7 RNA polymerase, which is the protein described in A1) or A2): A1) Mutations are made at amino acid sites based on the amino acid sequence of the wild-type T7 RNA polymerase, including M190D and A258D; 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).

[0013] 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.

[0014] In some embodiments of the present invention, the mutant T7 RNA polymerase further comprises at least one of the amino acid mutation sites S192P, V174K, H161E, K163W, M46L, N165P, Q169D, M219L, K363P, and A124E.

[0015] In some embodiments of the present invention, the amino acid mutation sites of the mutant T7 RNA polymerase are selected from the following combinations of mutations: M190D / A258D, M190D / A258D / S192P, M190D / A258D / V174K, M190D / A258D / H161E, M190D / A258D / K163W, M190D / A258D / M46L, M190D / A258D / N165P, M190D / A258D / Q169D, M190D / A258D / M219L, M190D / A258D / K363P, M190D / A258D / A124E.

[0016] 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.

[0017] In some embodiments of the present invention, the tag is a histidine tag.

[0018] 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. For example: tags that facilitate the dissolution of mutant T7 RNA polymerase include, but are not limited to, nus tags or maltose-binding protein tags; tags that facilitate 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 that facilitate 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.

[0019] 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).

[0020] 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 B1-13): B1-1) The nucleic acid molecule obtained by mutating nucleotides 595-597 of the nucleic acid sequence shown in SEQ ID NO: 2 from “ATG” to “GAT” and nucleotides 799-801 from “GCG” to “GAT”, is nucleotides 28-2676. B1-2) The nucleic acid molecule obtained by mutating nucleotides 595-597 of the nucleic acid sequence shown in SEQ ID NO: 2 from “ATG” to “GAT”, nucleotides 601-603 from “AGC” to “CCG”, and nucleotides 799-801 from “GCG” to “GAT” is nucleotide 28-2676. B1-3) The nucleic acid molecule obtained by mutating nucleotides 547-549 of the nucleic acid sequence shown in SEQ ID NO: 2 from “GTG” to “AAA”, nucleotides 595-597 from “ATG” to “GAT”, and nucleotides 799-801 from “GCG” to “GAT” is nucleotide 28-2676. B1-4) The nucleic acid molecule obtained by mutating nucleotides 508-510 from “CAT” to “GAA”, nucleotides 595-597 from “ATG” to “GAT”, and nucleotides 799-801 from “GCG” to “GAT” is nucleotide 28-2676. B1-5) The nucleic acid molecule obtained by mutating nucleotides 514-516 of the nucleic acid sequence shown in SEQ ID NO: 2 from “AAA” to “TGG”, nucleotides 595-597 from “ATG” to “GAT”, 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 595-597 from “ATG” to “GAT”, and nucleotides 799-801 from “GCG” to “GAT” is nucleotide 28-2676. B1-7) The nucleic acid molecule obtained by mutating nucleotides 520-522 of the nucleic acid sequence shown in SEQ ID NO: 2 from “AAC” to “CCG”, nucleotides 595-597 from “ATG” to “GAT”, and nucleotides 799-801 from “GCG” to “GAT” is nucleotide 28-2676. B1-8) The nucleic acid molecule obtained by mutating nucleotides 532-534 from “CAG” to “GAT”, nucleotides 595-597 from “ATG” to “GAT”, and nucleotides 799-801 from “GCG” to “GAT”, is obtained by mutating nucleotides 28-2676 of the nucleic acid sequence shown in SEQ ID NO: 2. B1-9) The nucleic acid molecule obtained by mutating nucleotides 595-597 of the nucleic acid sequence shown in SEQ ID NO: 2 from “ATG” to “GAT”, nucleotides 799-801 from “GCG” to “GAT”, and nucleotides 682-684 from “ATG” to “CTG” is nucleotide 28-2676. B1-10) The nucleic acid molecule obtained by mutating nucleotides 595-597 from “ATG” to “GAT”, nucleotides 799-801 from “GCG” to “GAT”, and nucleotides 1114-1116 from “AAA” to “CCG”, as shown in SEQ ID NO: 2, has 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”, nucleotides 595-597 from “ATG” to “GAT”, and nucleotides 799-801 from “GCG” to “GAT” is nucleotide 28-2676. The nucleic acid sequence shown in B1-12) has more than 70% homology with any of B1-1) to B1-11) and encodes the mutant T7 RNA polymerase; B1-13) hybridizes under stringent conditions with any of the specified nucleic acid sequences from B1-1) to B12) and encodes the mutant T7 RNA polymerase.

[0021] 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.

[0022] 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.

[0023] 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.

[0024] 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.

[0025] 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.

[0026] 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.

[0027] A third aspect of the present invention provides an enzyme preparation comprising the mutant T7 RNA polymerase described in any one of the first aspects.

[0028] In some embodiments of the present invention, the enzyme preparation further includes a reaction premix or an enzyme preservation solution.

[0029] 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.

[0030] In some embodiments of the present invention, the enzyme preservation solution contains at least one of KPB, NaCl, DTT, EDTA, and glycerol.

[0031] It is understood that the reaction premix and enzyme preservation solution should preferably not affect the activity of the mutant T7 RNA polymerase.

[0032] A fourth 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.

[0033] In some embodiments of the present invention, the biological cells include prokaryotic cells and eukaryotic cells.

[0034] 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).

[0035] In some embodiments of the present invention, the eukaryotic cells include fungi (such as yeast), mammalian cells (such as HEK293 cells), or insect cells.

[0036] A fifth aspect of the present invention provides a method comprising the following steps: RNA molecules were amplified using the mutant T7 RNA polymerase described in the first aspect embodiment or the enzyme preparation described in the third aspect embodiment.

[0037] A sixth aspect of the invention provides the use of any one of C1) to C3) 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; C3), the enzyme preparation described in the third aspect embodiment.

[0038] A seventh aspect of the present invention provides the application of the mutant T7 RNA polymerase described in the first aspect embodiment or the enzyme preparation described in the third aspect embodiment in nucleic acid transcription.

[0039] In an eighth aspect, the present invention provides a kit comprising the mutant T7 RNA polymerase described in the first aspect embodiment or the enzyme preparation described in the third aspect embodiment.

[0040] Other features and advantages of the present invention will be set forth in the following description. Attached Figure Description

[0041] Figure 1 To detect the standard curve constructed in Example 1; Figure 2 The results were analyzed to determine the percentage of dsRNA, a transcriptional byproduct of mutant T7 RNA polymerase M1-M12, in Example 1. Detailed Implementation

[0042] 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.

[0043] 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.

[0044] 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.

[0045] The following detailed description is provided with reference to specific embodiments. Unless otherwise specified, conditions in the following embodiments are performed under 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.

[0046] 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 G47S; the mutation sites of mutant T7 RNA polymerase M2 are A124E, G47S, and S192P; the mutation sites of mutant T7 RNA polymerase M3 are A124E, G47S, and V174K; the mutation sites of mutant T7 RNA polymerase M4 are A124E, G47S, and H161E; the mutation sites of mutant T7 RNA polymerase M5 are A124E, G47S, and K163W; the mutation sites of mutant T7 RNA polymerase M6 are A124E, G47S, and M46L; and the mutation sites of mutant T7 RNA polymerase M7 are A124E, G47S, and N165P. The mutation sites for RNA polymerase M8 are A124E, G47S, and Q169D; the mutation sites for mutant T7 RNA polymerase M9 are A124E, G47S, and M219L; the mutation sites for mutant T7 RNA polymerase M10 are A124E, G47S, and K363P; and the mutation sites for mutant T7 RNA polymerase M11 are A124E, G47S, and A258D.

[0047] MNTINIAKNDFSDIELAAIPFNTLADHYGERLAREQLALEHESYEMGEARFRKMFERQLKAGEVADNAAAKPLITTLLPKMIARINDWFEEVKAKRGKRPTAFQFLQEIKPEAVAYITIKTTLACLTSADNTTVQAVASAIGRAIEDEARFGRIRDLEAKHFKKNVEEQLNKRVGHVYKKAFMQVVEADMLSKGLLGGEAWSSWHKEDSIHVGVRCIEMLIESTGMVSLHRQNAGVVGQDSETIELAPEYAEAIATRAGALAGISPMFQPCVVPPKPWTGITGGGYWANGRRPLALVRTHSKKALMRYEDVYMPEVYKAINIAQNTAWKINKKVLAVANVITKWKHCPVEDIPAIEREELPMKPEDIDMNPEALTAWKRAAAAVYRKDKARKSRRISLEFMLEQANKFANHKAIWFPYNMDWRGRVYAVSMFNPQGNDMTKGLLTLAKGKPIGKEGYYWLKIHGANCAGVDKVPFPERIKFIEENHENIMACAKSPLENTWWAEQDSPFCFLAFCFEYAGVQHHGLSYNCSLPLAFDGSCSGIQHFSAMLRDEVGGRAVNLLPSETVQDIYGIVAKKVNEILQADAINGTDNEVVTVTDENTGEISEKVKLGTKALAGQWLAYGVTRSVTKRSVMTLAYGSKEFGFRQQVLEDTIQPAIDSGKGLMFTQPNQAAGYMAKLIWESVSVTVVAAVEAMNWLKSAAKLLAAEVKDKKTGEILRKRCAVHWVTPDGFPVWQEYKKPIQTRLNLMFLGQFRLQPTINTNKDSEIDAHKQESGIAPNFVHSQDGSHLRKTVVWAHEKYGIESFALIHDSFGTIPADAANLFKAVRETMVDTYESCDVLADFYDQFADQLHESQLDKMPALPAKGNLNLRDILESDFAFA (SEQ ID NO: 1).

[0048] 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.

[0049] The specific mutation details are as follows: Mutant T7 RNA polymerase M1 (M190D / A258D): Nucleotides 595-597 are mutated from "ATG" to "GAT", and nucleotides 799-801 are mutated from "GCG" to "GAT". Mutant T7 RNA polymerase M2 (M190D / A258D / S192P): nucleotides 595-597 are mutated from "ATG" to "GAT", nucleotides 601-603 are mutated from "AGC" to "CCG", and nucleotides 799-801 are mutated from "GCG" to "GAT". Mutant T7 RNA polymerase M3 (M190D / A258D / V174K): nucleotides 547-549 are mutated from “GTG” to “AAA”, nucleotides 595-597 are mutated from “ATG” to “GAT”, and nucleotides 799-801 are mutated from “GCG” to “GAT”. Mutant T7 RNA polymerase M4 (M190D / A258D / H161E): nucleotides 508-510 are mutated from "CAT" to "GAA", nucleotides 595-597 are mutated from "ATG" to "GAT", and nucleotides 799-801 are mutated from "GCG" to "GAT". Mutant T7 RNA polymerase M5 (M190D / A258D / K163W): nucleotides 514-516 are mutated from “AAA” to “TGG”, nucleotides 595-597 are mutated from “ATG” to “GAT”, and nucleotides 799-801 are mutated from “GCG” to “GAT”. Mutant T7 RNA polymerase M6 (M190D / A258D / M46L): Nucleotides 163-165 are mutated from "ATG" to "CTG", nucleotides 595-597 are mutated from "ATG" to "GAT", and nucleotides 799-801 are mutated from "GCG" to "GAT". Mutant T7 RNA polymerase M7 (M190D / A258D / N165P): Nucleotides 520-522 are mutated from "AAC" to "CCG", nucleotides 595-597 are mutated from "ATG" to "GAT", and nucleotides 799-801 are mutated from "GCG" to "GAT". Mutant T7 RNA polymerase M8 (M190D / A258D / Q169D): nucleotides 532-534 are mutated from “CAG” to “GAT”, nucleotides 595-597 are mutated from “ATG” to “GAT”, and nucleotides 799-801 are mutated from “GCG” to “GAT”. Mutant T7 RNA polymerase M9 (M190D / A258D / M219L): Nucleotides 595-597 are mutated from "ATG" to "GAT", nucleotides 682-684 are mutated from "ATG" to "CTG", and nucleotides 799-801 are mutated from "GCG" to "GAT". Mutant T7 RNA polymerase M10 (M190D / A258D / K363P): nucleotides 595-597 are mutated from "ATG" to "GAT", nucleotides 799-801 are mutated from "GCG" to "GAT", and nucleotides 1114-1116 are mutated from "AAA" to "CCG". Mutant T7 RNA polymerase M11 (M190D / A258D / A124E): nucleotides 397-399 are mutated from “GCG” to “GAA”, nucleotides 595-597 are mutated from “ATG” to “GAT”, and nucleotides 799-801 are mutated from “GCG” to “GAT”.

[0050]

[0051] 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, and PET-28a / M11. Each recombinant expression vector was transformed into E. coli BL21DE3 Star competent cells, and positive single colonies were screened for sequencing verification. Single colonies with valid verification results were obtained.

[0052] 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℃.

[0053] 3. Protein purification: (1) High-pressure disruption of induced expression cells: After induction of expression, bacterial cells were frozen at -80℃. Based on the recorded wet weight of the bacterial 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 bacterial 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.

[0054] (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 (50mM Tris-HCl, pH 7.8 at 25℃, 100mM NaCl, 80 mM Imidazole, 5% Glycerol); the elution buffer was buffer B (50mM Tris-HCl, pH 7.8 at 25℃, 100mM NaCl, 500 mM Imidazole, 5% Glycerol). The supernatant from step (1) was loaded into the chromatography column 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.

[0055] (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.

[0056] (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.

[0057] Detection Example 1 Using the nucleic acid sequence shown in SEQ ID NO: 3 as a template, 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-M11, respectively. Transcription was performed at 37℃ for 2 h. The transcribed product was digested with DNase I to inactivate the protein, and then 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 detection. The transcripts were quantified using a QubitRNA Quantification Kit (Thermo Fisher Scientific). The diluted RNA products were detected using the NovoFast dsRNA 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 as described in 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%).

[0058] Table 1

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

[0060] The constructed standard curve is as follows Figure 1 As shown. The results are as follows. Figure 2 (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-M11 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) Mutations are made at amino acid sites based on the amino acid sequence of the wild-type T7 RNA polymerase, including M190D and A258D; 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, The mutant T7 RNA polymerase also contains at least one of the following amino acid mutation sites: S192P, V174K, H161E, K163W, M46L, N165P, Q169D, M219L, K363P, and A124E.

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 B1-13): B1-1) The nucleic acid molecule obtained by mutating nucleotides 595-597 of the nucleic acid sequence shown in SEQ ID NO: 2 from "ATG" to "GAT" and nucleotides 799-801 from "GCG" to "GAT" is nucleotide 28-2676. B1-2) The nucleic acid molecule obtained by mutating nucleotides 595-597 from "ATG" to "GAT", nucleotides 601-603 from "AGC" to "CCG", and nucleotides 799-801 from "GCG" to "GAT" after mutating nucleotides 595-597 from "ATG" to "GAT"; B1-3) The nucleic acid molecule obtained by mutating nucleotides 547-549 from "GTG" to "AAA", nucleotides 595-597 from "ATG" to "GAT", and nucleotides 799-801 from "GCG" to "GAT" is nucleotide 28-2676. B1-4) The nucleic acid molecule obtained by mutating nucleotides 508-510 from "CAT" to "GAA", nucleotides 595-597 from "ATG" to "GAT", and nucleotides 799-801 from "GCG" to "GAT" after mutating nucleotides 508-510 from "CAT" to "GAA", nucleotides 595-597 from "ATG" to "GAT", and nucleotides 799-801 from "GCG" to "GAT" is nucleotide 28-2676. B1-5) The nucleic acid molecule obtained by mutating nucleotides 514-516 of the nucleic acid sequence shown in SEQ ID NO: 2 from "AAA" to "TGG", nucleotides 595-597 from "ATG" to "GAT", 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 595-597 from "ATG" to "GAT", and nucleotides 799-801 from "GCG" to "GAT" is nucleotide 28-2676. B1-7) The nucleic acid molecule obtained by mutating nucleotides 520-522 from "AAC" to "CCG", nucleotides 595-597 from "ATG" to "GAT", and nucleotides 799-801 from "GCG" to "GAT" after mutating nucleotides 520-522 from "AAC" to "CCG", nucleotides 595-597 from "ATG" to "GAT", and nucleotides 799-801 from "GCG" to "GAT" is nucleotide 28-2676. B1-8) The nucleic acid molecule obtained by mutating nucleotides 532-534 from "CAG" to "GAT", nucleotides 595-597 from "ATG" to "GAT", and nucleotides 799-801 from "GCG" to "GAT" is nucleotide 28-2676. B1-9) The nucleic acid molecule obtained by mutating nucleotides 595-597 from "ATG" to "GAT", nucleotides 799-801 from "GCG" to "GAT", and nucleotides 682-684 from "ATG" to "CTG" after mutating nucleotides 595-597 from "ATG" to "GAT" and nucleotides 799-801 from "GCG" to "GAT", and nucleotides 682-684 from "ATG" to "CTG" is nucleotide 28-2676. B1-10) The nucleic acid molecule obtained by mutating nucleotides 595-597 from "ATG" to "GAT", nucleotides 799-801 from "GCG" to "GAT", and nucleotides 1114-1116 from "AAA" to "CCG" after mutating nucleotides 28-2676 of the nucleic acid sequence shown in SEQ ID NO: 2; 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", nucleotides 595-597 from "ATG" to "GAT", and nucleotides 799-801 from "GCG" to "GAT" is nucleotide 28-2676. The nucleic acid sequence shown in B1-12) has more than 70% homology with any of B1-1) to B1-11) and encodes the mutant T7 RNA polymerase; B1-13) hybridizes under stringent conditions with any of the specified nucleic acid sequences from B1-1) to B12) and encodes the mutant T7 RNA polymerase.

5. An enzyme preparation, characterized in that, Includes the mutant T7 RNA polymerase as described in claim 1 or 2.

6. 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.

7. A method, characterized in that, Includes the following steps: RNA molecules are amplified using the mutant T7 RNA polymerase of claim 1 or 2 or the enzyme preparation of claim 5.

8. The application of any one of C1) to C3) in the preparation of nucleic acid transcription products; C1), the mutant T7 RNA polymerase as described in claim 1 or 2; C2), the biomaterial as described in claim 3 or 4; C3), the enzyme preparation according to claim 5.

9. The use of the mutant T7 RNA polymerase of claim 1 or 2 or the enzyme preparation of claim 5 in nucleic acid transcription.

10. A reagent kit, characterized in that, Includes the mutant T7 RNA polymerase of claim 1 or 2 or the enzyme preparation of claim 5.

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