A transcription reaction solution, a method for preparing locked nucleic acid-modified RNA, and application of a mutant T7 RNA polymerase

Abstract

The present application relates to the field of genetic engineering technology, and specifically, to a method for preparing a transcription reaction solution, a locked nucleic acid-modified RNA, and an application of a mutated T7 RNA polymerase. A transcription reaction solution for preparing locked nucleic acid-modified RNA comprises a DNA template, a mutated T7 RNA polymerase, LNA-NTPs, and a buffer; wherein the amino acid sequence of the mutated T7 RNA polymerase is shown in SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4, SEQ ID NO.5, SEQ ID NO.6, or SEQ ID NO.7. The present application can significantly improve the transcription yield of locked nucleic acid-modified RNA by adding the above-mentioned mutated T7 RNA polymerase to the transcription reaction solution for synthesizing locked nucleic acid-modified RNA, and the operation is simple, which is conducive to industrial production.

Description

Technical Field

[0001] The present application relates to the field of genetic engineering technology, and in particular to a transcription reaction solution, a method for preparing locked nucleic acid-modified RNA, and an application of a mutant T7 RNA polymerase. Background Art

[0002] Locked Nucleic Acid (LNA) is a modified nucleic acid derivative in which the 2'-O- and 4'-C-positions on the ribose are bridged by a methylene group to form a "locked" structure. This unique "locked" structure reduces the flexibility of the ribose structure and increases the stability of the local structure of the phosphate backbone.

[0003] RNA is often used as a therapeutic drug to regulate and limit the expression of mRNA of abnormal genes, such as antisense RNA, ribozymes, small interfering RNA (siRNA), microRNA and aptamers. After being modified with locked nucleic acid, the stability of RNA drugs in the body is greatly improved.

[0004] However, the current yield of locked nucleic acid-modified RNA is low, resulting in a high preparation cost of locked nucleic acid-modified RNA, which is not conducive to industrial production. Summary of the invention

[0005] The purpose of the present application is to provide a transcription reaction solution for preparing locked nucleic acid-modified RNA, a method for preparing locked nucleic acid-modified RNA, and the use of a modified T7 RNA polymerase in preparing locked nucleic acid-modified RNA, which aims to improve the existing technical problem of low yield of locked nucleic acid-modified RNA.

[0006] In a first aspect, the present application provides a transcription reaction solution for preparing locked nucleic acid modified RNA, comprising a DNA template, a mutant T7 RNA polymerase, LNA-NTPs and a buffer; wherein the mutant T7 RNA polymerase comprises at least one of a T7 RNA polymerase having an amino acid sequence as shown in SEQ ID NO.1, a T7 RNA polymerase having an amino acid sequence as shown in SEQ ID NO.2, a T7 RNA polymerase having an amino acid sequence as shown in SEQ ID NO.3, a T7 RNA polymerase having an amino acid sequence as shown in SEQ ID NO.4, a T7 RNA polymerase having an amino acid sequence as shown in SEQ ID NO.5, a T7 RNA polymerase having an amino acid sequence as shown in SEQ ID NO.6 and a T7 RNA polymerase having an amino acid sequence as shown in SEQ ID NO.7.

[0007] In the present application, when at least one of the mutant T7 RNA polymerases provided above is added to the transcription reaction solution for synthesizing locked nucleic acid-modified RNA, the transcription yield of locked nucleic acid-modified RNA can be significantly improved, the operation is simple, and it is conducive to industrial production.

[0008] The second aspect of the present application provides a method for preparing locked nucleic acid-modified RNA, comprising subjecting the transcription reaction solution for preparing locked nucleic acid-modified RNA provided in the first aspect to a transcription reaction.

[0009] Optionally, the method for preparing locked nucleic acid-modified RNA further comprises: adding a DNA template degrading enzyme to the transcribed system to obtain a first mixed system, and performing a digestion reaction; and then adding a precipitant to the first mixed system to obtain a second mixed system, and performing a purification treatment.

[0010] Optionally, the DNA template degrading enzyme is selected from DNase I enzyme, and the total amount of the DNA template degrading enzyme in the first mixed system is 1-3U.

[0011] Optionally, the precipitant is selected from ammonium acetate, and the final molar concentration of the ammonium acetate in the second mixed system is 3-6M.

[0012] Optionally, the precipitant is selected from lithium chloride, and the final molar concentration of the lithium chloride in the second mixed system is 6-8M.

[0013] The present application can significantly improve the transcription yield of locked nucleic acid modified RNA by performing a transcription reaction on a transcription reaction solution of a mutated T7 RNA polymerase containing at least one of the amino acid sequences shown in SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4, SEQ ID NO.5, SEQ ID NO.6 and SEQ ID NO.7; in addition, the yield of locked nucleic acid modified RNA can be further improved by combining the above-mentioned digestion reaction and purification treatment.

[0014] In a third aspect, the present application provides a mutated T7 RNA polymerase for use in preparing locked nucleic acid modified RNA, wherein the mutated T7 RNA polymerase includes at least one of a T7 RNA polymerase having an amino acid sequence as shown in SEQ ID NO.1, a T7 RNA polymerase having an amino acid sequence as shown in SEQ ID NO.2, a T7 RNA polymerase having an amino acid sequence as shown in SEQ ID NO.3, a T7 RNA polymerase having an amino acid sequence as shown in SEQ ID NO.4, a T7 RNA polymerase having an amino acid sequence as shown in SEQ ID NO.5, a T7 RNA polymerase having an amino acid sequence as shown in SEQ ID NO.6, and a T7 RNA polymerase having an amino acid sequence as shown in SEQ ID NO.7.

[0015] Using the mutant T7 RNA polymerase mentioned above for preparing locked nucleic acid-modified RNA can significantly improve the transcription yield of locked nucleic acid-modified RNA, is simple to operate, and is conducive to industrial production. DETAILED DESCRIPTION

[0016] In order to make the purpose, technical scheme and advantages of the embodiments of the present application clearer, the technical scheme in the embodiments of the present application will be described clearly and completely below. If the specific conditions are not specified in the embodiments, they are carried out according to the conventional conditions or the conditions recommended by the manufacturer. If the manufacturer is not specified for the reagents or instruments used, they are all conventional products that can be purchased commercially.

[0017] The following is a specific description of a transcription reaction solution for preparing locked nucleic acid-modified RNA, a method for preparing locked nucleic acid-modified RNA, and the use of a modified T7 RNA polymerase in preparing locked nucleic acid-modified RNA provided in the embodiments of the present application.

[0018] The present application provides a transcription reaction solution for preparing RNA, comprising a DNA template, a mutant T7 RNA polymerase, LNA-NTPs and a buffer; wherein the mutant T7 RNA polymerase comprises at least one of a T7 RNA polymerase with an amino acid sequence as shown in SEQ ID NO.1, a T7 RNA polymerase with an amino acid sequence as shown in SEQ ID NO.2, a T7 RNA polymerase with an amino acid sequence as shown in SEQ ID NO.3, a T7 RNA polymerase with an amino acid sequence as shown in SEQ ID NO.4, a T7 RNA polymerase with an amino acid sequence as shown in SEQ ID NO.5, a T7 RNA polymerase with an amino acid sequence as shown in SEQ ID NO.6 and a T7 RNA polymerase with an amino acid sequence as shown in SEQ ID NO.7.

[0019] LNA-NTPs (Nucleoside triphosphates) refers to a nucleoside triphosphate bridged between the 2'-O- and 4'-C-positions on the ribose by a methylene group, and is the raw material for synthesizing locked nucleic acid modified RNA. It should be noted that in the present application, LNA-NTPs refers to a base in the molecular structure that can be a natural or modified base, and LNA-NTPs refers to a triphosphate structure in the molecular structure that can also be a modified or unmodified triphosphate structure.

[0020] In this embodiment, the LNA-NTPs can be selected from LNA-ATP, LNA-GTP, LNA-CTP and LNA-UTP.

[0021] The transcription reaction solution can be used to perform a transcription reaction in a buffer system, using the middle template strand of the DNA template as a template and LNA-NTPs as a raw material, and performing in vitro transcription under the catalysis of a mutant T7 RNA polymerase to synthesize locked nucleic acid-modified RNA.

[0022] In the present application, when a mutated T7 RNA polymerase having an amino acid sequence such as at least one of SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4, SEQ ID NO.5, SEQ ID NO.6 and SEQ ID NO.7 is added to a transcription reaction solution for synthesizing a locked nucleic acid-modified RNA, the transcription yield of the locked nucleic acid-modified RNA can be significantly improved compared to using a natural T7 RNA polymerase as the RNA polymerase in the transcription system. The operation is simple and is conducive to industrial production.

[0023] In this embodiment, the DNA template includes a target sequence and a promoter corresponding to a mutated T7 RNA polymerase with an amino acid sequence such as SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4, SEQ ID NO.5, SEQ ID NO.6 or SEQ ID NO.7.

[0024] In this example, the DNA template is selected from linearized plasmid DNA.

[0025] It should be noted that the transcription reaction solution used to prepare RNA in the present application may include, in addition to LNA-NTPs, NTPs (i.e., modified or unmodified nucleoside triphosphates), such as natural ATP, natural GTP, natural CTP, natural UTP, modified ATP, modified GTP, modified CTP, and modified UTP, etc.

[0026] In this embodiment, the pH of the buffer is 6.5-7.5, which is conducive to better transcription reaction to synthesize locked nucleic acid-modified RNA. As an example, the pH of the buffer can be 6.5, 7.0, 7.2, 7.5, etc.

[0027] In this embodiment, the buffer comprises a biological buffer, an activator, a polyamine, a detergent and a reducing agent. The biological buffer is used to maintain the pH required for the transcription reaction. The activator is used to form a complex with LNA-NTPs and a DNA template to be recognized by the above-mentioned mutant RNA polymerase, so that the DNA template is stably transcribed to synthesize the RNA modified by the locked nucleic acid. The polyamine has a promoting effect on the recognition and binding of the DNA template and the above-mentioned mutant RNA polymerase, which is conducive to the transcription reaction to be more easily carried out to synthesize the RNA modified by the locked nucleic acid. The role of the detergent is to increase the stability of the enzyme in the transcription reaction solution. The reducing agent can inhibit the dimerization of DNA, which is conducive to promoting the efficient conduct of the transcription reaction to synthesize the RNA modified by the locked nucleic acid.

[0028] In this embodiment, the biological buffer in the buffer can be selected from Tris (tris(hydroxymethyl)aminomethane), HEPES (4-hydroxyethylpiperazineethanesulfonic acid), MOPS (3-(N-morpholino)propanesulfonic acid) or PIPES (piperazine-1,4-diethanesulfonic acid). The above substances can effectively maintain the pH required for the transcription reaction. It should be noted that in other embodiments of the present application, the biological buffer may not be limited to the above substances. For example, the biological buffer may be BICINE (N,N-dihydroxyethylglycine), CAPS (3-(cyclohexylamine)-1-propanesulfonic acid), TAPS (N-tris(hydroxymethyl)methyl-3-aminopropanesulfonic acid), EPPS (N-(2-hydroxyethyl)piperazine-N'-3-propanesulfonic acid), MOPSO (3-(N-morpholino)-2-hydroxypropanesulfonic acid) and other substances.

[0029] Further, in this embodiment, the final molar concentration of the biological buffer in the transcription reaction solution is 25-35 mM, which can maintain the stability of the transcription reaction solution and facilitate the transcription reaction to synthesize the locked nucleic acid-modified RNA. As an example, the final molar concentration of the biological buffer in the transcription reaction solution can be 25 mM, 27 mM, 30 mM, 32 mM, 35 mM, etc.

[0030] In this embodiment, the activator can be selected from magnesium ions or sodium ions, which can better form a complex with LNA-NTPs and DNA templates to be more easily recognized by the above-mentioned mutant RNA polymerase, thereby facilitating the transcription reaction to synthesize locked nucleic acid-modified RNA.

[0031] Further, in this embodiment, the final molar concentration of the activator in the transcription reaction solution is 4.5-5.5 mM, which can not only provide the ion concentration required for the transcription reaction, but also facilitate the complexation of magnesium ions or sodium ions with LNA-NTPs and DNA templates to form a complex that is easily recognized by the above-mentioned mutant RNA polymerase. As an example, the final molar concentration of the activator in the transcription reaction solution can be 4.5 mM, 4.8 mM, 5.0 mM, 5.5 mM, etc.

[0032] In this embodiment, the polyamine can be selected from spermidine, putrescine or spermine, which can better promote the recognition and binding of the DNA template and the mutant RNA polymerase, and facilitate the transcription reaction to synthesize the locked nucleic acid modified RNA. It should be noted that in other embodiments of the present application, the above substances can also be replaced by spermidine salts, such as spermidine phosphate or spermidine hydrochloride.

[0033] Furthermore, the final molar concentration of the polyamine in the transcription reaction solution is 0.8-1.2 mM, which can further promote the recognition and binding of the DNA template and the above-mentioned mutant RNA polymerase. As an example, the final molar concentration of the polyamine in the transcription reaction solution can be 0.8 mM, 1.0 mM, 1.2 mM, etc.

[0034] In this embodiment, the detergent can be selected from Triton X-100 (polyethylene glycol octylphenyl ether), which can better stabilize the mutant T7 RNA polymerase. Further, the mass fraction of the detergent in the transcription reaction solution is 0.01-0.03%, which can further achieve the effect of stabilizing the mutant T7 RNA polymerase. As an example, the mass fraction of the detergent in the transcription reaction solution can be 0.01%, 0.02% and 0.03%, etc.

[0035] In this embodiment, the reducing agent can be selected from DTT (dithiothreitol) or β-mercaptoethanol. The above substances as reducing agents can better inhibit the dimerization of DNA.

[0036] Furthermore, the final molar concentration of the reducing agent in the transcription reaction solution is 2.5-3.5 mM, which can further inhibit the dimerization of DNA. As an example, the final molar concentration of the reducing agent in the transcription reaction solution can be 2.5 mM, 3.0 mM, 3.5 mM, etc.

[0037] In addition, in this embodiment, the buffer further includes inorganic pyrophosphatase and nuclease inhibitor. Inorganic pyrophosphatase can promote the transcription reaction, which is beneficial to increase the yield of the locked nucleic acid modified RNA obtained by transcription. Nuclease inhibitor has the effect of inhibiting RNase.

[0038] Further, the final concentration of inorganic pyrophosphatase in the transcription reaction solution is 0.01-1U / μl, which can further promote the transcription reaction to synthesize locked nucleic acid-modified RNA; as an example, the final concentration of inorganic pyrophosphatase in the transcription reaction solution can be 0.01U / μl, 0.02U / μl, 0.05U / μl and 1U / μl, etc. The final concentration of nuclease inhibitor in the transcription reaction solution is 1-5U / μl, which can further inhibit RNase; as an example, the final concentration of nuclease inhibitor in the transcription reaction solution can be 1U / μl, 2U / μl, 4U / μl and 5U / μl, etc.

[0039] The present application also provides a method for preparing locked nucleic acid-modified RNA, comprising subjecting the above-provided transcription reaction solution for preparing locked nucleic acid-modified RNA to a transcription reaction.

[0040] The present application can significantly improve the transcription yield of locked nucleic acid modified RNA by performing a transcription reaction with a transcription reaction solution of a mutated T7 RNA polymerase containing at least one of the amino acid sequences shown in SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4, SEQ ID NO.5, SEQ ID NO.6 and SEQ ID NO.7.

[0041] In this embodiment, the temperature of the transcription reaction for preparing the locked nucleic acid-modified RNA is 30-40° C. and the time is 10-18 h. Further, the temperature of the transcription reaction is 37° C. and the time is 16 h.

[0042] In this embodiment, after the transcription reaction, a DNA template degrading enzyme is added to the transcribed system to obtain a first mixed system for a digestion reaction; the original DNA template can be digested, which is beneficial for the subsequent purification reaction and is beneficial for improving the purity of the locked nucleic acid-modified RNA in the in vitro transcribed system.

[0043] In some embodiments of the present application, the DNA template degrading enzyme is selected from DNase I enzyme (Chinese name: deoxyribonuclease I, a nuclease that can digest single-stranded or double-stranded DNA to produce monodeoxynucleotides or single-stranded or double-stranded oligodeoxynucleotides). DNase I enzyme can digest the original DNA template well, which is beneficial to the subsequent purification reaction.

[0044] In some embodiments of the present application, the total amount of the DNA template degrading enzyme in the first mixed system is 1-3 U, which can further promote the digestion of the original DNA template. As an example, the total amount of the DNA template degrading enzyme in the first mixed system can be 1U, 2U, 3U, etc.

[0045] In this embodiment, after the digestion reaction, a precipitant is added to the first mixed system after the digestion reaction to obtain a second mixed system for purification: the reaction product locked nucleic acid-modified RNA can be precipitated, while other impurities are not precipitated, thereby achieving purification.

[0046] In some embodiments of the present application, the precipitant may be selected from ammonium acetate or lithium chloride, which may better allow the reaction product locked nucleic acid-modified RNA to precipitate without precipitating other impurities.

[0047] Furthermore, the purification treatment includes adding a precipitant to the first mixed system after the digestion reaction and mixing thoroughly to obtain a second mixed system, then placing the mixture in an ice bath, centrifuging, and removing the supernatant to obtain a precipitate.

[0048] In the embodiment, after the reaction product locked nucleic acid-modified RNA is precipitated with a precipitant, the precipitate is washed to obtain the final locked nucleic acid-modified RNA. Further, in this embodiment, the precipitate is washed with 70% by mass ethanol.

[0049] The method for preparing locked nucleic acid-modified RNA provided in the present application has at least the following advantages:

[0050] The present application can significantly improve the transcription yield of locked nucleic acid modified RNA by performing a transcription reaction on a transcription reaction solution of a mutated T7 RNA polymerase containing at least one of the amino acid sequences shown in SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4, SEQ ID NO.5, SEQ ID NO.6 and SEQ ID NO.7; in addition, the yield of locked nucleic acid modified RNA can be further improved by combining the above-mentioned digestion reaction and purification treatment.

[0051] The present application also provides an application of a mutated T7 RNA polymerase in preparing locked nucleic acid modified RNA; wherein the mutated T7 RNA polymerase includes at least one of a T7 RNA polymerase having an amino acid sequence as shown in SEQ ID NO.1, a T7 RNA polymerase having an amino acid sequence as shown in SEQ ID NO.2, a T7 RNA polymerase having an amino acid sequence as shown in SEQ ID NO.3, a T7 RNA polymerase having an amino acid sequence as shown in SEQ ID NO.4, a T7 RNA polymerase having an amino acid sequence as shown in SEQ ID NO.5, a T7 RNA polymerase having an amino acid sequence as shown in SEQ ID NO.6, and a T7 RNA polymerase having an amino acid sequence as shown in SEQ ID NO.7.

[0052] Compared with using the natural T7 RNA polymerase as the RNA polymerase in the transcription system, using the mutated T7 RNA polymerase to prepare locked nucleic acid-modified RNA can significantly improve the transcription yield of locked nucleic acid-modified RNA, is simple to operate, and is conducive to industrial production.

[0053] The features and performance of the present application are further described in detail below in conjunction with the embodiments.

[0054] Example 1

[0055] This embodiment provides a transcription reaction solution for preparing locked nucleic acid-modified RNA and a method for preparing locked nucleic acid-modified RNA, which specifically includes the following steps:

[0056] (1) Preparation of transcription reaction solution:

[0057] 2 μl of 10× buffer, 2.5 μl of 20 mM LNA-ATP, 2.5 μl of 20 mM GTP, 2.5 μl of 20 mM CTP, 2.5 μl of 20 mM LNA-UTP, 2 μl of mixed enzyme solution and 2 μl of X μg / μl DNA template were mixed, and nuclease-free water was added to the total volume of the system to 20 μl to prepare a transcription reaction solution.

[0058] Among them, the configuration of 10× buffer is as follows: HEPES-sodium hydroxide (pH7.0@25°C), magnesium acetate, spermidine, Triton X-100 and DTT are mixed to prepare 10× buffer; the concentrations of HEPES-sodium hydroxide, magnesium acetate, spermidine, Triton X-100 and DTT in 10× buffer are 300mM, 50mM, 10mM, 0.02% (mass fraction) and 30mM, respectively.

[0059] Preparation of the mixed enzyme solution: a mixed enzyme system is prepared by mixing a mutant T7 RNA polymerase having an amino acid sequence as shown in SEQ ID NO.1, an inorganic pyrophosphatase and a nuclease inhibitor; the concentrations of the mutant T7 RNA polymerase having an amino acid sequence as shown in SEQ ID NO.1, the inorganic pyrophosphatase and the nuclease inhibitor in the mixed enzyme solution are 200 U / μl, 0.5 U / μl and 20 U / μl, respectively.

[0060] The sequence of the DNA template is as follows1: (Note: The underlined part in the sequence of the above DNA template is the primer, and the bold part is the promoter).

[0061] (2) Preparation method of locked nucleic acid modified RNA:

[0062] After the transcription reaction solution provided above was transcribed at 37°C for 16 hours, 1U of DNase I enzyme was added to digest the original DNA template at 37°C for 30 minutes. Then 20μl of 5M ammonium acetate was added and mixed thoroughly, ice-bathed for 30 minutes, centrifuged at 4°C for 15 minutes, the supernatant was removed, and the precipitate was retained. The precipitate was rinsed twice with 70% ethanol by mass to obtain locked nucleic acid-modified RNA.

[0063] Example 2

[0064] The difference between Example 2 and Example 1 is that HEPES-sodium hydroxide is replaced by Tris-HCl, and the concentration of Tris-HCl in 10× buffer is 350 mM.

[0065] Example 3

[0066] The difference between Example 3 and Example 1 is that magnesium acetate is replaced by sodium acetate, and the concentration of sodium acetate in 10× buffer is 55 mM.

[0067] Example 4

[0068] The difference between Example 4 and Example 1 is that spermidine is replaced by putrescine, and the concentration of putrescine in 10× buffer is 12 mM.

[0069] Example 5

[0070] The difference between Example 5 and Example 1 is that DTT is replaced by β-mercaptoethanol, and the concentration of β-mercaptoethanol in 10× buffer is 35 mM.

[0071] Example 6

[0072] The difference between Example 6 and Example 1 is that the amino acid sequence of the mutant T7 RNA polymerase is shown in SEQ ID NO.2.

[0073] Example 7

[0074] The difference between Example 7 and Example 1 is that the amino acid sequence of the mutant T7 RNA polymerase is shown in SEQ ID NO.3.

[0075] Example 8

[0076] The difference between Example 8 and Example 1 is that the amino acid sequence of the mutant T7 RNA polymerase is shown in SEQ ID NO.4.

[0077] Example 9

[0078] The difference between Example 9 and Example 1 is that the amino acid sequence of the mutant T7 RNA polymerase is shown in SEQ ID NO.5.

[0079] Example 10

[0080] The difference between Example 10 and Example 1 is that the amino acid sequence of the mutant T7 RNA polymerase is shown in SEQ ID NO.6.

[0081] Embodiment 11

[0082] The difference between Example 11 and Example 1 is that the amino acid sequence of the mutant T7 RNA polymerase is shown in SEQ ID NO.7.

[0083] Comparative Example 1

[0084] The difference between Comparative Example 1 and Example 1 is that the RNA polymerase is natural T7 RNA polymerase.

[0085] Comparative Example 2

[0086] The difference between Comparative Example 2 and Example 1 is that the concentration of HEPES-sodium hydroxide in the 10× buffer is 100 mM.

[0087] Comparative Example 3

[0088] The difference between Comparative Example 3 and Example 1 is that magnesium acetate is replaced by sodium acetate, and the concentration of sodium acetate in 10× buffer is 30 mM.

[0089] Comparative Example 4

[0090] The difference between Comparative Example 4 and Example 1 is that spermidine is replaced by putrescine, and the concentration of putrescine in 10× buffer is 5 mM.

[0091] Comparative Example 5

[0092] The difference between Comparative Example 5 and Example 1 is that DTT is replaced by β-mercaptoethanol, and the concentration of β-mercaptoethanol in 10× buffer is 10 mM.

[0093] Test example

[0094] The locked nucleic acid modified RNA obtained by the transcription reaction solution for preparing locked nucleic acid modified RNA and the locked nucleic acid modified RNA preparation method provided in Examples 1-5 and Comparative Examples 1-5 was tested for yield. The locked nucleic acid modified RNA precipitate obtained in Examples 1-5 and Comparative Examples 1-5 was dried, nuclease-free water was added to dissolve the precipitate, and the OD was measured. 260 The absorbance M of the locked nucleic acid was measured, and the yield of RNA modified with locked nucleic acid was calculated (RNA yield = M * dilution factor * 0.04 μg / μl * volume of the system, where 0.04 μg / μl is OD 260 =1 is the corresponding RNA concentration value), and the results are shown in Table 1.

[0095] Table 1

[0096] Yield (μg) Yield (μg) Yield (μg) Example 1 18 Example 7 11 Comparative Example 1 4 Example 2 15 Example 8 10 Comparative Example 2 10 Example 3 10 Example 9 8 Comparative Example 3 6 Example 4 14 Example 10 6 Comparative Example 4 9 Example 5 12 Embodiment 11 6 Comparative Example 5 8 Example 6 10

[0097] As can be seen from Table 1: the locked nucleic acid modified RNA yields of Examples 1 and Examples 6-11 are higher than the locked nucleic acid modified RNA yield of Comparative Example 1, indicating that the mutant T7 RNA polymerases with amino acid sequences such as SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4, SEQ ID NO.5, SEQ ID NO.6 and SEQ ID NO.7 can effectively increase the locked nucleic acid modified RNA yield relative to the natural T7 RNA polymerase; at the same time, the locked nucleic acid modified RNA yields of Examples 1 and 2 are higher than the locked nucleic acid modified yield of Comparative Example 2, the locked nucleic acid modified RNA yields of Examples 1 and 3 are higher than the locked nucleic acid modified yield of Comparative Example 3, the locked nucleic acid modified RNA yields of Examples 1 and 4 are higher than the locked nucleic acid modified yield of Comparative Example 4, and the locked nucleic acid modified RNA yields of Examples 1 and 5 are higher than the locked nucleic acid modified yield of Comparative Example 5, indicating that the substance concentration in the buffer of the present application has an advantage and can further increase the locked nucleic acid modified RNA yield.

[0098] In addition, the mutant T7 RNA polymerase used in the transcription reaction solution for preparing locked nucleic acid-modified RNA and the method for preparing locked nucleic acid-modified RNA provided in Example 1 is a polymerase with a amino acid sequence such as SEQ ID NO.1, and combined with specific buffer substances and concentrations, the yield of locked nucleic acid-modified RNA can be as high as 18 μg.

[0099] Therefore, the transcription reaction solution for preparing locked nucleic acid-modified RNA and the method for preparing locked nucleic acid-modified RNA provided in the present application can effectively improve the yield of locked nucleic acid-modified RNA, is simple to operate, and is conducive to industrial production.

[0100] The above description is only the preferred embodiment of the present application and is not intended to limit the present application. For those skilled in the art, the present application may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application shall be included in the protection scope of the present application. SEQUENCE LISTING <110> Shanghai Zhaowei Technology Development Co., Ltd. Shanghai Zhaowei Bioengineering Co., Ltd. <120> A transcription reaction solution, a method for preparing locked nucleic acid-modified RNA, and application of mutant T7 RNA polymerase use <130> 2022.01.28 <160> 7 <170> PatentIn version 3.5 <210> 1 <211> 883 <212> PRT <213> artificial sequence <400> 1 Met Asn Thr Ile Asn Ile Ala Lys Asn Asp Phe Ser Asp Ile Glu Leu 1 5 10 15 Ala Ala Ile Pro Phe Asn Thr Leu Ala Asp His Tyr Gly Glu Arg Leu 20 25 30 Ala Arg Glu Gln Leu Ala Leu Glu His Glu Ser Tyr Glu Met Gly Glu 35 40 45 Ala Arg Phe Arg Lys Met Phe Glu Arg Gln Leu Lys Ala Gly Glu Val 50 55 60 Ala Asp Asn Ala Ala Ala Lys Pro Leu Ile Thr Thr Leu Leu Pro Lys 65 70 75 80 Met Ile Ala Arg Ile Asn Asp Trp Phe Glu Glu Val Lys Ala Lys Arg 85 90 95 Gly Lys Arg Pro Thr Ala Phe Gln Phe Leu Gln Glu Ile Lys Pro Glu 100 105 110 Ala Val Ala Tyr Ile Thr Ile Lys Thr Thr Leu Ala Cys Leu Thr Ser 115 120 125 Ala Asp Asn Thr Thr Val Gln Ala Val Ala Ser Ala Ile Gly Arg Ala 130 135 140 Ile Glu Asp Glu Ala Arg Phe 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Leu Pro Met Lys Pro Glu Asp Ile Asp 355 360 365 Met Asn Pro Glu Ala Leu Thr Ala Trp Arg Arg Ala Ala Ala Ala Val 370 375 380 Tyr Arg Lys Asp Lys Ala Arg Lys Ser Arg Arg Ile Ser Leu Glu Phe 385 390 395 400 Met Leu Glu Gln Ala Asn Lys Phe Ala Asn His Lys Ala Ile Trp Phe 405 410 415 Pro Tyr Asn Met Asp Trp Arg Gly Arg Val Tyr Ala Val Ser Met Phe 420 425 430 Asn Pro Gln Gly Asn Asp Met Thr Lys Gly Leu Leu Thr Leu Ala Lys 435 440 445 Gly Lys Pro Ile Gly Lys Glu Gly Tyr Tyr Trp Leu Lys Ile His Gly 450 455 460 Ala Asn Cys Ala Gly Val Asp Lys Val Pro Phe Pro Glu Arg Ile Lys 465 470 475 480 Phe Ile Glu Glu Asn His Glu Asn Ile Met Ala Cys Ala Lys Ser Pro 485 490 495 Leu Glu Asn Thr Trp Trp Ala Glu Gln Asp Ser Pro Phe Cys Phe Leu 500 505 510 Ala Phe Cys Phe Glu Tyr Ala Gly Val Gln His His Gly Leu Ser Tyr 515 520 525 Asn Cys Ser Leu Pro Leu Ala Phe Asp Gly Ser Cys Ser Gly Ile Gln 530 535 540 His Phe Ser Ala Met Leu Arg Asp Glu Val Gly Gly Arg Ala Val Asn 545 550 555 560 Leu Leu Pro Ser Glu Thr Val Gln Asp Ile Tyr Gly Ile Val Ala Lys 565 570 575 Lys Val Asn Glu Ile Leu Gln Ala Asp Ala Ile Asn Gly Thr Asp Asn 580 585 590 Glu Val Val Thr Val Thr Asp Glu Asn Thr Gly Glu Ile Ser Glu Lys 595 600 605 Val Lys Leu Gly Thr Lys Ala Leu Ala Gly Gln Trp Leu Ala Tyr Gly 610 615 620 Val Thr Arg Ser Val Thr Lys Arg Ser Val Met Thr Leu Ala Leu Gly 625 630 635 640 Ser Lys Glu Phe Gly Phe Arg Gln Gln Val Leu Glu Asp Thr Ile Gln 645 650 655 Pro Ala Ile Asp Ser Gly Lys Gly Leu Met Phe Thr Gln Pro Asn Gln 660 665 670 Ala Ala Gly Tyr Met Ala Lys Leu Ile Trp Glu Ser Val Ser Val Thr 675 680 685 Val Val Ala Ala Val Glu Ala Met Asn Trp Leu Lys Ser Ala Ala Lys 690 695 700 Leu Leu Ala Ala Glu Val Lys Asp Lys Lys Thr Gly Glu Ile Leu Arg 705 710 715 720 Lys Arg Cys Ala Val His Trp Val Thr Pro Asp Gly Phe Pro Val Trp 725 730 735 Gln Glu Tyr Lys Lys Pro Ile Gln Thr Arg Leu Asn Leu Met Phe Leu 740 745 750 Gly Gln Phe Arg Leu Gln Pro Thr Ile Asn Thr Asn Lys Asp Ser Glu 755 760 765 Ile Asp Ala His Lys Gln Glu Ser Gly Ile Ala Pro Asn Phe Val Ala 770 775 780 Ser Gln Asp Gly Ser His Leu Arg Lys Thr Val Val Trp Ala His Glu 785 790 795 800 Lys Tyr Gly Ile Glu Ser Phe Ala Leu Ile His Asp Ser Phe Gly Thr 805 810 815 Ile Pro Ala Asp Ala Ala Asn Leu Phe Lys Ala Val Arg Glu Thr Met 820 825 830 Val Asp Thr Tyr Glu Ser Cys Asp Val Leu Ala Asp Phe Tyr Asp Gln 835 840 845 Phe Ala Asp Gln Leu His Glu Ser Gln Leu Asp Lys Met Pro Ala Leu 850 855 860 Pro Ala Lys Gly Asn Leu Asn Leu Arg Asp Ile Leu Glu Ser Asp Phe 865 870 875 880 Ala Phe Ala <210> 2 <211> 883 <212> PRT <213> Artificial Sequence <400> 2 Met Asn Thr Ile Asn Ile Ala Lys Asn Asp Phe Ser Asp Ile Glu Leu 1 5 10 15 Ala Ala Ile Pro Phe Asn Thr Leu Ala Asp His Tyr Gly Glu Arg Leu 20 25 30 Ala Arg Glu Gln Leu Ala Leu Glu His Glu Ser Tyr Glu Met Gly Glu 35 40 45 Ala Arg Phe Arg Lys Met Phe Glu Arg Gln Leu Lys Ala Gly Glu Val 50 55 60 Ala Asp Asn Ala Ala Ala Lys Pro Leu Ile Thr Thr Leu Leu Pro Lys 65 70 75 80 Met Ile Ala Arg Ile Asn Asp Trp Phe Glu Glu Val Lys Ala Lys Arg 85 90 95 Gly Lys Arg Pro Thr Ala Phe Gln Phe Leu Gln Glu Ile Lys Pro Glu 100 105 110 Ala Val Ala Tyr Ile Thr Ile Lys Thr Thr Leu Ala Cys Leu Thr Ser 115 120 125 Ala Asp Asn Thr Thr Val Gln Ala Val Ala Ser Ala Ile Gly Arg Ala 130 135 140 Ile Glu Asp Glu Ala Arg Phe Gly Arg Ile Arg Asp Leu Glu Ala Lys 145 150 155 160 His Phe Lys Lys Asn Val Glu Glu Gln Leu Asn Lys Arg Val Gly His 165 170 175 Val Tyr Lys Lys Ala Phe Met Gln Val Val Glu Ala Asp Met Leu Ser 180 185 190 Lys Gly Leu Leu Gly Gly Glu Ala Trp Ser Ser Trp His Lys Glu Asp 195 200 205 Ser Ile His Val Gly Val Arg Cys Ile Glu Met Leu Ile Glu Ser Thr 210 215 220 Gly Met Val Ser Leu His Arg Gln Asn Ala Gly Val Val Gly Gln Asp 225 230 235 240 Ser Glu Thr Ile Glu Leu Ala Pro Glu Tyr Ala Glu Ala Ile Ala Thr 245 250 255 Arg Ala Gly Ala Leu Ala Gly Ile Ser Pro Met Phe Gln Pro Cys Val 260 265 270 Val Pro Pro Lys Pro Trp Thr Gly Ile Thr Gly Gly Gly Tyr Trp Ala 275 280 285 Asn Gly Arg Arg Pro Leu Ala Leu Val Arg Thr His Ser Lys Lys Ala 290 295 300 Leu Met Arg Tyr Glu Asp Val Tyr Met Pro Glu Val Tyr Lys Ala Ile 305 310 315 320 Asn Ile Ala Gln Asn Thr Ala Trp Lys Ile Asn Lys Lys Val Leu Ala 325 330 335 Val Ala Asn Val Ile Thr Lys Trp Lys His Cys Pro Val Glu Asp Ile 340 345 350 Pro Ala Ile Glu Arg Glu Glu Leu Pro Met Lys Pro Glu Asp Ile Asp 355 360 365 Met Asn Pro Glu Ala Leu Thr Ala Trp Lys Arg Ala Ala Ala Ala Val 370 375 380 Tyr Arg Lys Asp Lys Ala Arg Lys Ser Arg Arg Ile Ser Leu Glu Phe 385 390 395 400 Met Leu Glu Gln Ala Asn Lys Phe Ala Asn His Lys Ala Ile Trp Phe 405 410 415 Pro Tyr Asn Met Asp Trp Arg Gly Arg Val Tyr Ala Val Ser Met Phe 420 425 430 Asn Pro Gln Gly Asn Asp Met Thr Lys Gly Leu Leu Thr Leu Ala Lys 435 440 445 Gly Lys Pro Ile Gly Lys Glu Gly Tyr Tyr Trp Leu Lys Ile His Gly 450 455 460 Ala Asn Cys Ala Gly Val Asp Lys Val Pro Phe Pro Glu Arg Ile Lys 465 470 475 480 Phe Ile Glu Glu Asn His Glu Asn Ile Met Ala Cys Ala Lys Ser Pro 485 490 495 Leu Glu Asn Thr Trp Trp Ala Glu Gln Asp Ser Pro Phe Cys Phe Leu 500 505 510 Ala Phe Cys Phe Glu Tyr Ala Gly Val Gln His His Gly Leu Ser Tyr 515 520 525 Asn Cys Ser Leu Pro Leu Ala Phe Asp Gly Ser Cys Ser Gly Ile Gln 530 535 540 His Phe Ser Ala Met Leu Arg Asp Glu Val Gly Gly Arg Ala Val Asn 545 550 555 560 Leu Leu Pro Ser Glu Thr Val Gln Asp Ile Tyr Gly Ile Val Ala Lys 565 570 575 Lys Val Asn Glu Ile Leu Gln Ala Asp Ala Ile Asn Gly Thr Asp Asn 580 585 590 Glu Val Val Thr Val Thr Asp Glu Asn Thr Gly Glu Ile Ser Glu Lys 595 600 605 Val Lys Leu Gly Thr Lys Ala Leu Ala Gly Gln Trp Leu Ala Tyr Gly 610 615 620 Val Thr Arg Ser Val Thr Lys Arg Ser Val Met Thr Leu Ala Leu Gly 625 630 635 640 Ser Lys Glu Phe Gly Phe Arg Gln Gln Val Leu Glu Asp Thr Ile Gln 645 650 655 Pro Ala Ile Asp Ser Gly Lys Gly Leu Met Phe Thr Gln Pro Asn Gln 660 665 670 Ala Ala Gly Tyr Met Ala Lys Leu Ile Trp Glu Ser Val Ser Val Thr 675 680 685 Val Val Ala Ala Val Glu Ala Met Asn Trp Leu Lys Ser Ala Ala Lys 690 695 700 Leu Leu Ala Ala Glu Val Lys Asp Lys Lys Thr Gly Glu Ile Leu Arg 705 710 715 720 Lys Arg Cys Ala Val His Trp Val Thr Pro Asp Gly Phe Pro Val Trp 725 730 735 Gln Glu Tyr Lys Lys Pro Ile Gln Thr Arg Leu Asn Leu Met Phe Leu 740 745 750 Gly Gln Phe Arg Leu Gln Pro Thr Ile Asn Thr Asn Lys Asp Ser Glu 755 760 765 Ile Asp Ala His Lys Gln Glu Ser Gly Ile Ala Pro Asn Phe Val Ala 770 775 780 Ser Gln Asp Gly Ser His Leu Arg Lys Thr Val Val Trp Ala His Glu 785 790 795 800 Lys Tyr Gly Ile Glu Ser Phe Ala Leu Ile His Asp Ser Phe Gly Thr 805 810 815 Ile Pro Ala Asp Ala Ala Asn Leu Phe Lys Ala Val Arg Glu Thr Met 820 825 830 Val Asp Thr Tyr Glu Ser Cys Asp Val Leu Ala Asp Phe Tyr Asp Gln 835 840 845 Phe Ala Asp Gln Leu His Glu Ser Gln Leu Asp Lys Met Pro Ala Leu 850 855 860 Pro Ala Lys Gly Asn Leu Asn Leu Arg Asp Ile Leu Glu Ser Asp Phe 865 870 875 880 Ala Phe Ala <210> 3 <211> 883 <212> PRT <213> artificial sequence <400> 3 Met Asn Thr Ile Asn Ile Ala Lys Asn Asp Phe Ser Asp Ile Glu Leu 1 5 10 15 Ala Ala Ile Pro Phe Asn Thr Leu Ala Asp His Tyr Gly Glu Arg Leu 20 25 30 Ala Arg Glu Gln Leu Ala Leu Glu His Glu Ser Tyr Glu Met Gly Glu 35 40 45 Ala Arg Phe Arg Lys Met Phe Glu Arg Gln Leu Lys Ala Gly Glu Val 50 55 60 Ala Asp Asn Ala Ala Ala Lys Pro Leu Ile Thr Thr Leu Leu Pro Lys 65 70 75 80 Met Ile Ala Arg Ile Asn Asp Trp Phe Glu Glu Val Lys Ala Lys Arg 85 90 95 Gly Lys Arg Pro Thr Ala Phe Gln Phe Leu Gln Glu Ile Lys Pro Glu 100 105 110 Ala Val Ala Tyr Ile Thr Ile Lys Thr Thr Leu Ala Cys Leu Thr Ser 115 120 125 Ala Asp Asn Thr Thr Val Gln Ala Val Ala Ser Ala Ile Gly Arg Ala 130 135 140 Ile Glu Asp Glu Ala Arg Phe Gly Arg Ile Arg Asp Leu Glu Ala Lys 145 150 155 160 His Phe Lys Lys Asn Val Glu Glu Gln Leu Asn Lys Arg Val Gly His 165 170 175 Val Tyr Lys Lys Ala Phe Met Gln Val Val Glu Ala Asp Met Leu Ser 180 185 190 Lys Gly Leu Leu Gly Gly Glu Ala Trp Ser Ser Trp His Lys Glu Asp 195 200 205 Ser Ile His Val Gly Val Arg Cys Ile Glu Met Leu Ile Glu Ser Thr 210 215 220 Gly Met Val Ser Leu His Arg Gln Asn Ala Gly Val Val Gly Gln Asp 225 230 235 240 Ser Glu Thr Ile Glu Leu Ala Pro Glu Tyr Ala Glu Ala Ile Ala Thr 245 250 255 Arg Ala Gly Ala Leu Ala Gly Ile Ser Pro Met Phe Gln Pro Cys Val 260 265 270 Val Pro Pro Lys Pro Trp Thr Gly Ile Thr Gly Gly Gly Tyr Trp Ala 275 280 285 Asn Gly Arg Arg Pro Leu Ala Leu Val Arg Thr His Ser Lys Lys Ala 290 295 300 Leu Met Arg Tyr Glu Asp Val Tyr Met Pro Glu Val Tyr Lys Ala Ile 305 310 315 320 Asn Ile Ala Gln Asn Thr Ala Trp Lys Ile Asn Lys Lys Val Leu Ala 325 330 335 Val Ala Asn Val Ile Thr Lys Trp Lys His Cys Pro Val Glu Asp Ile 340 345 350 Pro Ala Ile Glu Arg Glu Glu Leu Pro Met Lys Pro Glu Asp Ile Asp 355 360 365 Met Asn Pro Glu Ala Leu Thr Ala Trp Arg Arg Ala Ala Ala Ala Val 370 375 380 Tyr Arg Lys Asp Lys Ala Arg Lys Ser Arg Arg Ile Ser Leu Glu Phe 385 390 395 400 Met Leu Glu Gln Ala Asn Lys Phe Ala Asn His Lys Ala Ile Trp Phe 405 410 415 Pro Tyr Asn Met Asp Trp Arg Gly Arg Val Tyr Ala Val Ser Met Phe 420 425 430 Asn Pro Gln Gly Asn Asp Met Thr Lys Gly Leu Leu Thr Leu Ala Lys 435 440 445 Gly Lys Pro Ile Gly Lys Glu Gly Tyr Tyr Trp Leu Lys Ile His Gly 450 455 460 Ala Asn Cys Ala Gly Val Asp Lys Val Pro Phe Pro Glu Arg Ile Lys 465 470 475 480 Phe Ile Glu Glu Asn His Glu Asn Ile Met Ala Cys Ala Lys Ser Pro 485 490 495 Leu Glu Asn Thr Trp Trp Ala Glu Gln Asp Ser Pro Phe Cys Phe Leu 500 505 510 Ala Phe Cys Phe Glu Tyr Ala Gly Val Gln His His Gly Leu Ser Tyr 515 520 525 Asn Cys Ser Leu Pro Leu Ala Phe Asp Gly Ser Cys Ser Gly Ile Gln 530 535 540 His Phe Ser Ala Met Leu Arg Asp Glu Val Gly Gly Arg Ala Val Asn 545 550 555 560 Leu Leu Pro Ser Glu Thr Val Gln Asp Ile Tyr Gly Ile Val Ala Lys 565 570 575 Lys Val Asn Glu Ile Leu Gln Ala Asp Ala Ile Asn Gly Thr Asp Asn 580 585 590 Glu Val Val Thr Val Thr Asp Glu Asn Thr Gly Glu Ile Ser Glu Lys 595 600 605 Val Lys Leu Gly Thr Lys Ala Leu Ala Gly Gln Trp Leu Ala Tyr Gly 610 615 620 Val Thr Arg Ser Val Thr Lys Arg Ser Val Met Thr Leu Ala Leu Gly 625 630 635 640 Ser Lys Glu Phe Gly Phe Arg Gln Gln Val Leu Glu Asp Thr Ile Gln 645 650 655 Pro Ala Ile Asp Ser Gly Lys Gly Leu Met Phe Thr Gln Pro Asn Gln 660 665 670 Ala Ala Gly Tyr Met Ala Lys Leu Ile Trp Glu Ser Val Ser Val Thr 675 680 685 Val Val Ala Ala Val Glu Ala Met Asn Trp Leu Lys Ser Ala Ala Lys 690 695 700 Leu Leu Ala Ala Glu Val Lys Asp Lys Lys Thr Gly Glu Ile Leu Arg 705 710 715 720 Lys Arg Cys Ala Val His Trp Val Thr Pro Asp Gly Phe Pro Val Trp 725 730 735 Gln Glu Tyr Lys Lys Pro Ile Gln Thr Arg Leu Asn Leu Met Phe Leu 740 745 750 Gly Gln Phe Arg Leu Gln Pro Thr Ile Asn Thr Asn Lys Asp Ser Glu 755 760 765 Ile Asp Ala His Lys Gln Glu Ser Gly Ile Ala Pro Asn Phe Val His 770 775 780 Ser Gln Asp Gly Ser His Leu Arg Lys Thr Val Val Trp Ala His Glu 785 790 795 800 Lys Tyr Gly Ile Glu Ser Phe Ala Leu Ile His Asp Ser Phe Gly Thr 805 810 815 Ile Pro Ala Asp Ala Ala Asn Leu Phe Lys Ala Val Arg Glu Thr Met 820 825 830 Val Asp Thr Tyr Glu Ser Cys Asp Val Leu Ala Asp Phe Tyr Asp Gln 835 840 845 Phe Ala Asp Gln Leu His Glu Ser Gln Leu Asp Lys Met Pro Ala Leu 850 855 860 Pro Ala Lys Gly Asn Leu Asn Leu Arg Asp Ile Leu Glu Ser Asp Phe 865 870 875 880 Ala Phe Ala <210> 4 <211> 883 <212> PRT <213> artificial sequence <400> 4 Met Asn Thr Ile Asn Ile Ala Lys Asn Asp Phe Ser Asp Ile Glu Leu 1 5 10 15 Ala Ala Ile Pro Phe Asn Thr Leu Ala Asp His Tyr Gly Glu Arg Leu 20 25 30 Ala Arg Glu Gln Leu Ala Leu Glu His Glu Ser Tyr Glu Met Gly Glu 35 40 45 Ala Arg Phe Arg Lys Met Phe Glu Arg Gln Leu Lys Ala Gly Glu Val 50 55 60 Ala Asp Asn Ala Ala Ala Lys Pro Leu Ile Thr Thr Leu Leu Pro Lys 65 70 75 80 Met Ile Ala Arg Ile Asn Asp Trp Phe Glu Glu Val Lys Ala Lys Arg 85 90 95 Gly Lys Arg Pro Thr Ala Phe Gln Phe Leu Gln Glu Ile Lys Pro Glu 100 105 110 Ala Val Ala Tyr Ile Thr Ile Lys Thr Thr Leu Ala Cys Leu Thr Ser 115 120 125 Ala Asp Asn Thr Thr Val Gln Ala Val Ala Ser Ala Ile Gly Arg Ala 130 135 140 Ile Glu Asp Glu Ala Arg Phe Gly Arg Ile Arg Asp Leu Glu Ala Lys 145 150 155 160 His Phe Lys Lys Asn Val Glu Glu Gln Leu Asn Lys Arg Val Gly His 165 170 175 Val Tyr Lys Lys Ala Phe Met Gln Val Val Glu Ala Asp Met Leu Ser 180 185 190 Lys Gly Leu Leu Gly Gly Glu Ala Trp Ser Ser Trp His Lys Glu Asp 195 200 205 Ser Ile His Val Gly Val Arg Cys Ile Glu Met Leu Ile Glu Ser Thr 210 215 220 Gly Met Val Ser Leu His Arg Gln Asn Ala Gly Val Val Gly Gln Asp 225 230 235 240 Ser Glu Thr Ile Glu Leu Ala Pro Glu Tyr Ala Glu Ala Ile Ala Thr 245 250 255 Arg Ala Gly Ala Leu Ala Gly Ile Ser Pro Met Phe Gln Pro Cys Val 260 265 270 Val Pro Pro Lys Pro Trp Thr Gly Ile Thr Gly Gly Gly Tyr Trp Ala 275 280 285 Asn Gly Arg Arg Pro Leu Ala Leu Val Arg Thr His Ser Lys Lys Ala 290 295 300 Leu Met Arg Tyr Glu Asp Val Tyr Met Pro Glu Val Tyr Lys Ala Ile 305 310 315 320 Asn Ile Ala Gln Asn Thr Ala Trp Lys Ile Asn Lys Lys Val Leu Ala 325 330 335 Val Ala Asn Val Ile Thr Lys Trp Lys His Cys Pro Val Glu Asp Ile 340 345 350 Pro Ala Ile Glu Arg Glu Glu Leu Pro Met Lys Pro Glu Asp Ile Asp 355 360 365 Met Asn Pro Glu Ala Leu Thr Ala Trp Arg Arg Ala Ala Ala Ala Val 370 375 380 Tyr Arg Lys Asp Lys Ala Arg Lys Ser Arg Arg Ile Ser Leu Glu Phe 385 390 395 400 Met Leu Glu Gln Ala Asn Lys Phe Ala Asn His Lys Ala Ile Trp Phe 405 410 415 Pro Tyr Asn Met Asp Trp Arg Gly Arg Val Tyr Ala Val Ser Met Phe 420 425 430 Asn Pro Gln Gly Asn Asp Met Thr Lys Gly Leu Leu Thr Leu Ala Lys 435 440 445 Gly Lys Pro Ile Gly Lys Glu Gly Tyr Tyr Trp Leu Lys Ile His Gly 450 455 460 Ala Asn Cys Ala Gly Val Asp Lys Val Pro Phe Pro Glu Arg Ile Lys 465 470 475 480 Phe Ile Glu Glu Asn His Glu Asn Ile Met Ala Cys Ala Lys Ser Pro 485 490 495 Leu Glu Asn Thr Trp Trp Ala Glu Gln Asp Ser Pro Phe Cys Phe Leu 500 505 510 Ala Phe Cys Phe Glu Tyr Ala Gly Val Gln His His Gly Leu Ser Tyr 515 520 525 Asn Cys Ser Leu Pro Leu Ala Phe Asp Gly Ser Cys Ser Gly Ile Gln 530 535 540 His Phe Ser Ala Met Leu Arg Asp Glu Val Gly Gly Arg Ala Val Asn 545 550 555 560 Leu Leu Pro Ser Glu Thr Val Gln Asp Ile Tyr Gly Ile Val Ala Lys 565 570 575 Lys Val Asn Glu Ile Leu Gln Ala Asp Ala Ile Asn Gly Thr Asp Asn 580 585 590 Glu Val Val Thr Val Thr Asp Glu Asn Thr Gly Glu Ile Ser Glu Lys 595 600 605 Val Lys Leu Gly Thr Lys Ala Leu Ala Gly Gln Trp Leu Ala Tyr Gly 610 615 620 Val Thr Arg Ser Val Thr Lys Arg Ser Val Met Thr Leu Ala Tyr Gly 625 630 635 640 Ser Lys Glu Phe Gly Phe Arg Gln Gln Val Leu Glu Asp Thr Ile Gln 645 650 655 Pro Ala Ile Asp Ser Gly Lys Gly Leu Met Phe Thr Gln Pro Asn Gln 660 665 670 Ala Ala Gly Tyr Met Ala Lys Leu Ile Trp Glu Ser Val Ser Val Thr 675 680 685 Val Val Ala Ala Val Glu Ala Met Asn Trp Leu Lys Ser Ala Ala Lys 690 695 700 Leu Leu Ala Ala Glu Val Lys Asp Lys Lys Thr Gly Glu Ile Leu Arg 705 710 715 720 Lys Arg Cys Ala Val His Trp Val Thr Pro Asp Gly Phe Pro Val Trp 725 730 735 Gln Glu Tyr Lys Lys Pro Ile Gln Thr Arg Leu Asn Leu Met Phe Leu 740 745 750 Gly Gln Phe Arg Leu Gln Pro Thr Ile Asn Thr Asn Lys Asp Ser Glu 755 760 765 Ile Asp Ala His Lys Gln Glu Ser Gly Ile Ala Pro Asn Phe Val Ala 770 775 780 Ser Gln Asp Gly Ser His Leu Arg Lys Thr Val Val Trp Ala His Glu 785 790 795 800 Lys Tyr Gly Ile Glu Ser Phe Ala Leu Ile His Asp Ser Phe Gly Thr 805 810 815 Ile Pro Ala Asp Ala Ala Asn Leu Phe Lys Ala Val Arg Glu Thr Met 820 825 830 Val Asp Thr Tyr Glu Ser Cys Asp Val Leu Ala Asp Phe Tyr Asp Gln 835 840 845 Phe Ala Asp Gln Leu His Glu Ser Gln Leu Asp Lys Met Pro Ala Leu 850 855 860 Pro Ala Lys Gly Asn Leu Asn Leu Arg Asp Ile Leu Glu Ser Asp Phe 865 870 875 880 Ala Phe Ala <210> 5 <211> 883 <212> PRT <213> artificial sequence <400> 5 Met Asn Thr Ile Asn Ile Ala Lys Asn Asp Phe Ser Asp Ile Glu Leu 1 5 10 15 Ala Ala Ile Pro Phe Asn Thr Leu Ala Asp His Tyr Gly Glu Arg Leu 20 25 30 Ala Arg Glu Gln Leu Ala Leu Glu His Glu Ser Tyr Glu Met Gly Glu 35 40 45 Ala Arg Phe Arg Lys Met Phe Glu Arg Gln Leu Lys Ala Gly Glu Val 50 55 60 Ala Asp Asn Ala Ala Ala Lys Pro Leu Ile Thr Thr Leu Leu Pro Lys 65 70 75 80 Met Ile Ala Arg Ile Asn Asp Trp Phe Glu Glu Val Lys Ala Lys Arg 85 90 95 Gly Lys Arg Pro Thr Ala Phe Gln Phe Leu Gln Glu Ile Lys Pro Glu 100 105 110 Ala Val Ala Tyr Ile Thr Ile Lys Thr Thr Leu Ala Cys Leu Thr Ser 115 120 125 Ala Asp Asn Thr Thr Val Gln Ala Val Ala Ser Ala Ile Gly Arg Ala 130 135 140 Ile Glu Asp Glu Ala Arg Phe Gly Arg Ile Arg Asp Leu Glu Ala Lys 145 150 155 160 His Phe Lys Lys Asn Val Glu Glu Gln Leu Asn Lys Arg Val Gly His 165 170 175 Val Tyr Lys Lys Ala Phe Met Gln Val Val Glu Ala Asp Met Leu Ser 180 185 190 Lys Gly Leu Leu Gly Gly Glu Ala Trp Ser Ser Trp His Lys Glu Asp 195 200 205 Ser Ile His Val Gly Val Arg Cys Ile Glu Met Leu Ile Glu Ser Thr 210 215 220 Gly Met Val Ser Leu His Arg Gln Asn Ala Gly Val Val Gly Gln Asp 225 230 235 240 Ser Glu Thr Ile Glu Leu Ala Pro Glu Tyr Ala Glu Ala Ile Ala Thr 245 250 255 Arg Ala Gly Ala Leu Ala Gly Ile Ser Pro Met Phe Gln Pro Cys Val 260 265 270 Val Pro Pro Lys Pro Trp Thr Gly Ile Thr Gly Gly Gly Tyr Trp Ala 275 280 285 Asn Gly Arg Arg Pro Leu Ala Leu Val Arg Thr His Ser Lys Lys Ala 290 295 300 Leu Met Arg Tyr Glu Asp Val Tyr Met Pro Glu Val Tyr Lys Ala Ile 305 310 315 320 Asn Ile Ala Gln Asn Thr Ala Trp Lys Ile Asn Lys Lys Val Leu Ala 325 330 335 Val Ala Asn Val Ile Thr Lys Trp Lys His Cys Pro Val Glu Asp Ile 340 345 350 Pro Ala Ile Glu Arg Glu Glu Leu Pro Met Lys Pro Glu Asp Ile Asp 355 360 365 Met Asn Pro Glu Ala Leu Thr Ala Trp Lys Arg Ala Ala Ala Ala Val 370 375 380 Tyr Arg Lys Asp Lys Ala Arg Lys Ser Arg Arg Ile Ser Leu Glu Phe 385 390 395 400 Met Leu Glu Gln Ala Asn Lys Phe Ala Asn His Lys Ala Ile Trp Phe 405 410 415 Pro Tyr Asn Met Asp Trp Arg Gly Arg Val Tyr Ala Val Ser Met Phe 420 425 430 Asn Pro Gln Gly Asn Asp Met Thr Lys Gly Leu Leu Thr Leu Ala Lys 435 440 445 Gly Lys Pro Ile Gly Lys Glu Gly Tyr Tyr Trp Leu Lys Ile His Gly 450 455 460 Ala Asn Cys Ala Gly Val Asp Lys Val Pro Phe Pro Glu Arg Ile Lys 465 470 475 480 Phe Ile Glu Glu Asn His Glu Asn Ile Met Ala Cys Ala Lys Ser Pro 485 490 495 Leu Glu Asn Thr Trp Trp Ala Glu Gln Asp Ser Pro Phe Cys Phe Leu 500 505 510 Ala Phe Cys Phe Glu Tyr Ala Gly Val Gln His His Gly Leu Ser Tyr 515 520 525 Asn Cys Ser Leu Pro Leu Ala Phe Asp Gly Ser Cys Ser Gly Ile Gln 530 535 540 His Phe Ser Ala Met Leu Arg Asp Glu Val Gly Gly Arg Ala Val Asn 545 550 555 560 Leu Leu Pro Ser Glu Thr Val Gln Asp Ile Tyr Gly Ile Val Ala Lys 565 570 575 Lys Val Asn Glu Ile Leu Gln Ala Asp Ala Ile Asn Gly Thr Asp Asn 580 585 590 Glu Val Val Thr Val Thr Asp Glu Asn Thr Gly Glu Ile Ser Glu Lys 595 600 605 Val Lys Leu Gly Thr Lys Ala Leu Ala Gly Gln Trp Leu Ala Tyr Gly 610 615 620 Val Thr Arg Ser Val Thr Lys Arg Ser Val Met Thr Leu Ala Leu Gly 625 630 635 640 Ser Lys Glu Phe Gly Phe Arg Gln Gln Val Leu Glu Asp Thr Ile Gln 645 650 655 Pro Ala Ile Asp Ser Gly Lys Gly Leu Met Phe Thr Gln Pro Asn Gln 660 665 670 Ala Ala Gly Tyr Met Ala Lys Leu Ile Trp Glu Ser Val Ser Val Thr 675 680 685 Val Val Ala Ala Val Glu Ala Met Asn Trp Leu Lys Ser Ala Ala Lys 690 695 700 Leu Leu Ala Ala Glu Val Lys Asp Lys Lys Thr Gly Glu Ile Leu Arg 705 710 715 720 Lys Arg Cys Ala Val His Trp Val Thr Pro Asp Gly Phe Pro Val Trp 725 730 735 Gln Glu Tyr Lys Lys Pro Ile Gln Thr Arg Leu Asn Leu Met Phe Leu 740 745 750 Gly Gln Phe Arg Leu Gln Pro Thr Ile Asn Thr Asn Lys Asp Ser Glu 755 760 765 Ile Asp Ala His Lys Gln Glu Ser Gly Ile Ala Pro Asn Phe Val His 770 775 780 Ser Gln Asp Gly Ser His Leu Arg Lys Thr Val Val Trp Ala His Glu 785 790 795 800 Lys Tyr Gly Ile Glu Ser Phe Ala Leu Ile His Asp Ser Phe Gly Thr 805 810 815 Ile Pro Ala Asp Ala Ala Asn Leu Phe Lys Ala Val Arg Glu Thr Met 820 825 830 Val Asp Thr Tyr Glu Ser Cys Asp Val Leu Ala Asp Phe Tyr Asp Gln 835 840 845 Phe Ala Asp Gln Leu His Glu Ser Gln Leu Asp Lys Met Pro Ala Leu 850 855 860 Pro Ala Lys Gly Asn Leu Asn Leu Arg Asp Ile Leu Glu Ser Asp Phe 865 870 875 880 Ala Phe Ala <210> 6 <211> 883 <212> PRT <213> Artificial Sequence <400> 6 Met Asn Thr Ile Asn Ile Ala Lys Asn Asp Phe Ser Asp Ile Glu Leu 1 5 10 15 Ala Ala Ile Pro Phe Asn Thr Leu Ala Asp His Tyr Gly Glu Arg Leu 20 25 30 Ala Arg Glu Gln Leu Ala Leu Glu His Glu Ser Tyr Glu Met Gly Glu 35 40 45 Ala Arg Phe Arg Lys Met Phe Glu Arg Gln Leu Lys Ala Gly Glu Val 50 55 60 Ala Asp Asn Ala Ala Ala Lys Pro Leu Ile Thr Thr Leu Leu Pro Lys 65 70 75 80 Met Ile Ala Arg Ile Asn Asp Trp Phe Glu Glu Val Lys Ala Lys Arg 85 90 95 Gly Lys Arg Pro Thr Ala Phe Gln Phe Leu Gln Glu Ile Lys Pro Glu 100 105 110 Ala Val Ala Tyr Ile Thr Ile Lys Thr Thr Leu Ala Cys Leu Thr Ser 115 120 125 Ala Asp Asn Thr Thr Val Gln Ala Val Ala Ser Ala Ile Gly Arg Ala 130 135 140 Ile Glu Asp Glu Ala Arg Phe Gly Arg Ile Arg Asp Leu Glu Ala Lys 145 150 155 160 His Phe Lys Lys Asn Val Glu Glu Gln Leu Asn Lys Arg Val Gly His 165 170 175 Val Tyr Lys Lys Ala Phe Met Gln Val Val Glu Ala Asp Met Leu Ser 180 185 190 Lys Gly Leu Leu Gly Gly Glu Ala Trp Ser Ser Trp His Lys Glu Asp 195 200 205 Ser Ile His Val Gly Val Arg Cys Ile Glu Met Leu Ile Glu Ser Thr 210 215 220 Gly Met Val Ser Leu His Arg Gln Asn Ala Gly Val Val Gly Gln Asp 225 230 235 240 Ser Glu Thr Ile Glu Leu Ala Pro Glu Tyr Ala Glu Ala Ile Ala Thr 245 250 255 Arg Ala Gly Ala Leu Ala Gly Ile Ser Pro Met Phe Gln Pro Cys Val 260 265 270 Val Pro Pro Lys Pro Trp Thr Gly Ile Thr Gly Gly Gly Tyr Trp Ala 275 280 285 Asn Gly Arg Arg Pro Leu Ala Leu Val Arg Thr His Ser Lys Lys Ala 290 295 300 Leu Met Arg Tyr Glu Asp Val Tyr Met Pro Glu Val Tyr Lys Ala Ile 305 310 315 320 Asn Ile Ala Gln Asn Thr Ala Trp Lys Ile Asn Lys Lys Val Leu Ala 325 330 335 Val Ala Asn Val Ile Thr Lys Trp Lys His Cys Pro Val Glu Asp Ile 340 345 350 Pro Ala Ile Glu Arg Glu Glu Leu Pro Met Lys Pro Glu Asp Ile Asp 355 360 365 Met Asn Pro Glu Ala Leu Thr Ala Trp Lys Arg Ala Ala Ala Ala Val 370 375 380 Tyr Arg Lys Asp Lys Ala Arg Lys Ser Arg Arg Ile Ser Leu Glu Phe 385 390 395 400 Met Leu Glu Gln Ala Asn Lys Phe Ala Asn His Lys Ala Ile Trp Phe 405 410 415 Pro Tyr Asn Met Asp Trp Arg Gly Arg Val Tyr Ala Val Ser Met Phe 420 425 430 Asn Pro Gln Gly Asn Asp Met Thr Lys Gly Leu Leu Thr Leu Ala Lys 435 440 445 Gly Lys Pro Ile Gly Lys Glu Gly Tyr Tyr Trp Leu Lys Ile His Gly 450 455 460 Ala Asn Cys Ala Gly Val Asp Lys Val Pro Phe Pro Glu Arg Ile Lys 465 470 475 480 Phe Ile Glu Glu Asn His Glu Asn Ile Met Ala Cys Ala Lys Ser Pro 485 490 495 Leu Glu Asn Thr Trp Trp Ala Glu Gln Asp Ser Pro Phe Cys Phe Leu 500 505 510 Ala Phe Cys Phe Glu Tyr Ala Gly Val Gln His His Gly Leu Ser Tyr 515 520 525 Asn Cys Ser Leu Pro Leu Ala Phe Asp Gly Ser Cys Ser Gly Ile Gln 530 535 540 His Phe Ser Ala Met Leu Arg Asp Glu Val Gly Gly Arg Ala Val Asn 545 550 555 560 Leu Leu Pro Ser Glu Thr Val Gln Asp Ile Tyr Gly Ile Val Ala Lys 565 570 575 Lys Val Asn Glu Ile Leu Gln Ala Asp Ala Ile Asn Gly Thr Asp Asn 580 585 590 Glu Val Val Thr Val Thr Asp Glu Asn Thr Gly Glu Ile Ser Glu Lys 595 600 605 Val Lys Leu Gly Thr Lys Ala Leu Ala Gly Gln Trp Leu Ala Tyr Gly 610 615 620 Val Thr Arg Ser Val Thr Lys Arg Ser Val Met Thr Leu Ala Tyr Gly 625 630 635 640 Ser Lys Glu Phe Gly Phe Arg Gln Gln Val Leu Glu Asp Thr Ile Gln 645 650 655 Pro Ala Ile Asp Ser Gly Lys Gly Leu Met Phe Thr Gln Pro Asn Gln 660 665 670 Ala Ala Gly Tyr Met Ala Lys Leu Ile Trp Glu Ser Val Ser Val Thr 675 680 685 Val Val Ala Ala Val Glu Ala Met Asn Trp Leu Lys Ser Ala Ala Lys 690 695 700 Leu Leu Ala Ala Glu Val Lys Asp Lys Lys Thr Gly Glu Ile Leu Arg 705 710 715 720 Lys Arg Cys Ala Val His Trp Val Thr Pro Asp Gly Phe Pro Val Trp 725 730 735 Gln Glu Tyr Lys Lys Pro Ile Gln Thr Arg Leu Asn Leu Met Phe Leu 740 745 750 Gly Gln Phe Arg Leu Gln Pro Thr Ile Asn Thr Asn Lys Asp Ser Glu 755 760 765 Ile Asp Ala His Lys Gln Glu Ser Gly Ile Ala Pro Asn Phe Val Ala 770 775 780 Ser Gln Asp Gly Ser His Leu Arg Lys Thr Val Val Trp Ala His Glu 785 790 795 800 Lys Tyr Gly Ile Glu Ser Phe Ala Leu Ile His Asp Ser Phe Gly Thr 805 810 815 Ile Pro Ala Asp Ala Ala Asn Leu Phe Lys Ala Val Arg Glu Thr Met 820 825 830 Val Asp Thr Tyr Glu Ser Cys Asp Val Leu Ala Asp Phe Tyr Asp Gln 835 840 845 Phe Ala Asp Gln Leu His Glu Ser Gln Leu Asp Lys Met Pro Ala Leu 850 855 860 Pro Ala Lys Gly Asn Leu Asn Leu Arg Asp Ile Leu Glu Ser Asp Phe 865 870 875 880 Ala Phe Ala <210> 7 <211> 883 <212> PRT <213> artificial sequence <400> 7 Met Asn Thr Ile Asn Ile Ala Lys Asn Asp Phe Ser Asp Ile Glu Leu 1 5 10 15 Ala Ala Ile Pro Phe Asn Thr Leu Ala Asp His Tyr Gly Glu Arg Leu 20 25 30 Ala Arg Glu Gln Leu Ala Leu Glu His Glu Ser Tyr Glu Met Gly Glu 35 40 45 Ala Arg Phe Arg Lys Met Phe Glu Arg Gln Leu Lys Ala Gly Glu Val 50 55 60 Ala Asp Asn Ala Ala Ala Lys Pro Leu Ile Thr Thr Leu Leu Pro Lys 65 70 75 80 Met Ile Ala Arg Ile Asn Asp Trp Phe Glu Glu Val Lys Ala Lys Arg 85 90 95 Gly Lys Arg Pro Thr Ala Phe Gln Phe Leu Gln Glu Ile Lys Pro Glu 100 105 110 Ala Val Ala Tyr Ile Thr Ile Lys Thr Thr Leu Ala Cys Leu Thr Ser 115 120 125 Ala Asp Asn Thr Thr Val Gln Ala Val Ala Ser Ala Ile Gly Arg Ala 130 135 140 Ile Glu Asp Glu Ala Arg Phe Gly Arg Ile Arg Asp Leu Glu Ala Lys 145 150 155 160 His Phe Lys Lys Asn Val Glu Glu Gln Leu Asn Lys Arg Val Gly His 165 170 175 Val Tyr Lys Lys Ala Phe Met Gln Val Val Glu Ala Asp Met Leu Ser 180 185 190 Lys Gly Leu Leu Gly Gly Glu Ala Trp Ser Ser Trp His Lys Glu Asp 195 200 205 Ser Ile His Val Gly Val Arg Cys Ile Glu Met Leu Ile Glu Ser Thr 210 215 220 Gly Met Val Ser Leu His Arg Gln Asn Ala Gly Val Val Gly Gln Asp 225 230 235 240 Ser Glu Thr Ile Glu Leu Ala Pro Glu Tyr Ala Glu Ala Ile Ala Thr 245 250 255 Arg Ala Gly Ala Leu Ala Gly Ile Ser Pro Met Phe Gln Pro Cys Val 260 265 270 Val Pro Pro Lys Pro Trp Thr Gly Ile Thr Gly Gly Gly Tyr Trp Ala 275 280 285 Asn Gly Arg Arg Pro Leu Ala Leu Val Arg Thr His Ser Lys Lys Ala 290 295 300 Leu Met Arg Tyr Glu Asp Val Tyr Met Pro Glu Val Tyr Lys Ala Ile 305 310 315 320 Asn Ile Ala Gln Asn Thr Ala Trp Lys Ile Asn Lys Lys Val Leu Ala 325 330 335 Val Ala Asn Val Ile Thr Lys Trp Lys His Cys Pro Val Glu Asp Ile 340 345 350 Pro Ala Ile Glu Arg Glu Glu Leu Pro Met Lys Pro Glu Asp Ile Asp 355 360 365 Met Asn Pro Glu Ala Leu Thr Ala Trp Arg Arg Ala Ala Ala Ala Val 370 375 380 Tyr Arg Lys Asp Lys Ala Arg Lys Ser Arg Arg Ile Ser Leu Glu Phe 385 390 395 400 Met Leu Glu Gln Ala Asn Lys Phe Ala Asn His Lys Ala Ile Trp Phe 405 410 415 Pro Tyr Asn Met Asp Trp Arg Gly Arg Val Tyr Ala Val Ser Met Phe 420 425 430 Asn Pro Gln Gly Asn Asp Met Thr Lys Gly Leu Leu Thr Leu Ala Lys 435 440 445 Gly Lys Pro Ile Gly Lys Glu Gly Tyr Tyr Trp Leu Lys Ile His Gly 450 455 460 Ala Asn Cys Ala Gly Val Asp Lys Val Pro Phe Pro Glu Arg Ile Lys 465 470 475 480 Phe Ile Glu Glu Asn His Glu Asn Ile Met Ala Cys Ala Lys Ser Pro 485 490 495 Leu Glu Asn Thr Trp Trp Ala Glu Gln Asp Ser Pro Phe Cys Phe Leu 500 505 510 Ala Phe Cys Phe Glu Tyr Ala Gly Val Gln His His Gly Leu Ser Tyr 515 520 525 Asn Cys Ser Leu Pro Leu Ala Phe Asp Gly Ser Cys Ser Gly Ile Gln 530 535 540 His Phe Ser Ala Met Leu Arg Asp Glu Val Gly Gly Arg Ala Val Asn 545 550 555 560 Leu Leu Pro Ser Glu Thr Val Gln Asp Ile Tyr Gly Ile Val Ala Lys 565 570 575 Lys Val Asn Glu Ile Leu Gln Ala Asp Ala Ile Asn Gly Thr Asp Asn 580 585 590 Glu Val Val Thr Val Thr Asp Glu Asn Thr Gly Glu Ile Ser Glu Lys 595 600 605 Val Lys Leu Gly Thr Lys Ala Leu Ala Gly Gln Trp Leu Ala Tyr Gly 610 615 620 Val Thr Arg Ser Val Thr Lys Arg Ser Val Met Thr Leu Ala Tyr Gly 625 630 635 640 Ser Lys Glu Phe Gly Phe Arg Gln Gln Val Leu Glu Asp Thr Ile Gln 645 650 655 Pro Ala Ile Asp Ser Gly Lys Gly Leu Met Phe Thr Gln Pro Asn Gln 660 665 670 Ala Ala Gly Tyr Met Ala Lys Leu Ile Trp Glu Ser Val Ser Val Thr 675 680 685 Val Val Ala Ala Val Glu Ala Met Asn Trp Leu Lys Ser Ala Ala Lys 690 695 700 Leu Leu Ala Ala Glu Val Lys Asp Lys Lys Thr Gly Glu Ile Leu Arg 705 710 715 720 Lys Arg Cys Ala Val His Trp Val Thr Pro Asp Gly Phe Pro Val Trp 725 730 735 Gln Glu Tyr Lys Lys Pro Ile Gln Thr Arg Leu Asn Leu Met Phe Leu 740 745 750 Gly Gln Phe Arg Leu Gln Pro Thr Ile Asn Thr Asn Lys Asp Ser Glu 755 760 765 Ile Asp Ala His Lys Gln Glu Ser Gly Ile Ala Pro Asn Phe Val His 770 775 780 Ser Gln Asp Gly Ser His Leu Arg Lys Thr Val Val Trp Ala His Glu 785 790 795 800 Lys Tyr Gly Ile Glu Ser Phe Ala Leu Ile His Asp Ser Phe Gly Thr 805 810 815 Ile Pro Ala Asp Ala Ala Asn Leu Phe Lys Ala Val Arg Glu Thr Met 820 825 830 Val Asp Thr Tyr Glu Ser Cys Asp Val Leu Ala Asp Phe Tyr Asp Gln 835 840 845 Phe Ala Asp Gln Leu His Glu Ser Gln Leu Asp Lys Met Pro Ala Leu 850 855 860 Pro Ala Lys Gly Asn Leu Asn Leu Arg Asp Ile Leu Glu Ser Asp Phe 865 870 875 880 Ala Phe Ala

Claims

1. A transcription reaction solution for preparing locked nucleic acid-modified RNA, characterized in that: Includes DNA template, mutant T7 RNA polymerase, LNA-NTPs and buffer; Wherein, the mutant T7 RNA polymerase includes a T7 RNA polymerase with an amino acid sequence as shown in SEQ ID NO.1; The buffer comprises a biological buffer, an activator, a polyamine, a detergent and a reducing agent; The final molar concentration of the biological buffer in the transcription reaction solution is 25-35 mM; the biological buffer is selected from Tris and HEPES; The final molar concentration of the activator in the transcription reaction solution is 4.5-5.5 mM; the activator is selected from magnesium ions; The final molar concentration of the polyamine in the transcription reaction solution is 0.8-1.2 mM; the polyamine is selected from spermidine and putrescine; The final molar concentration of the reducing agent in the transcription reaction solution is 2.5-3.5 mM; the reducing agent is selected from DTT.

2. The transcription reaction solution according to claim 1, characterized in that The pH of the buffer is 6.5-7.

5.

3. The transcription reaction solution according to claim 1, characterized in that The detergent is selected from Triton X-100; The mass fraction of the detergent in the transcription reaction solution is 0.01-0.03%.

4. The transcription reaction solution according to claim 1, characterized in that The buffer also includes inorganic pyrophosphatase and nuclease inhibitors; The final concentration of the inorganic pyrophosphatase in the transcription reaction solution is 0.01-1 U / μl; The final concentration of the nuclease inhibitor in the transcription reaction solution is 1-5 U / μl.

5. A method for preparing locked nucleic acid-modified RNA, characterized in that: The method comprises subjecting the transcription reaction solution for preparing locked nucleic acid-modified RNA according to any one of claims 1 to 4 to a transcription reaction.

6. The method for preparing locked nucleic acid-modified RNA according to claim 5, characterized in that: The method for preparing the locked nucleic acid modified RNA further comprises: adding a DNA template degrading enzyme to the transcribed system to obtain a first mixed system, and performing a digestion reaction; and then adding a precipitant to the first mixed system to obtain a second mixed system, and performing a purification treatment.

7. The method for preparing locked nucleic acid-modified RNA according to claim 6, characterized in that: The DNA template degrading enzyme is selected from DNase I enzyme, and the total amount of the DNA template degrading enzyme in the first mixed system is 1-3U.

8. Use of the transcription reaction solution according to any one of claims 1 to 4 in the transcription for preparing locked nucleic acid-modified RNA, characterized in that: The mutant T7 RNA polymerase includes a T7 RNA polymerase having an amino acid sequence as shown in SEQ ID NO.1.

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