CN113862235A - Chimeric enzyme and application and method thereof in synthesis of Cap0mRNA by in vitro one-step reaction - Google Patents

Chimeric enzyme and application and method thereof in synthesis of Cap0mRNA by in vitro one-step reaction Download PDF

Info

Publication number
CN113862235A
CN113862235A CN202111123695.9A CN202111123695A CN113862235A CN 113862235 A CN113862235 A CN 113862235A CN 202111123695 A CN202111123695 A CN 202111123695A CN 113862235 A CN113862235 A CN 113862235A
Authority
CN
China
Prior art keywords
enzyme
leu
lys
chimeric
rna polymerase
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Granted
Application number
CN202111123695.9A
Other languages
Chinese (zh)
Other versions
CN113862235B (en
Inventor
崔利兰
王凡
邹媛华
徐志豪
张静华
郑紫君
王明明
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Suzhou Inshore Protein Technology Co ltd
Original Assignee
Suzhou Inshore Protein Technology Co ltd
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Suzhou Inshore Protein Technology Co ltd filed Critical Suzhou Inshore Protein Technology Co ltd
Priority to CN202111123695.9A priority Critical patent/CN113862235B/en
Publication of CN113862235A publication Critical patent/CN113862235A/en
Application granted granted Critical
Publication of CN113862235B publication Critical patent/CN113862235B/en
Active legal-status Critical Current
Anticipated expiration legal-status Critical

Links

Images

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/12—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
    • C12N9/1241—Nucleotidyltransferases (2.7.7)
    • C12N9/1247—DNA-directed RNA polymerase (2.7.7.6)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/70—Vectors or expression systems specially adapted for E. coli
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/1003—Transferases (2.) transferring one-carbon groups (2.1)
    • C12N9/1007—Methyltransferases (general) (2.1.1.)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/12—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
    • C12N9/1241—Nucleotidyltransferases (2.7.7)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y201/00—Transferases transferring one-carbon groups (2.1)
    • C12Y201/01—Methyltransferases (2.1.1)
    • C12Y201/01056—Methyltransferases (2.1.1) mRNA (guanine-N7-)-methyltransferase (2.1.1.56)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y207/00—Transferases transferring phosphorus-containing groups (2.7)
    • C12Y207/07—Nucleotidyltransferases (2.7.7)
    • C12Y207/07006—DNA-directed RNA polymerase (2.7.7.6)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y207/00—Transferases transferring phosphorus-containing groups (2.7)
    • C12Y207/07—Nucleotidyltransferases (2.7.7)
    • C12Y207/0705—Nucleotidyltransferases (2.7.7) mRNA guanylyltransferase (2.7.7.50)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y306/00—Hydrolases acting on acid anhydrides (3.6)
    • C12Y306/01—Hydrolases acting on acid anhydrides (3.6) in phosphorus-containing anhydrides (3.6.1)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide

Landscapes

  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Organic Chemistry (AREA)
  • Genetics & Genomics (AREA)
  • Engineering & Computer Science (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Wood Science & Technology (AREA)
  • Zoology (AREA)
  • General Engineering & Computer Science (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Molecular Biology (AREA)
  • Biomedical Technology (AREA)
  • Biotechnology (AREA)
  • Microbiology (AREA)
  • Medicinal Chemistry (AREA)
  • Physics & Mathematics (AREA)
  • Biophysics (AREA)
  • Plant Pathology (AREA)
  • Crystallography & Structural Chemistry (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Enzymes And Modification Thereof (AREA)

Abstract

The invention relates to the field of molecular biology, in particular to a chimeric enzyme, and application and a method thereof in one-step synthesis of Cap0mRNA in-vitro transcription, wherein the method comprises the steps of mixing the chimeric enzyme and an in-vitro transcription template, carrying out a co-reaction, and carrying out a one-step reaction to obtain a capped and tailed mRNA product, wherein the chimeric enzyme comprises an RNA polymerase structural domain and a capped enzyme catalytic structural domain, and the chimeric enzyme has dual functions of in-vitro transcription and capping through a chimeric design. The invention opens up a method for synthesizing mRNA in one step by an in vitro enzyme method, and improves the synthesis efficiency and the mRNA yield.

Description

Chimeric enzyme and application and method thereof in synthesis of Cap0mRNA by in vitro one-step reaction
Technical Field
The invention relates to the field of molecular biology, in particular to a chimeric enzyme and application and a method thereof in synthesizing Cap0mRNA by one-step reaction in vitro.
Background
In recent years, infectious diseases mainly in respiratory tracts seriously threaten public health, under the background, the mRNA-based in vitro transcription technology only needs to provide a gene sequence of an antigen in research and development design without isolated culture of highly pathogenic pathogens, has obvious advantages in coping with viruses with high transmission risk, has simple production process, quick synthesis and lower cost, provides a natural antigen epitope, and becomes a novel vaccine with great advantages.
The mRNA in vitro transcription system adopts the working principle that DNA is used as a template, and RNA is synthesized by RNA polymerase under the action of a series of transcription factors. The capped RNA synthesized in the transcription reaction can be used for in vitro translation, transfection and other tests. Commonly used polymerases are bacteriophage-derived RNA polymerases such as T7, SP6, T3, etc., wherein T7RNA Polymerase (T7 RNA Polymerase) is a DNA-dependent 5'→ 3' RNA Polymerase that highly specifically recognizes the T7 promoter sequence. The T7RNA polymerase can catalyze the incorporation of NTPs downstream of the T7 promoter, either single-stranded or double-stranded DNA, to synthesize RNA complementary to the template DNA downstream of the T7 promoter. Because the RNA obtained by transcription is unstable, the RNA needs to be processed and modified, and the RNA comprises a cap structure formed at the 5 'end and a poly (A) tail structure added at the 3' end, so that the effects of stabilizing the RNA, preventing degradation, assisting translation and the like are achieved. Two common approaches to in vitro mRNA capping exist, the first is the addition of a regular m7 gppppg cap analog structure or an anti-reverse cap analog (ARCA) to the mRNA transcription system to achieve mRNA capping and in vitro transcription. Second, in the early stage of in vitro transcription, mRNA capping is accomplished by a capping enzyme reaction, which is an effective enzyme catalyzing the formation of a cap structure, consisting of two subunits, D1 and D12, having RNA triphosphatase activity, guanylyl transferase activity, and guanine methyltransferase activity, and can attach a 7-methylguanine cap structure (m7Gppp) to the 5 'end of RNA (m7Gppp5' N).
At present, in vitro transcription kits on the market are mainly developed aiming at the two methods, the first method of generating a cap structure in the transcription process by relying on a cap analogue has low efficiency and low yield, and the generated transcription product has high immunogenicity and causes difficulty in the application of subsequent transcription product mRNA. Secondly, a capping structure is added to the 5' end of RNA by using capping enzyme, products of the type are capped on the basis of obtaining in vitro transcribed RNA, the method involves multiple enzyme adding operations from transcription, capping to tailing, the time consumption is long, the procedure is complicated, the degradation and loss of intermediate products can be caused in the circulating process, and the overall efficiency and yield are all to be improved.
In view of the technical problems of the products in the market, especially in view of how to synchronously perform transcription capping and simultaneously produce high-quality transcription products, many professionals make related modifications and researches, such as preparing mRNA by using related transcription tool enzymes in a prokaryotic system, selecting RNA polymerase and capping enzyme after cracking prokaryotic cells, but the cracking products of the method are all enzymes in the prokaryotic cells, wherein other foreign nucleases or nucleic acids originated from the large intestine are not lacked, so that the specificity of the enzyme system is not high, and the subsequent production efficiency, quality and yield of the mRNA are reduced. Even directly degrade RNA and fail to effectively transcribe the desired fragment.
Therefore, the existing mRNA production technology with simple and convenient process and short flow for large-scale mass production is lacked, and the establishment of the mRNA preparation method with simple and convenient operation, good stability and high efficiency can promote an mRNA vaccine platform and assist the future vaccine research and development process.
Disclosure of Invention
In view of the above-mentioned disadvantages of the prior art, the present invention aims to provide a chimeric enzyme and a method for synthesizing Cap0mRNA by one-step reaction in vitro, which solves the problems of the prior art.
To achieve the above and other related objects, the present invention provides a chimeric enzyme for one-step synthesis of mRNA by an in vitro enzymatic method, the chimeric enzyme being a transcription-capped chimeric enzyme, characterized in that the chimeric enzyme comprises a capping enzyme domain and an RNA polymerase domain, the RNA polymerase domain comprising RNA polymerase and Spy Catcher, and the capping enzyme catalytic domain comprising viral capping enzyme and Spy Tag; or, the RNA polymerase domain comprises RNA polymerase and Spy Tag, and the capping enzyme catalytic domain comprises viral capping enzyme and Spy Catcher; in the chimeric enzyme, Spy Tag and Spy Catcher are covalently bound; both the RNA polymerase domain and the capping enzyme catalytic domain further comprise a protein purification tag.
The invention also provides a method for synthesizing mRNA by in vitro transcription in one step, which is characterized by comprising the following steps: firstly, obtaining a template DNA transcribed by mRNA; ② adding the mosaic enzyme to carry out in vitro transcription to obtain mRNA with Cap0 structure.
The invention also provides a polynucleotide encoding the RNA polymerase domain and/or the capping enzyme catalytic domain.
The invention also provides a nucleic acid construct comprising the polynucleotide.
The invention also provides a cell comprising said nucleic acid construct or having exogenous said polynucleotide integrated into its genome.
The invention also provides a preparation method of the chimeric enzyme, which comprises the following steps:
1) co-transfecting a construct comprising the RNA polymerase domain and a construct comprising a capping enzyme catalytic domain into a host cell, and culturing the host cell;
2) the chimeric enzyme was extracted from the culture system.
The invention also provides a kit for synthesizing mRNA with a 5' end cap structure in one step in vitro, wherein the kit comprises the chimeric enzyme.
The invention also provides the use of the chimeric enzyme, the polynucleotide, the nucleic acid construct and the kit in vitro synthesis of mRNA with a 5' end cap structure.
As described above, the method for synthesizing mRNA having a 5' end cap structure in one step in vitro according to the present invention has the following beneficial effects: the T7RNA polymerase and the capping enzyme are embedded, so that the capping work can be simultaneously finished in the in vitro transcription process, the vaccinia virus capping enzyme can cap mRNA within one hour, the efficiency reaches nearly 100%, and the direction is correct. T7RNA polymerase and capping enzyme are chimeric based on connecting peptide and prokaryotes are used as a production platform to generate chimeric enzyme, and the chimeric enzyme can complete RNA transcription and 5' end capping in one step in the protein translation process. Through cooperative work, the post-transcriptional modification of RNA is simplified, and the efficiency of protein expression is improved. The chimeric enzyme simplifies the step-by-step and fractional process of the traditional mRNA synthesis, shortens the whole synthesis time, improves the reaction efficiency, reduces the loss of intermediate products in the step-by-step reaction process, improves the mRNA yield, and can obtain stable mRNA on an expression vector with a corresponding original. Saves the production process and cost of mRNA, can meet the requirement of industrial production, and lays a foundation for economic and efficient production of mRNA.
Drawings
FIG. 1 shows SDS-PAGE electrophoresis of T7RNA polymerase and vaccinia virus capping enzyme chimeric enzyme, MK:180KD protein Marker, R: SDS-PAGE reduction electrophoresis;
FIG. 2 is a graph showing the comparison of chimeric enzymes with stepwise transcribed and capped mRNA product production; wherein 1 and 2 are the electrophoresis images of the chimeric enzyme and the mRNA nucleic acid detection by step-by-step transcription and capping respectively.
FIG. 3 shows the expression verification of EGFP mRNA transcribed in vitro under a fluorescence microscope, wherein A is a negative control, B is a fluorescent signal of a stepwise method, and C is a fluorescent signal of a chimeric enzyme.
FIG. 4 shows a comparison of chimeric enzymes with stepwise transcription and capping for detecting fluorescence signal intensity after cell transfection in a flow cytometer, where A is a negative control, B is a flow analysis diagram of the stepwise method, and C is a flow analysis diagram of the chimeric enzymes;
Detailed Description
The invention provides a chimeric enzyme for synthesizing mRNA in one step by an in vitro enzymatic method, which is a transcription capping chimeric enzyme and is characterized in that the chimeric enzyme comprises a capping enzyme domain and an RNA polymerase domain, wherein the RNA polymerase domain comprises RNA polymerase and Spy Catcher, and the capping enzyme catalytic domain comprises virus capping enzyme and Spy Tag; or, the RNA polymerase domain comprises RNA polymerase and Spy Tag, and the capping enzyme catalytic domain comprises viral capping enzyme and Spy Catcher; in the chimeric enzyme, Spy Tag and Spy Catcher are covalently bound; both the RNA polymerase domain and the capping enzyme catalytic domain further comprise a protein purification tag.
The invention also provides a method for synthesizing mRNA with a 5' end cap structure in one step in vitro, which comprises the step of mixing the chimeric enzyme and template DNA and then carrying out a coreaction, wherein the chimeric enzyme comprises an RNA polymerase domain and a capping enzyme catalysis domain, the RNA polymerase domain and the capping enzyme catalysis domain are chimeric through flexible connecting peptide, the nitrogen end of the chimeric enzyme contains a histidine purification tag, and the template DNA comprises a PolyA tail element.
The template DNA is selected from plasmid DNA or an isolated DNA fragment of interest.
The plasmid DNA includes a target DNA and a vector.
In one embodiment, the plasmid DNA is a linearizable plasmid DNA. Because the transcription reaction continues to the end of the DNA template, the linearizable plasmid DNA ensures that RNA transcripts of defined length and sequence are obtained.
In one embodiment, the vector sequence of the linearizable plasmid DNA is set forth in SEQ ID NO 13. The integration of the target DNA into the vector to obtain linearized plasmid DNA as the transcribed template DNA can increase the amount of the template DNA and facilitate transcription. Meanwhile, the in vitro transcription of the linearized plasmid DNA can complete the simultaneous capping and tailing of RNA in one step.
The usage amount of the template DNA is 20ng-1 mu g.
The separated target DNA fragment is not integrated into a vector, but is amplified and directly used as a template DNA for transcription.
The method further comprises adding a buffer to the co-reaction system of the chimeric enzyme and the template DNA. The buffer solution comprises Tris-HCl and MgCl2、KCl、DTT、Spermidine。
In one embodiment, the concentration of each reagent in the buffer is 50mM Tris-HCl, pH 8.0; 5mM KCl; 6mM MgCl2；1mM DTT；2mM Spermidine。
In one embodiment, the method further comprises adding dntps to a co-reaction system of the chimeric enzyme and the template DNA. The dNTP is usually used as a raw material in RNA synthesis and is a mixture, and specifically includes dATP, dGTP, dUTP, dCTP, and the like.
The effects of other substances in the buffer are: the buffer solution is usually used for stabilizing the acid-base balance of a reaction system, and the magnesium ions are beneficial to stabilizing nucleotide and stabilizing an amplification system, so that the enzyme activity is improved; DTT can protect the activity of an enzyme of which the active center contains-SH; spermidine is used to increase the efficiency of restriction enzyme reactions to low purity DNA.
In one embodiment, the buffer is used in a volume of 1.5 to 2.5. mu.L based on the total volume of the co-reaction system. Specifically, one selected from the following ranges: 1.5 to 1.7. mu.L, 1.7 to 1.9. mu.L, 1.9 to 2.1. mu.L, 2.1 to 2.3. mu.L, 2.3 to 2.5. mu.L.
In one embodiment, the method further comprises adding a methylated donor reagent to the co-reaction system of the chimeric enzyme and the template DNA.
In one embodiment, the substrate for the capping reaction is SAM (S-adenosylmethionine). The methylated donor reagent serves as a substrate for the capping reaction.
In one embodiment, the total volume of the co-reaction system can be made up with RNase Free Water.
In one embodiment, the co-reaction conditions are from 35 ℃ to 38 ℃ for 2 to 3 hours.
In one embodiment, the method further comprises purifying the in vitro transcription product obtained from the co-reaction. The purification method may be any RNA purification method known in the art, for example, phenol/chloroform purification.
In the chimeric enzyme, the RNA polymerase is bacteriophage-derived RNA polymerase, prokaryotic RNA polymerase, eukaryotic RNA polymerase, and other mitochondrial (mitochondrial single subunit RNA polymerase can be used for in vitro transcription) and chloroplast RNA polymerase species.
The bacteriophage-derived RNA Polymerase is selected from T7RNA Polymerase (T7 RNA Polymerase), SP6 RNA Polymerase, or T3 RNA Polymerase.
The RNA polymerase was linked to the Spy Catcher via a first Linker peptide (i.e., Linker 1).
The first connecting peptide sequence is: GGGGSGGGGSGGGGS.
The term "catalytic domain" refers to a protein domain that is necessary and sufficient (especially in terms of its three-dimensional structure) to ensure enzyme function.
The viral capping enzyme is selected from one or more of the following: vaccinia virus capping enzyme, bluetongue virus capping enzyme, bamboo mosaic virus capping enzyme, African swine fever virus capping enzyme, organic lake algae type 1 DNA virus capping enzyme, organic lake algae type 2 DNA virus capping enzyme, spherical synechocystis virus capping enzyme or golden algae virus capping enzyme.
The viral capping enzyme may also be an enzyme subunit of any of the above viral capping enzymes.
In a preferred embodiment, the chimeric enzyme is a T7RNA polymerase-vaccinia virus capping enzyme chimeric enzyme.
The vaccinia virus capping enzyme is an effective enzyme catalyzing formation of a cap structure, is composed of two subunits of D1 and D12, has RNA triphosphatase activity, guanylyl transferase activity and guanine methyltransferase activity, and can be used for connecting a 7-methylguanine cap structure (m7Gppp) to the 5 'end of RNA (m7Gppp5' N).
The Spy Tag-Spy Catcher system is derived from a CnaB2 domain (a domain separated from fibronectin FbaB), Spy Tag is a polypeptide segment, Spy Catcher is a polypeptide segment combined with Spy Catcher, and Asp in Spy Tag and Lys in Spy Catcher can spontaneously form a stable isopeptide covalent bond under various conditions to generate a stable molecular self-assembly.
The invention compounds RNA polymerase and virus capping enzyme by using a Spy Tag-Spy Catcher protein self-assembly system, and the Spy Tag and the Spy Catcher can be randomly assembled. The Spy Tag and Spy Catcher can be complete polypeptide fragments or partial polypeptide fragments.
The viral capping enzyme is linked to Spy Tag via a second Linker peptide (i.e., Linker 2). The second connecting peptide sequence is: GGGGS.
Both the RNA polymerase domain and the capping enzyme catalytic domain further comprise a protein purification tag.
The protein purification Tag is selected from HIS-Tag (histidine Tag), GST-Tag (glutathione mercaptotransferase Tag), MBP-Tag (maltose binding protein Tag), NusA-Tag (transcription termination/anti-termination protein Tag) or SUMO (small ubiquitin related modifier).
The invention also provides a polynucleotide encoding the RNA polymerase domain and/or the capping enzyme catalytic domain.
In one embodiment, the polynucleotide encodes the RNA polymerase domain, the capping enzyme catalytic domain, or the chimeric enzyme.
The invention also provides a nucleic acid construct comprising the polynucleotide.
The nucleic acid construct may be an expression vector, such as a plasmid.
The invention also provides a cell comprising said nucleic acid construct or having exogenous said polynucleotide integrated into its genome.
The cell may be a mammalian cell, an insect cell, a bacterium or a yeast. In one embodiment, the cell is an escherichia coli cell. The cells may be wild-type or artificially engineered.
The invention also provides a preparation method of the chimeric enzyme, which comprises the following steps:
1) co-transfecting a construct comprising the RNA polymerase domain and a construct comprising a capping enzyme catalytic domain into a host cell, and culturing the host cell;
2) the chimeric enzyme was extracted from the culture system.
The host cell is selected from mammalian cells, insect cells, bacteria or yeast. In one embodiment, the cell is an escherichia coli cell. The cells may be wild-type or artificially engineered.
The Escherichia coli cell is BL21(DE 3).
In one embodiment, after co-transfecting the construct comprising the RNA polymerase domain and the construct comprising the capping enzyme catalytic domain into a host cell, the host cell is induced to express the chimeric enzyme during culture by the addition of an inducer and/or at low temperature.
The inducer is a beta-galactosidase inducer. The inducer is, for example, IPTG.
The low-temperature induction conditions are as follows: carrying out induction expression for 10-18 h at 10-18 ℃.
In one embodiment, the step 2) further comprises purifying the chimeric enzyme after extraction from the culture system.
The invention also provides a kit for synthesizing mRNA with a 5' end cap structure in one step in vitro, wherein the kit comprises the chimeric enzyme.
The invention also provides the use of the chimeric enzyme, the polynucleotide, the nucleic acid construct and the kit in vitro synthesis of mRNA with a 5' end cap structure.
The embodiments of the present invention are described below with reference to specific embodiments, and other advantages and effects of the present invention will be easily understood by those skilled in the art from the disclosure of the present specification. The invention is capable of other and different embodiments and of being practiced or of being carried out in various ways, and its several details are capable of modification in various respects, all without departing from the spirit and scope of the present invention.
Before the present embodiments are further described, it is to be understood that the scope of the invention is not limited to the particular embodiments described below; it is also to be understood that the terminology used in the examples is for the purpose of describing particular embodiments, and is not intended to limit the scope of the present invention; in the description and claims of the present application, the singular forms "a", "an" and "the" include plural referents unless the context clearly dictates otherwise.
When numerical ranges are given in the examples, it is understood that both endpoints of each of the numerical ranges and any value therebetween can be selected unless the invention otherwise indicated. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In addition to the specific methods, devices, and materials used in the examples, any methods, devices, and materials similar or equivalent to those described in the examples may be used in the practice of the invention in addition to the specific methods, devices, and materials used in the examples, in keeping with the knowledge of one skilled in the art and with the description of the invention.
Example 1: preparation of chimeric enzyme of T7RNA polymerase and vaccinia RNA capping enzyme
Vector construction and product expression of T7RNA polymerase and vaccinia RNA capping enzyme chimeric enzyme
(1) Construction of fusion expression plasmid of T7RNA polymerase gene, Spy Catcher element and 6His
T7RNA polymerase (T7RP) was fused to the Spy Catcher gene by Linker1 to construct recombinant plasmid A, as follows: the T7RNA polymerase fragment was first amplified using primers pQE30-T7RP-F (SEQ ID NO:1) and T7RP-R (SEQ ID NO:2), and the gene sequence of the Linker1-Spy Catcher was then amplified using T7RP-Linker1-F (SEQ ID NO:3) and T7RP-SC-R (SEQ ID NO: 4). After amplification, the amplified DNA is used
Figure BDA0003278085840000071
The PCR one-step directional cloning kit (self-produced by Suzhou near-shore protein science and technology Co., Ltd.) splices the gene sequence of T7RP + Linker1+ Spy Catcher and seamlessly clones the gene sequence to pQE30 expression vector (Shanghai Ying Jun Biotechnology Co., Ltd.) which is linearized by NdeI (CATATG) and XbaI (TCTAGA) (Shanghai Ying Jun Biotechnology Co., Ltd.) and is transformed into Escherichia coli DH5 alpha. After culturing in LB culture medium (containing ampicillin), selecting single colony, extracting recombinant plasmid, sequencing, and selecting recombinant plasmid A with correct sequencing. The amino acid sequence expressed by the recombinant plasmid A is shown as SEQ ID NO. 11.
(2) Construction of vaccinia virus capping enzyme gene, Spy Tag element and 6His fusion expression plasmid
Vaccinia virus capping enzyme (two subunits D1 and D12) is fused with Spy Tag gene and 6His gene to form recombinant plasmid B, and the steps are as follows:
the large subunit of vaccinia virus capping enzyme D1 was first amplified using primers pRSFDuet1-D1-F (SEQ ID NO:5) and D1-His-R (SEQ ID NO:6) to give the gene sequence D1+ His. The gene sequence was cloned seamlessly into pRSFDuet1 double expression vector (Shanghai Yingjun Biotechnology Co., Ltd.) linearized with BamHI (GGATTC) and hindIII (AAGCTT).
The small subunit of vaccinia virus capping enzyme D12 was amplified by pRSFDuet1-D12-F (SEQ ID NO:7) and D12-R (SEQ ID NO:8), and the gene sequence of Linker2-ST was amplified using D12-Linker2-F (SEQ ID NO:9) and D12-ST-R (SEQ ID NO: 10). After amplification, the amplified DNA is used
Figure BDA0003278085840000081
The PCR one-step directional cloning kit (self-produced by Suzhou near-shore protein science and technology Co., Ltd.) splices the gene sequence of D12+ Linker2+ Spy Tag and seamlessly clones the sequence to the NdeI (CATATG) and Xho I (CTCGAG) linearized pRSFDuet1 double expression vector.
The above-mentioned double expression vector was transformed into E.coli DH5 alpha. After culturing in LB culture medium (containing kanamycin), selecting single colony, extracting recombinant plasmid B, sequencing, and selecting the recombinant plasmid B with correct sequencing.
The amino acid sequence expressed by the recombinant plasmid B is shown as SEQ ID NO. 12. These include vaccinia virus capping enzymes (two subunits, D1 and D12), Spy Tag, and 6 His.
The two recombinant plasmids with correct sequencing are co-transfected into a high-efficiency competent Escherichia coli expression strain BL21(DE3) to obtain a strain of chimeric enzyme capable of expressing T7RNA polymerase and vaccinia virus capping enzyme, the obtained strain 1:100 is inoculated into LB culture medium (containing ampicillin and kanamycin), shaking culture is carried out at 37 ℃ and 250rpm until OD600 reaches 0.6, 0.1mM IPTG is added for induction expression, the culture temperature is adjusted to 16 ℃ at the same time for low-temperature induction expression overnight (16h) for improving the expression ratio of soluble protein, and centrifugal collection is carried out at 5000 rpm.
Purification of chimeric enzyme of T7RNA polymerase and vaccinia RNA capping enzyme
And (2) breaking the bacteria of the bacteria obtained in the step (1), mixing the total amount of the bacteria with a bacteria breaking buffer solution (50mM Tris-HCl, pH8.0,500mM NaCl) according to a ratio of 1:10, and carrying out ultrasonic disruption. Centrifuging at 12000rpm for 30min, collecting supernatant, discarding precipitate, collecting supernatant, and purifying chimeric enzyme by nickel column affinity chromatography according to 10His purification tag.
(1) Purification with Ni affinity chromatography column
Column balancing: equilibrating with equilibration buffer (20mM Tris-HCl, pH8.0,500mM NaCl,10mM imidazole) for 5-10 column volumes, wherein the buffer and sample do not contain chelating agents such as EDTA, etc., pH8.0, and detecting, zeroing and loading after the UV monitoring reading is stable.
Sampling: the sample should be kept clear, and if turbidity, precipitation and the like occur, centrifugation is needed, and effluent liquid is collected.
Column balancing: after the loading is finished, the chromatographic column needs to be re-equilibrated by an equilibration buffer solution, and the reading returns to a baseline or is relatively stable by ultraviolet detection.
③ prewashing the sample: the fractions eluted were collected by prewashing with prewashing buffer (20mM Tris-HCl, pH8.0,500mM NaCl,50mM imidazole).
Elution of the sample: the target protein was subjected to gradient elution with 20mM Tris-HCl, pH8.0,500mM NaCl buffer solution containing a linear imidazole gradient (concentrations of 100mM imidazole, 250mM imidazole and 500mM imidazole, respectively) eluent, and the fractions obtained by the elution were collected to obtain a preliminarily purified chimeric enzyme.
(2) Heparin affinity chromatography column purification
Column balancing: 5-10 column volumes are equilibrated with equilibration buffer (50mM NaCl, 20mM Tris-HCl, pH8.0), pH reaches 8.0, detection zeroing and loading are carried out after ultraviolet monitoring reading is stable, and the specific steps are as follows:
sampling: and loading the initial product purified by the Ni affinity chromatographic column to a heparin affinity chromatographic column.
Column balancing: after the loading is finished, the chromatographic column needs to be re-equilibrated by an equilibration buffer solution, and the reading returns to a baseline or is relatively stable by ultraviolet detection.
③ eluting the sample: the target protein was subjected to gradient elution using 20mM Tris-HCl, pH8.0,1mM DTT, 0.1mM EDTA buffer solution containing a linear salt gradient (concentration: 50mM NaCl, 100mM NaCl, 250mM NaCl and 500mM NaCl) as an eluent, and the fractions obtained by the elution were collected to obtain a purified chimeric enzyme.
The obtained chimeric enzyme eluate was subjected to ultrafiltration concentration and then dialyzed and replaced into a storage buffer (20mM Tris-HCl pH8.0, 100mM NaCl, 1mM DTT, 0.1mM EDTA, 0.1% Triton X-100, 50% Glycerol).
SDS-PAGE electrophoretic detection of purity of chimeric enzyme
FIG. 1 shows SDS-PAGE of the final purification of chimeric enzyme fused with T7RNA polymerase and vaccinia RNA capping enzyme. The results show that under reducing (R) conditions, the non-covalent bond of the chimeric enzyme is opened, the T7RNA polymerase and the D12 domain of the vaccinia virus capping enzyme are successfully covalently coupled, the size of the coupled enzyme is about 150KD as detected by SDS-PAGE, and the size of the D1 subunit domain of the vaccinia virus capping enzyme is about 110KD as detected by SDS-PAGE. It can be seen that T7RNA polymerase and vaccinia virus capping enzyme chimeric enzyme preparation.
Example 2: recombinant mRNA is obtained by one step by using chimeric enzyme fused by T7RNA polymerase and vaccinia RNA capping enzyme
1. Design of recombinant mRNA transcription templates
In the present invention, the transcription template of the recombinant mRNA is plasmid DNA which can be linearized, and the plasmid is characterized in that: contains a T7 promoter, a 5 'UTR region, a multiple cloning site region, a 3' UTR region, a polyadenylation sequence and a linearization site Esp 3I. Polyadenylation sequence and linearization site: the polyadenylation sequence consists of 150 consecutive adenosines, which are synthesized by the third-party GenBank, and a restriction enzyme Esp3I site is added after the sequence, 5' -CGTCTC (N)1-3 '/3 ' -GCAGAG (N)5-5 ", Esp3I in the present invention: 5'-AAAAAGAGACG-3' this site was used for subsequent linearization of the vector and to ensure that no nucleic acid remains from the polyA tail after linearization. The basic skeleton of the plasmid was selected from the vector pUC19, all the sequences were synthesized and cloned seamlessly into the vector pUC19 by restriction enzyme sites EcoR I (GAATTC) and HindIII (AAGCTT), and Esp3I was used as the linearization site of the vector. The resulting plasmid was designated pRTT and its sequence is SEQ ID NO: 13.
2. preparation of linearized Green fluorescent protein (EGFP) transcription template DNA
The sequence of EGFP synthesized by the gene (SEQ ID NO: 14) was used as a template and cloned into pRTT vector by restriction enzyme sites Kpn I (CCATGG) and Pst I (GATATC) using the overlap PCR technique. Transforming to Escherichia coli DH5alpha competent cells, culturing in LB culture medium, selecting single colony, extracting recombinant plasmid, sequencing, selecting the recombinant plasmid with correct sequencing, and naming the recombinant plasmid as pRTT-EGFP.
The recombinant plasmid pRTT-EGFP is linearized at the Esp3I site, and meanwhile, the linearized pRTT idle plasmid is used as a negative control, and a transcription template after enzyme digestion is obtained through gel recovery to prepare in-vitro transcription capping reaction.
3. One-step obtaining of recombinant mRNA by in vitro transcription using chimeric enzyme
RNA was obtained by in vitro transcription of the linearized plasmids pRTT-EGFP and pRTT (1. mu.g) obtained in example 2, step 2 with chimeric enzyme fused with vaccinia RNA capping enzyme by T7RNA polymerase and by simultaneous capping and tailing. And performing gel running verification on the product. The 20. mu.L reaction system included the following: reaction buffer (50mM Tris-HCl, pH 8.0; 5mM KCl; 6mM MgCl 2; 1mM DTT; 2mM Spermidine) 2. mu. L, ATP (100mM) 1.5. mu. L, UTP (100mM) 1.5. mu. L, CTP (100mM) 1.5. mu. L, GTP (120mM) 1.5. mu. L, SAM (2mM) 2. mu.L, Template DNA 20 ng-1. mu.g, T7RNA Polymerase and clamping Enzyme-linked complex 1. mu.L, and RNase Free Water to 20. mu.L. Reacting at 37 ℃ for 2-3 hours.
Meanwhile, the in vitro transcription product is purified by a phenol/chloroform method, and the steps are as follows:
add 160. mu.l RNase-free Water to dilute the product to 180. mu.l.
② adding 20 mul of 3M sodium acetate (pH 5.2) into the diluted product, and fully mixing by using a pipette.
③ adding 200. mu.l of phenol/chloroform mixture (1:1) for extraction, centrifuging at room temperature of 10000rpm for 5min, and transferring the upper solution (aqueous phase) to a new RNase-free EP tube.
Adding chloroform with the same volume as the water phase for extraction for 2 times, and collecting the upper water phase.
Adding 2 times volume of absolute ethyl alcohol and mixing evenly, incubating at-20 ℃ for at least 30min, and centrifuging at 4 ℃ and 15000rpm for 15 min.
Sixthly, abandoning the supernatant, adding 500 mul of precooled 70% ethanol to wash the RNA sediment, centrifuging at the temperature of 4 ℃ and the speed of 15000rpm, and abandoning the supernatant.
Seventhly, drying the cover for 2min, and adding 20-50 mu l of RNase-free Water or other buffer solution to dissolve the RNA precipitate. Storing at-80 deg.C.
Example 3: comparison of in vitro transcription efficiency and yield of chimeric enzyme fused from T7RNA polymerase and vaccinia RNA capping enzyme and combination of T7RNA polymerase and vaccinia capping enzyme monomer
This example is substantially the same as the method of step 3 in example 2, except that T7RNA polymerase and the vaccinia virus capping enzyme chimeric enzyme in example 2 were replaced with a transcription reaction using T7RNA polymerase, and a 20. mu.L reaction system included the following: ATP (100mM) 1.5. mu. L, UTP (100mM) 1.5. mu. L, CTP (100mM) 1.5. mu. L, GTP (120mM) 1.5. mu.L, Reaction buffer (50mM Tris-HCl, pH 8.0L; 6mM MgCl 2; 1mM DTT; 2mM Spermidine) 2. mu.L, Template DNA 20 ng-1. mu.g, T7RNA polymerase 1. mu.L, RNase Free Water Up to 20. mu.L, and Reaction at 37 ℃ for 2-3 hours. The resulting RNA samples to be transcribed in vitro were incubated at 65 ℃ for 5 minutes to denature the RNA and then immediately transferred to ice, and a 20. mu.L reaction included the following: 50mM Tris-HCl, GTP (120mM) 1.5. mu. L, SAM (2mM) 2. mu.L, 1mM DTT, vaccinia RNA capping enzyme, and a exported in vitro transcribed RNA sample. The reaction was carried out at 37 ℃ for 30 min.
1. And (3) comparing efficiencies:
table 1 shows the main steps and duration comparison of chimeric enzymes and step-by-step transcription and capping, and it can be seen from Table 1 that chimeric enzymes show the advantages of simple operation and short reaction time in comparison with step-by-step transcription and capping during the process of in vitro transcription to generate mRNA; the step-by-step steps are omitted, so that the loss of the intermediate product can be reduced, and the yield of the subsequent product is improved.
TABLE 1
Figure BDA0003278085840000111
2. And (3) yield comparison:
FIG. 2 is a graph comparing the production of chimeric enzyme with stepwise transcribed and capped mRNA, wherein 1 and 2 are the nucleic acid electrophoresis of chimeric enzyme with stepwise transcribed and capped mRNA, respectively, and it can be seen from the graph that the brightness of the target band transcribed by chimeric enzyme is significantly higher than that of the product obtained by stepwise method, and the yield of chimeric enzyme product is calculated from the mRNA obtained after 1 μ g linearized DNA is transcribed in vitro: 83%, fractional transcription and capping yields were: 70 percent. Meanwhile, the results shown in Table 1 show that the chimeric enzyme has a shortened reaction time and an improved reaction efficiency by simplifying the reaction procedure, thereby increasing the product yield.
Example 4: quality verification of recombinant mRNA obtained by chimeric enzyme
1. Transfection of cells with synthetic mRNA
HEK293 cells were plated in 24-well plates at a cell density of 0.25 × 106cells/ml and cultured at 37 ℃ in 5% CO 2. When the confluency of the cells was about 80%, the OPTI-MEM medium was replaced before transfection to a total amount of 0.45ml per well. Mu.g of the mRNA obtained in example 3, including the negative control, was added to a certain amount of OPTI-MEM medium, and mixed well to prepare an RNA dilution of 25. mu.l. Mu.l of lipoMessenger Max is taken, then 23.5 mu.l of serum-free diluent is added, and the mixture is fully mixed to prepare lipoMessenger Max diluent with the final volume of 25 mu.l. Standing at room temperature for 10 min. The lipoMessenger Max diluent and the RNA diluent were mixed well (aspirated 10 times or more with a sample injector) and allowed to stand at room temperature for 5 min. The transfection complex preparation is complete. 50 μ l of the transfection complex was added dropwise to the cells with 0.45ml of OPTI-MEM, and the plates were moved back and forth and mixed well (gently shaking, slow throughout the addition). Culturing at 37 deg.C and 5% CO2 for 24 hr, observing culture result after 24 hr, and taking pictures.
2. Detection of cellular fluorescence signals after transfection
Placing the cells cultured after transfection in the step 1 under a fluorescence microscope, as shown in fig. 3, fig. 3A is a fluorescence signal of an HEK293 cell transfected with negative control mRNA, fig. 3B is a fluorescence signal of a cell transfected with a step-by-step in vitro transcription product, and fig. 3C is a fluorescence signal of an HEK293 cell transfected with a chimeric enzyme in vitro transcription product and EGFP recombinant mRNA. Comparison shows that HEK293 cells transfected with EGFP recombinant mRNA can correctly recognize and successfully translate green fluorescent protein. The cell transfection effect of mRNA obtained by transcription of the chimeric enzyme and the cell transfection effect of stepwise in vitro transcription are not obviously different, which shows that the chimeric enzyme can ensure the enzyme activities of RNA polymerase and capping enzyme on the basis of improving the reaction efficiency, and simultaneously combines with yield analysis. The results demonstrate that the chimeric enzyme fused with T7RNA polymerase and vaccinia RNA capping enzyme can successfully perform efficient transcription capping work in vitro.
3. Flow cytometry for detecting cell transfection efficiency
The transfection complex of step 1 was aspirated along the tube wall to remove the medium, 500. mu.l trypsin was added and placed at 37 ℃ with 5% CO2Digest for 1 min, shake front and back, and add 500 μ l of 10% calf serum in DMEM. Sucking, mixing, placing in a 1.5ml centrifuge tube, centrifuging at 2050rpm for 5min, pouring the supernatant, resuspending in 1 XPBS 500. mu.l, and detecting by flow. FIG. 4 shows detection of fluorescence after cell transfection under chimeric enzymes and flow cytometer with stepwise transcription and cappingIn the light signal intensity detection, compared with the negative, the chimeric enzyme has the green fluorescence signal intensity of 71.5 percent, the stepwise green fluorescence signal intensity of 67.2 percent, and the green fluorescence signals of the two have no significant difference.
The above examples are intended to illustrate the disclosed embodiments of the invention and are not to be construed as limiting the invention. In addition, various modifications of the invention set forth herein, as well as variations of the methods of the invention, will be apparent to persons skilled in the art without departing from the scope and spirit of the invention. While the invention has been specifically described in connection with various specific preferred embodiments thereof, it should be understood that the invention should not be unduly limited to such specific embodiments. Indeed, various modifications of the above-described embodiments which are obvious to those skilled in the art to which the invention pertains are intended to be covered by the scope of the present invention.
Sequence listing
<110> Suzhou near shore protein science and technology GmbH
<120> chimeric enzyme and application and method thereof in synthesis of Cap0mRNA by one-step reaction in vitro
<160> 14
<170> SIPOSequenceListing 1.0
<210> 1
<211> 44
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 1
cgcatcacca tcaccatcac aacacgatta acatcgctaa gaac 44
<210> 2
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 2
cgcgaacgcg tagtccgact c 21
<210> 3
<211> 86
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 3
gagtcggact acgcgttcgc gggtggtggt ggtagcggtg gtggtggtag cggtggtggt 60
ggtagcatgg ttgatacctt atcagg 86
<210> 4
<211> 44
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 4
ccaagctcag ctaattaagc ttaaatatga gcgtcacctt tagt 44
<210> 5
<211> 50
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 5
cagccatcac catcatcacc acagccaatg gatgccaatg tggtgagcag 50
<210> 6
<211> 60
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 6
cattatgcgg ccgcaagctt agtggtggtg gtggtggtgg cgcttactaa aaacataaac 60
<210> 7
<211> 46
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 7
gtataagaag gagatataca tatggatgaa atcgtgaaaa acatcc 46
<210> 8
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 8
cagcagcagt ttcaccaggc g 21
<210> 9
<211> 57
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 9
cgcctggtga aactgctgct gggtggtggt ggtagcgtgc cgaccattgt gatggtg 57
<210> 10
<211> 67
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 10
cggtttcttt accagactcg agttatttat agcgtttata cgcatccacc atcacaatgg 60
tcggcac 67
<210> 11
<211> 1021
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 11
Met Arg Gly Ser His His His His His His Asn Thr Ile Asn Ile Ala
1 5 10 15
Lys Asn Asp Phe Ser Asp Ile Glu Leu Ala Ala Ile Pro Phe Asn Thr
20 25 30
Leu Ala Asp His Tyr Gly Glu Arg Leu Ala Arg Glu Gln Leu Ala Leu
35 40 45
Glu His Glu Ser Tyr Glu Met Gly Glu Ala Arg Phe Arg Lys Met Phe
50 55 60
Glu Arg Gln Leu Lys Ala Gly Glu Val Ala Asp Asn Ala Ala Ala Lys
65 70 75 80
Pro Leu Ile Thr Thr Leu Leu Pro Lys Met Ile Ala Arg Ile Asn Asp
85 90 95
Trp Phe Glu Glu Val Lys Ala Lys Arg Gly Lys Arg Pro Thr Ala Phe
100 105 110
Gln Phe Leu Gln Glu Ile Lys Pro Glu Ala Val Ala Tyr Ile Thr Ile
115 120 125
Lys Thr Thr Leu Ala Cys Leu Thr Ser Ala Asp Asn Thr Thr Val Gln
130 135 140
Ala Val Ala Ser Ala Ile Gly Arg Ala Ile Glu Asp Glu Ala Arg Phe
145 150 155 160
Gly Arg Ile Arg Asp Leu Glu Ala Lys His Phe Lys Lys Asn Val Glu
165 170 175
Glu Gln Leu Asn Lys Arg Val Gly His Val Tyr Lys Lys Ala Phe Met
180 185 190
Gln Val Val Glu Ala Asp Met Leu Ser Lys Gly Leu Leu Gly Gly Glu
195 200 205
Ala Trp Ser Ser Trp His Lys Glu Asp Ser Ile His Val Gly Val Arg
210 215 220
Cys Ile Glu Met Leu Ile Glu Ser Thr Gly Met Val Ser Leu His Arg
225 230 235 240
Gln Asn Ala Gly Val Val Gly Gln Asp Ser Glu Thr Ile Glu Leu Ala
245 250 255
Pro Glu Tyr Ala Glu Ala Ile Ala Thr Arg Ala Gly Ala Leu Ala Gly
260 265 270
Ile Ser Pro Met Phe Gln Pro Cys Val Val Pro Pro Lys Pro Trp Thr
275 280 285
Gly Ile Thr Gly Gly Gly Tyr Trp Ala Asn Gly Arg Arg Pro Leu Ala
290 295 300
Leu Val Arg Thr His Ser Lys Lys Ala Leu Met Arg Tyr Glu Asp Val
305 310 315 320
Tyr Met Pro Glu Val Tyr Lys Ala Ile Asn Ile Ala Gln Asn Thr Ala
325 330 335
Trp Lys Ile Asn Lys Lys Val Leu Ala Val Ala Asn Val Ile Thr Lys
340 345 350
Trp Lys His Cys Pro Val Glu Asp Ile Pro Ala Ile Glu Arg Glu Glu
355 360 365
Leu Pro Met Lys Pro Glu Asp Ile Asp Met Asn Pro Glu Ala Leu Thr
370 375 380
Ala Trp Lys Arg Ala Ala Ala Ala Val Tyr Arg Lys Asp Lys Ala Arg
385 390 395 400
Lys Ser Arg Arg Ile Ser Leu Glu Phe Met Leu Glu Gln Ala Asn Lys
405 410 415
Phe Ala Asn His Lys Ala Ile Trp Phe Pro Tyr Asn Met Asp Trp Arg
420 425 430
Gly Arg Val Tyr Ala Val Pro Met Phe Asn Pro Gln Gly Asn Asp Met
435 440 445
Thr Lys Gly Leu Leu Thr Leu Ala Lys Gly Lys Pro Ile Gly Lys Glu
450 455 460
Gly Tyr Tyr Trp Leu Lys Ile His Gly Ala Asn Cys Ala Gly Val Asp
465 470 475 480
Lys Val Pro Phe Pro Glu Arg Ile Lys Phe Ile Glu Glu Asn His Glu
485 490 495
Asn Ile Met Ala Cys Ala Lys Ser Pro Leu Glu Asn Thr Trp Trp Ala
500 505 510
Glu Gln Asp Ser Pro Phe Cys Phe Leu Ala Phe Cys Phe Glu Tyr Ala
515 520 525
Gly Val Gln His His Gly Leu Ser Tyr Asn Cys Ser Leu Pro Leu Ala
530 535 540
Phe Asp Gly Ser Cys Ser Gly Ile Gln His Phe Ser Ala Met Leu Arg
545 550 555 560
Asp Glu Val Gly Gly Arg Ala Val Asn Leu Leu Pro Ser Glu Thr Val
565 570 575
Gln Asp Ile Tyr Gly Ile Val Ala Lys Lys Val Asn Glu Ile Leu Gln
580 585 590
Ala Asp Ala Ile Asn Gly Thr Asp Asn Glu Val Val Thr Val Thr Asp
595 600 605
Glu Asn Thr Gly Glu Ile Ser Glu Lys Val Lys Leu Gly Thr Lys Ala
610 615 620
Leu Ala Gly Gln Trp Leu Ala Tyr Gly Val Thr Arg Ser Val Thr Lys
625 630 635 640
Arg Ser Val Met Thr Leu Ala Tyr Gly Ser Lys Glu Phe Gly Phe Arg
645 650 655
Gln Gln Val Leu Glu Asp Thr Ile Gln Pro Ala Ile Asp Ser Gly Lys
660 665 670
Gly Leu Met Phe Thr Gln Pro Asn Gln Ala Ala Gly Tyr Met Ala Lys
675 680 685
Leu Ile Trp Glu Ser Val Ser Val Thr Val Val Ala Ala Val Glu Ala
690 695 700
Met Asn Trp Leu Lys Ser Ala Ala Lys Leu Leu Ala Ala Glu Val Lys
705 710 715 720
Asp Lys Lys Thr Gly Glu Ile Leu Arg Lys Arg Cys Ala Val His Trp
725 730 735
Val Thr Pro Asp Gly Phe Pro Val Trp Gln Glu Tyr Lys Lys Pro Ile
740 745 750
Leu Thr Arg Leu Asn Leu Met Phe Leu Gly Gln Phe Arg Leu Gln Pro
755 760 765
Thr Ile Asn Thr Asn Lys Asp Ser Glu Ile Asp Ala His Lys Gln Glu
770 775 780
Ser Gly Ile Ala Pro Asn Phe Val His Ser Gln Asp Gly Ser His Leu
785 790 795 800
Arg Lys Thr Val Val Trp Ala His Glu Lys Tyr Gly Ile Glu Ser Phe
805 810 815
Ala Leu Ile His Asp Ser Phe Gly Thr Ile Pro Ala Asp Ala Ala Asn
820 825 830
Leu Phe Lys Ala Val Arg Glu Thr Met Val Asp Thr Tyr Glu Ser Cys
835 840 845
Asp Val Leu Ala Asp Phe Tyr Asp Gln Ile Ala Asp Gln Leu His Glu
850 855 860
Ser Gln Leu Asp Lys Met Pro Ala Leu Pro Ala Lys Gly Asn Leu Asn
865 870 875 880
Leu Arg Asp Ile Leu Glu Ser Asp Tyr Ala Phe Ala Gly Gly Gly Gly
885 890 895
Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Met Val Asp Thr Leu
900 905 910
Ser Gly Leu Ser Ser Glu Gln Gly Gln Ser Gly Asp Met Thr Ile Glu
915 920 925
Glu Asp Ser Ala Thr His Ile Lys Phe Ser Lys Arg Asp Glu Asp Gly
930 935 940
Lys Glu Leu Ala Gly Ala Thr Met Glu Leu Arg Asp Ser Ser Gly Lys
945 950 955 960
Thr Ile Ser Thr Trp Ile Ser Asp Gly Gln Val Lys Asp Phe Tyr Leu
965 970 975
Tyr Pro Gly Lys Tyr Thr Phe Val Glu Thr Ala Ala Pro Asp Gly Tyr
980 985 990
Glu Val Ala Thr Ala Ile Thr Phe Thr Val Asn Glu Gln Gly Gln Val
995 1000 1005
Thr Val Asn Gly Lys Ala Thr Lys Gly Asp Ala His Ile
1010 1015 1020
<210> 12
<211> 1156
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 12
Met Asp Ala Asn Val Val Ser Ser Ser Thr Ile Ala Thr Tyr Ile Asp
1 5 10 15
Ala Leu Ala Lys Asn Ala Ser Glu Leu Glu Gln Arg Ser Thr Ala Tyr
20 25 30
Glu Ile Asn Asn Glu Leu Glu Leu Val Phe Ile Lys Pro Pro Leu Ile
35 40 45
Thr Leu Thr Asn Val Val Asn Ile Ser Thr Ile Gln Glu Ser Phe Ile
50 55 60
Arg Phe Thr Val Thr Asn Lys Glu Gly Val Lys Ile Arg Thr Lys Ile
65 70 75 80
Pro Leu Ser Lys Val His Gly Leu Asp Val Lys Asn Val Gln Leu Val
85 90 95
Asp Ala Ile Asp Asn Ile Val Trp Glu Lys Lys Ser Leu Val Thr Glu
100 105 110
Asn Arg Leu His Lys Glu Cys Leu Leu Arg Leu Ser Thr Glu Glu Arg
115 120 125
His Ile Phe Leu Asp Tyr Lys Lys Tyr Gly Ser Ser Ile Arg Leu Glu
130 135 140
Leu Val Asn Leu Ile Gln Ala Lys Thr Lys Asn Phe Thr Ile Asp Phe
145 150 155 160
Lys Leu Lys Tyr Phe Leu Gly Ser Gly Ala Gln Ser Lys Ser Ser Leu
165 170 175
Leu His Ala Ile Asn His Pro Lys Ser Arg Pro Asn Thr Ser Leu Glu
180 185 190
Ile Glu Phe Thr Pro Arg Asp Asn Glu Thr Val Pro Tyr Asp Glu Leu
195 200 205
Ile Lys Glu Leu Thr Thr Leu Ser Arg His Ile Phe Met Ala Ser Pro
210 215 220
Glu Asn Val Ile Leu Ser Pro Pro Ile Asn Ala Pro Ile Lys Thr Phe
225 230 235 240
Met Leu Pro Lys Gln Asp Ile Val Gly Leu Asp Leu Glu Asn Leu Tyr
245 250 255
Ala Val Thr Lys Thr Asp Gly Ile Pro Ile Thr Ile Arg Val Thr Ser
260 265 270
Asn Gly Leu Tyr Cys Tyr Phe Thr His Leu Gly Tyr Ile Ile Arg Tyr
275 280 285
Pro Val Lys Arg Ile Ile Asp Ser Glu Val Val Val Phe Gly Glu Ala
290 295 300
Val Lys Asp Lys Asn Trp Thr Val Tyr Leu Ile Lys Leu Ile Glu Pro
305 310 315 320
Val Asn Ala Ile Asn Asp Arg Leu Glu Glu Ser Lys Tyr Val Glu Ser
325 330 335
Lys Leu Val Asp Ile Cys Asp Arg Ile Val Phe Lys Ser Lys Lys Tyr
340 345 350
Glu Gly Pro Phe Thr Thr Thr Ser Glu Val Val Asp Met Leu Ser Thr
355 360 365
Tyr Leu Pro Lys Gln Pro Glu Gly Val Ile Leu Phe Tyr Ser Lys Gly
370 375 380
Pro Lys Ser Asn Ile Asp Phe Lys Ile Lys Lys Glu Asn Thr Ile Asp
385 390 395 400
Gln Thr Ala Asn Val Val Phe Arg Tyr Met Ser Ser Glu Pro Ile Ile
405 410 415
Phe Gly Glu Ser Ser Ile Phe Val Glu Tyr Lys Lys Phe Ser Asn Asp
420 425 430
Lys Gly Phe Pro Lys Glu Tyr Gly Ser Gly Lys Ile Val Leu Tyr Asn
435 440 445
Gly Val Asn Tyr Leu Asn Asn Ile Tyr Cys Leu Glu Tyr Ile Asn Thr
450 455 460
His Asn Glu Val Gly Ile Lys Ser Val Val Val Pro Ile Lys Phe Ile
465 470 475 480
Ala Glu Phe Leu Val Asn Gly Glu Ile Leu Lys Pro Arg Ile Asp Lys
485 490 495
Thr Met Lys Tyr Ile Asn Ser Glu Asp Tyr Tyr Gly Asn Gln His Asn
500 505 510
Ile Ile Val Glu His Leu Arg Asp Gln Ser Ile Lys Ile Gly Asp Ile
515 520 525
Phe Asn Glu Asp Lys Leu Ser Asp Val Gly His Gln Tyr Ala Asn Asn
530 535 540
Asp Lys Phe Arg Leu Asn Pro Glu Val Ser Tyr Phe Thr Asn Lys Arg
545 550 555 560
Thr Arg Gly Pro Leu Gly Ile Leu Ser Asn Tyr Val Lys Thr Leu Leu
565 570 575
Ile Ser Met Tyr Cys Ser Lys Thr Phe Leu Asp Asp Ser Asn Lys Arg
580 585 590
Lys Val Leu Ala Ile Asp Phe Gly Asn Gly Ala Asp Leu Glu Lys Tyr
595 600 605
Phe Tyr Gly Glu Ile Ala Leu Leu Val Ala Thr Asp Pro Asp Ala Asp
610 615 620
Ala Ile Ala Arg Gly Asn Glu Arg Tyr Asn Lys Leu Asn Ser Gly Ile
625 630 635 640
Lys Thr Lys Tyr Tyr Lys Phe Asp Tyr Ile Gln Glu Thr Ile Arg Ser
645 650 655
Asp Thr Phe Val Ser Ser Val Arg Glu Val Phe Tyr Phe Gly Lys Phe
660 665 670
Asn Ile Ile Asp Trp Gln Phe Ala Ile His Tyr Ser Phe His Pro Arg
675 680 685
His Tyr Ala Thr Val Met Asn Asn Leu Ser Glu Leu Thr Ala Ser Gly
690 695 700
Gly Lys Val Leu Ile Thr Thr Met Asp Gly Asp Lys Leu Ser Lys Leu
705 710 715 720
Thr Asp Lys Lys Thr Phe Ile Ile His Lys Asn Leu Pro Ser Ser Glu
725 730 735
Asn Tyr Met Ser Val Glu Lys Ile Ala Asp Asp Arg Ile Val Val Tyr
740 745 750
Asn Pro Ser Thr Met Ser Thr Pro Met Thr Glu Tyr Ile Ile Lys Lys
755 760 765
Asn Asp Ile Val Arg Val Phe Asn Glu Tyr Gly Phe Val Leu Val Asp
770 775 780
Asn Val Asp Phe Ala Thr Ile Ile Glu Arg Ser Lys Lys Phe Ile Asn
785 790 795 800
Gly Ala Ser Thr Met Glu Asp Arg Pro Ser Thr Arg Asn Phe Phe Glu
805 810 815
Leu Asn Arg Gly Ala Ile Lys Cys Glu Gly Leu Asp Val Glu Asp Leu
820 825 830
Leu Ser Tyr Tyr Val Val Tyr Val Phe Ser Lys Arg His His His His
835 840 845
His His Met Asp Glu Ile Val Lys Asn Ile Arg Glu Gly Thr His Val
850 855 860
Leu Leu Pro Phe Tyr Glu Thr Leu Pro Glu Leu Asn Leu Ser Leu Gly
865 870 875 880
Lys Ser Pro Leu Pro Ser Leu Glu Tyr Gly Ala Asn Tyr Phe Leu Gln
885 890 895
Ile Ser Arg Val Asn Asp Leu Asn Arg Met Pro Thr Asp Met Leu Lys
900 905 910
Leu Phe Thr His Asp Ile Met Leu Pro Glu Ser Asp Leu Asp Lys Val
915 920 925
Tyr Glu Ile Leu Lys Ile Asn Ser Val Lys Tyr Tyr Gly Arg Ser Thr
930 935 940
Lys Ala Asp Ala Val Val Ala Asp Leu Ser Ala Arg Asn Lys Leu Phe
945 950 955 960
Lys Arg Glu Arg Asp Ala Ile Lys Ser Asn Asn His Leu Thr Glu Asn
965 970 975
Asn Leu Tyr Ile Ser Asp Tyr Lys Met Leu Thr Phe Asp Val Phe Arg
980 985 990
Pro Leu Phe Asp Phe Val Asn Glu Lys Tyr Cys Ile Ile Lys Leu Pro
995 1000 1005
Thr Leu Phe Gly Arg Gly Val Ile Asp Thr Met Arg Ile Tyr Cys Ser
1010 1015 1020
Leu Phe Lys Asn Val Arg Leu Leu Lys Cys Val Ser Asp Ser Trp Leu
1025 1030 1035 1040
Lys Asp Ser Ala Ile Met Val Ala Ser Asp Val Cys Lys Lys Asn Leu
1045 1050 1055
Asp Leu Phe Met Ser His Val Lys Ser Val Thr Lys Ser Ser Ser Trp
1060 1065 1070
Lys Asp Val Asn Ser Val Gln Phe Ser Ile Leu Asn Asn Pro Val Asp
1075 1080 1085
Thr Glu Phe Ile Asn Lys Phe Leu Glu Phe Ser Asn Arg Val Tyr Glu
1090 1095 1100
Ala Leu Tyr Tyr Val His Ser Leu Leu Tyr Ser Ser Met Thr Ser Asp
1105 1110 1115 1120
Ser Lys Ser Ile Glu Asn Lys His Gln Arg Arg Leu Val Lys Leu Leu
1125 1130 1135
Leu Gly Gly Gly Gly Ser Val Pro Thr Ile Val Met Val Asp Ala Tyr
1140 1145 1150
Lys Arg Tyr Lys
1155
<210> 13
<211> 3133
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 13
tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat gcagctcccg gagacggtca 60
cagcttgtct gtaagcggat gccgggagca gacaagcccg tcagggcgcg tcagcgggtg 120
ttggcgggtg tcggggctgg cttaactatg cggcatcaga gcagattgta ctgagagtgc 180
accatatgcg gtgtgaaata ccgcacagat gcgtaaggag aaaataccgc atcaggcgcc 240
attcgccatt caggctgcgc aactgttggg aagggcgatc ggtgcgggcc tcttcgctat 300
tacgccagct ggcgaaaggg ggatgtgctg caaggcgatt aagttgggta acgccagggt 360
tttcccagtc acgacgttgt aaaacgacgg ccagtgaatt ctaatacgac tcactatagg 420
acctcgagtt tttattttta attttctttc aaatacttcc atcccgccgc caccatgggt 480
accccgctgc agtaacagac atgataagat acattgatga gtttggacaa accacaacta 540
gaatgcagtg aaaaaaatgc tttatttgtg aaatttgtga tgctattgct ttatttgtaa 600
ccattataag ctgcaataaa caagttaaca acaacaattg cattcatttt atgtttcagg 660
ttcaggggga ggtgtgggag gttttttaaa gcaagtaaaa cctctacaaa tgtggtagga 720
tcctctagag tcgacctaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 780
aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 840
aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaagag acgaagcttg 900
gcgtaatcat ggtcatagct gtttcctgtg tgaaattgtt atccgctcac aattccacac 960
aacatacgag ccggaagcat aaagtgtaaa gcctggggtg cctaatgagt gagctaactc 1020
acattaattg cgttgcgctc actgcccgct ttccagtcgg gaaacctgtc gtgccagctg 1080
cattaatgaa tcggccaacg cgcggggaga ggcggtttgc gtattgggcg ctcttccgct 1140
tcctcgctca ctgactcgct gcgctcggtc gttcggctgc ggcgagcggt atcagctcac 1200
tcaaaggcgg taatacggtt atccacagaa tcaggggata acgcaggaaa gaacatgtga 1260
gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc gtttttccat 1320
aggctccgcc cccctgacga gcatcacaaa aatcgacgct caagtcagag gtggcgaaac 1380
ccgacaggac tataaagata ccaggcgttt ccccctggaa gctccctcgt gcgctctcct 1440
gttccgaccc tgccgcttac cggatacctg tccgcctttc tcccttcggg aagcgtggcg 1500
ctttctcata gctcacgctg taggtatctc agttcggtgt aggtcgttcg ctccaagctg 1560
ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg ccttatccgg taactatcgt 1620
cttgagtcca acccggtaag acacgactta tcgccactgg cagcagccac tggtaacagg 1680
attagcagag cgaggtatgt aggcggtgct acagagttct tgaagtggtg gcctaactac 1740
ggctacacta gaagaacagt atttggtatc tgcgctctgc tgaagccagt taccttcgga 1800
aaaagagttg gtagctcttg atccggcaaa caaaccaccg ctggtagcgg tggttttttt 1860
gtttgcaagc agcagattac gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt 1920
tctacggggt ctgacgctca gtggaacgaa aactcacgtt aagggatttt ggtcatgaga 1980
ttatcaaaaa ggatcttcac ctagatcctt ttaaattaaa aatgaagttt taaatcaatc 2040
taaagtatat atgagtaaac ttggtctgac agttaccaat gcttaatcag tgaggcacct 2100
atctcagcga tctgtctatt tcgttcatcc atagttgcct gactccccgt cgtgtagata 2160
actacgatac gggagggctt accatctggc cccagtgctg caatgatacc gcgagaccca 2220
cgctcaccgg ctccagattt atcagcaata aaccagccag ccggaagggc cgagcgcaga 2280
agtggtcctg caactttatc cgcctccatc cagtctatta attgttgccg ggaagctaga 2340
gtaagtagtt cgccagttaa tagtttgcgc aacgttgttg ccattgctac aggcatcgtg 2400
gtgtcacgct cgtcgtttgg tatggcttca ttcagctccg gttcccaacg atcaaggcga 2460
gttacatgat cccccatgtt gtgcaaaaaa gcggttagct ccttcggtcc tccgatcgtt 2520
gtcagaagta agttggccgc agtgttatca ctcatggtta tggcagcact gcataattct 2580
cttactgtca tgccatccgt aagatgcttt tctgtgactg gtgagtactc aaccaagtca 2640
ttctgagaat agtgtatgcg gcgaccgagt tgctcttgcc cggcgtcaat acgggataat 2700
accgcgccac atagcagaac tttaaaagtg ctcatcattg gaaaacgttc ttcggggcga 2760
aaactctcaa ggatcttacc gctgttgaga tccagttcga tgtaacccac tcgtgcaccc 2820
aactgatctt cagcatcttt tactttcacc agcgtttctg ggtgagcaaa aacaggaagg 2880
caaaatgccg caaaaaaggg aataagggcg acacggaaat gttgaatact catactcttc 2940
ctttttcaat attattgaag catttatcag ggttattgtc tcatgagcgg atacatattt 3000
gaatgtattt agaaaaataa acaaataggg gttccgcgca catttccccg aaaagtgcca 3060
cctgacgtct aagaaaccat tattatcatg acattaacct ataaaaatag gcgtatcacg 3120
aggccctttc gtc 3133
<210> 14
<211> 720
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 14
atggtgagca agggcgagga gctgttcacc ggggtggtgc ccatcctggt cgagctggac 60
ggcgacgtaa acggccacaa gttcagcgtg tccggcgagg gcgagggcga tgccacctac 120
ggcaagctga ccctgaagtt catctgcacc accggcaagc tgcccgtgcc ctggcccacc 180
ctcgtgacca ccctgaccta cggcgtgcag tgcttcagcc gctaccccga ccacatgaag 240
cagcacgact tcttcaagtc cgccatgccc gaaggctacg tccaggagcg caccatcttc 300
ttcaaggacg acggcaacta caagacccgc gccgaggtga agttcgaggg cgacaccctg 360
gtgaaccgca tcgagctgaa gggcatcgac ttcaaggagg acggcaacat cctggggcac 420
aagctggagt acaactacaa cagccacaac gtctatatca tggccgacaa gcagaagaac 480
ggcatcaagg tgaacttcaa gatccgccac aacatcgagg acggcagcgt gcagctcgcc 540
gaccactacc agcagaacac ccccatcggc gacggccccg tgctgctgcc cgacaaccac 600
tacctgagca cccagtccgc cctgagcaaa gaccccaacg agaagcgcga tcacatggtc 660
ctgctggagt tcgtgaccgc cgccgggatc actctcggca tggacgagct gtacaagtaa 720

Claims (17)

1. A chimeric enzyme for one-step synthesis of mRNA by an in vitro enzymatic method, wherein the chimeric enzyme is a transcription capped chimeric enzyme, and the chimeric enzyme comprises a capping enzyme domain and an RNA polymerase domain, wherein the RNA polymerase domain comprises RNA polymerase and Spy Catcher, and the capping enzyme catalytic domain comprises viral capping enzyme and Spy Tag; or, the RNA polymerase domain comprises RNA polymerase and Spy Tag, and the capping enzyme catalytic domain comprises viral capping enzyme and Spy Catcher; in the chimeric enzyme, Spy Tag and Spy Catcher are covalently bound; both the RNA polymerase domain and the capping enzyme catalytic domain further comprise a protein purification tag.
2. Use of the chimeric enzyme of claim 1 for the one-step synthesis of mRNA in vitro transcription.
3. A method for synthesizing mRNA in one step through in vitro transcription is characterized by comprising the following steps: firstly, obtaining a template DNA transcribed by mRNA; ② adding the chimeric enzyme of claim 1 to carry out in vitro transcription to obtain mRNA with Cap0 structure in one step.
4. The method of claim 3, further comprising any one or more of the following features:
1) the template DNA is selected from plasmid DNA or an isolated DNA fragment of interest;
2) the method also comprises the steps of adding the buffer solution into a coreaction system of the chimeric enzyme and the template DNA;
3) the method further comprises adding dNTP into a coreaction system of the chimeric enzyme and the template DNA;
4) the method further comprises adding a methylated donor reagent to the co-reaction system of the chimeric enzyme and the template DNA;
5) the co-reaction condition is that the reaction is carried out for 2 to 3 hours at the temperature of between 35 and 38 ℃;
6) the method also comprises purifying mRNA with Cap structure of Cap0 obtained from the co-reaction.
5. The method according to claim 4, wherein the plasmid DNA in step 1) comprises a DNA of interest and a vector comprising the T7 promoter, 5 'and 3' UTR and PolyA tail elements.
6. The method according to claim 4, wherein the plasmid DNA is a linearized plasmid DNA, and the vector of the linearized plasmid DNA has a sequence shown in SEQ ID NO. 13.
7. The method of claim 3, wherein the chimeric enzyme RNA polymerase is phage-derived RNA polymerase, prokaryotic RNA polymerase, eukaryotic RNA polymerase, or mitochondrial and chloroplast RNA polymerase;
and/or the presence of a gas in the gas,
the virus capping enzyme in the chimeric enzyme is selected from one or more of the following: vaccinia virus capping enzyme, bluetongue virus capping enzyme, bamboo mosaic virus capping enzyme, African swine fever virus capping enzyme, organic lake algae type 1 DNA virus capping enzyme, organic lake algae type 2 DNA virus capping enzyme, spherical synechocystis virus capping enzyme or golden algae virus capping enzyme.
8. The method of claim 7, wherein the bacteriophage-derived RNA polymerase is selected from the group consisting of T7RNA polymerase, SP6 RNA polymerase, and T3 RNA polymerase.
9. The method of claim 3, wherein the chimeric enzyme comprises an RNA polymerase linked to a Spy Catcher via a first linker peptide and/or the viral capping enzyme linked to a Spy Tag via a second linker peptide.
10. The method of claim 3, wherein the chimeric enzyme is T7RNA polymerase-vaccinia virus capping enzyme chimeric enzyme.
11. The method of claim 3, wherein the RNA polymerase domain and the capping enzyme catalytic domain of the chimeric enzyme each further comprise a protein purification tag; preferably, the protein purification Tag is selected from HIS-Tag, GST-Tag, MBP-Tag, NusA-Tag or SUMO.
12. The method of claim 3, wherein the amino acid sequence of the chimeric enzyme comprises the amino acid sequences shown in SEQ ID NO. 11 and SEQ ID NO. 12.
13. A polynucleotide encoding the chimeric enzyme of claim 1, or encoding the RNA polymerase domain, or encoding the capping enzyme catalytic domain.
14. A nucleic acid construct comprising the polynucleotide of claim 13.
15. A cell comprising the nucleic acid construct of claim 14 or having an exogenous polynucleotide of claim 13 integrated into its genome.
16. A kit for the in vitro one-step synthesis of mRNA having a 5' end cap structure, comprising the chimeric enzyme of claim 1.
17. The process for producing a chimeric enzyme according to claim 1, which comprises the steps of:
1) co-transfecting a nucleic acid construct comprising the RNA polymerase domain and a nucleic acid construct comprising a capping enzyme catalytic domain into a host cell, and culturing the host cell;
2) extracting the chimeric enzyme from the culture system in the step 1).
CN202111123695.9A 2021-09-24 2021-09-24 Chimeric enzyme and application and method thereof in one-step reaction synthesis of Cap0 mRNA in vitro Active CN113862235B (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN202111123695.9A CN113862235B (en) 2021-09-24 2021-09-24 Chimeric enzyme and application and method thereof in one-step reaction synthesis of Cap0 mRNA in vitro

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN202111123695.9A CN113862235B (en) 2021-09-24 2021-09-24 Chimeric enzyme and application and method thereof in one-step reaction synthesis of Cap0 mRNA in vitro

Publications (2)

Publication Number Publication Date
CN113862235A true CN113862235A (en) 2021-12-31
CN113862235B CN113862235B (en) 2024-07-05

Family

ID=78993972

Family Applications (1)

Application Number Title Priority Date Filing Date
CN202111123695.9A Active CN113862235B (en) 2021-09-24 2021-09-24 Chimeric enzyme and application and method thereof in one-step reaction synthesis of Cap0 mRNA in vitro

Country Status (1)

Country Link
CN (1) CN113862235B (en)

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN114574466A (en) * 2022-05-09 2022-06-03 翌圣生物科技(上海)股份有限公司 Chimeric capping enzyme and preparation method and application thereof
CN116836974A (en) * 2023-09-01 2023-10-03 苏州近岸蛋白质科技股份有限公司 Method for synthesizing capped mRNA in vitro
WO2024199310A1 (en) * 2023-03-31 2024-10-03 苏州近岸蛋白质科技股份有限公司 Method for enzymatic synthesis of capped mrna in one tube

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20130042334A1 (en) * 2010-04-16 2013-02-14 Eukarys Capping-Prone RNA Polymerase Enzymes and Their Applications
US20160024547A1 (en) * 2013-03-15 2016-01-28 Moderna Therapeutics, Inc. Manufacturing methods for production of rna transcripts
CN108026148A (en) * 2015-06-05 2018-05-11 牛津大学创新有限公司 Fusion protein synthetic method and product
CN111164207A (en) * 2017-07-27 2020-05-15 优卡瑞斯 Novel chimeric enzyme and use thereof

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20130042334A1 (en) * 2010-04-16 2013-02-14 Eukarys Capping-Prone RNA Polymerase Enzymes and Their Applications
US20160024547A1 (en) * 2013-03-15 2016-01-28 Moderna Therapeutics, Inc. Manufacturing methods for production of rna transcripts
CN108026148A (en) * 2015-06-05 2018-05-11 牛津大学创新有限公司 Fusion protein synthetic method and product
CN111164207A (en) * 2017-07-27 2020-05-15 优卡瑞斯 Novel chimeric enzyme and use thereof

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
TAN LL等: "Kinetic Controlled Tag-Catcher Interactions for Directed Covalent Protein Assembly", PLOS ONE, vol. 11, no. 10, pages 1 - 15 *
王梦珂等: "通过SpyTag/SpyCatcher介导的自发环化提高酿酒酵母S288C中Ulp1的稳定性", 农业生物技术学报, vol. 28, no. 2, pages 369 - 380 *
谢露露: "利用Tag-Catcher构建自组装双酶复合物高效制备D-氨基酸", 中国优秀硕士学位论文全文数据库基础科学辑, no. 12, pages 006 - 257 *

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN114574466A (en) * 2022-05-09 2022-06-03 翌圣生物科技(上海)股份有限公司 Chimeric capping enzyme and preparation method and application thereof
WO2024199310A1 (en) * 2023-03-31 2024-10-03 苏州近岸蛋白质科技股份有限公司 Method for enzymatic synthesis of capped mrna in one tube
CN116836974A (en) * 2023-09-01 2023-10-03 苏州近岸蛋白质科技股份有限公司 Method for synthesizing capped mRNA in vitro
CN116836974B (en) * 2023-09-01 2024-02-20 苏州近岸蛋白质科技股份有限公司 Method for synthesizing capped mRNA in vitro

Also Published As

Publication number Publication date
CN113862235B (en) 2024-07-05

Similar Documents

Publication Publication Date Title
CN109777761B (en) Construction and application of engineering bacteria for secretory expression of chitobiose deacetylase
CN113862235B (en) Chimeric enzyme and application and method thereof in one-step reaction synthesis of Cap0 mRNA in vitro
Alting-Mees et al. [42] pBluescriptII: Multifunctional cloning and mapping vectors
CA2520811C (en) Method of cdna synthesis
CN111088405A (en) Primer probe composition, kit and method for detecting coronavirus 2019-nCoV
CN103215372B (en) Primer sequence for detecting Brucella based on dual priming oligonucleotide (DPO) primer, and detection kit thereof
CN108220176B (en) Method for improving fermentation production of glucaric acid by saccharomyces cerevisiae engineering strain
CN107604004A (en) Tracer target practice plasmid for vaccinia virus Tiantan strain TK genes and preparation method thereof
CN110944656B (en) Novel polynucleotides encoding human FKRP proteins
CN108718529B (en) Mutant microorganism for producing L-cysteine and method for producing L-cysteine using the same
WO1992017581A1 (en) Mammalian expression vector
CN114032217A (en) Novel coronavirus compound vaccine based on DNA vector and replicative vaccinia virus vector
KR20200086902A (en) A vector for monitoring differentiation of neural cells and method for monitoring the differentiation into neural cells using the same
CN111718953B (en) CRISPR/Cas9 vector for sugarcane and construction method and application thereof
CN101538611B (en) Real-time quantitative fluorescence PCR test method based on double external references of RNA and DNA and application thereof
CN107034233B (en) Method for driving exogenous gene expression by endogenous promoter
CN103952483A (en) DPO (Dual Priming Oligonucleotide) primer sequences and detection kit for detecting Vibrio alginolyticus by DPO-PCR (Polymerase Chain Reaction) method
CN109022469B (en) Construction method of yeast expression vector, method for preparing xylanase by using yeast expression vector and application of xylanase
CN111239385B (en) Screening method and application of small molecule compound for targeted inhibition of vitamin K epoxide reductase
CN114277047B (en) Application of high-throughput screening tool for obtaining effective NHEJ system from escherichia coli in escherichia coli gene editing
CN108385170B (en) Regulatory sequence library of Bacillus subtilis F4 promoter
CN111560392B (en) MiRNA expression vector and application thereof
CN114164225B (en) High-throughput screening tool for enabling escherichia coli to obtain effective NHEJ system and application of high-throughput screening tool
CN114907997B (en) Construction and application of diosgenin synthetic strain
CN114457113B (en) Method for inhibiting haploid embryonic stem cell doubling

Legal Events

Date Code Title Description
PB01 Publication
PB01 Publication
SE01 Entry into force of request for substantive examination
SE01 Entry into force of request for substantive examination
GR01 Patent grant
GR01 Patent grant