WO2024092768A1 - Transcriptase inverse thermostable et son utilisation - Google Patents

Transcriptase inverse thermostable et son utilisation Download PDF

Info

Publication number
WO2024092768A1
WO2024092768A1 PCT/CN2022/130031 CN2022130031W WO2024092768A1 WO 2024092768 A1 WO2024092768 A1 WO 2024092768A1 CN 2022130031 W CN2022130031 W CN 2022130031W WO 2024092768 A1 WO2024092768 A1 WO 2024092768A1
Authority
WO
WIPO (PCT)
Prior art keywords
reverse transcriptase
thermostable
present
thermostable reverse
reaction
Prior art date
Application number
PCT/CN2022/130031
Other languages
English (en)
Chinese (zh)
Inventor
张晓红
刘晓晨
陈雪梅
谢庆庆
郑越
董宇亮
章文蔚
曹如茵
Original Assignee
深圳华大生命科学研究院
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 深圳华大生命科学研究院 filed Critical 深圳华大生命科学研究院
Priority to PCT/CN2022/130031 priority Critical patent/WO2024092768A1/fr
Publication of WO2024092768A1 publication Critical patent/WO2024092768A1/fr

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
    • 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
    • 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)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6844—Nucleic acid amplification reactions
    • C12Q1/686—Polymerase chain reaction [PCR]

Definitions

  • the invention relates to the field of enzyme engineering, and in particular to a thermostable reverse transcriptase and an application thereof.
  • Reverse transcriptase is a special DNA polymerase, also known as RNA-dependent DNA polymerase, which can synthesize DNA using RNA as a template. Reverse transcriptase was first discovered in RNA viruses. Reverse transcriptases derived from RNA viruses are also the most intensively studied and widely used reverse transcriptases, mainly including Moloney murine leukemia virus reverse transcriptase (M-MLV RT), human immunodeficiency virus reverse transcriptase (HIV RT) and avian myeloblastosis virus reverse transcriptase (AMV RT).
  • M-MLV RT Moloney murine leukemia virus reverse transcriptase
  • HAV RT human immunodeficiency virus reverse transcriptase
  • AMV RT avian myeloblastosis virus reverse transcriptase
  • Reverse transcription is a key step in many RNA reaction workflows.
  • reverse transcription is usually used to convert RNA into a more stable complementary DNA (cDNA), and then cloning, PCR, gene expression chips, sequencing and other technical research and analysis are carried out.
  • cDNA complementary DNA
  • RT-PCR technology has been widely used in technologies such as detecting gene expression levels in cells and detecting the content of RNA viruses in cells.
  • One-step Reverse Transcriptase-Quantitative Real-time Polymerase Chain Reaction is a time-saving and labor-saving nucleic acid detection technology with broad application prospects in the fields of rapid food detection, pathogen detection, and infectious disease prevention and control.
  • the One-step RT-qPCR system has problems such as insufficient detection sensitivity and low specificity.
  • reverse transcriptase plays an important role in controlling detection sensitivity and specificity. Improving reverse transcriptase is conducive to improving the detection sensitivity and specificity of One-step RT-qPCR.
  • the present invention aims to solve one of the technical problems in the related art at least to a certain extent.
  • one object of the present invention is to provide a reverse transcriptase that can improve the sensitivity of RT-qPCR detection, and the reverse transcriptase provided has high thermal stability.
  • reverse transcriptase is a room temperature enzyme and is easily denatured and inactivated at high temperatures. Therefore, improving the heat resistance of reverse transcriptase can not only effectively synthesize cDNA, but also facilitate the storage, packaging and transportation of the enzyme.
  • the inventors screened and obtained a reverse transcriptase with high thermal stability by mutating the reverse transcriptase derived from the wild-type M-MLV, which is of great significance to related industries and research that require the application of reverse transcription reactions.
  • the present invention provides a thermostable reverse transcriptase.
  • the thermostable reverse transcriptase has at least one of the mutation sites of L333, P65, Q68, P175, E176, F334, G178, G331, T332, P127 and G105, wherein the amino acid site positioning of the thermostable reverse transcriptase is based on the amino acid sequence shown in SEQ ID NO: 2.
  • the inventors studied multiple sites of the wild-type M-MLV reverse transcriptase, and found through experimental screening that the above-mentioned sites have a great influence on improving the thermostability of the reverse transcriptase. Therefore, the reverse transcriptase obtained after mutating the above-mentioned sites has high thermostability, and can effectively improve the yield or reaction efficiency when applied to reverse transcription and related technologies.
  • thermostable reverse transcriptase may further include at least one of the following additional technical features:
  • thermostable reverse transcriptase further has at least one of the mutation sites Q63, Y64, M66, D200, E562 and D583.
  • thermostable reverse transcriptase has the following mutation sites or site combinations: 1) Q63; 2) Y64; 3) P65; 4) M665) Q68; 6) P127; 7) P175; 8) E176; 9) G178; 10) G331; 11) T332; 12) L333; 13) F334; 14) E176, E562 and D583; 15) L333, E562 and D583; 16) M66, Q68, G105, E562 and D583; 17) M66, Q68, E176, D200, E562 and D583; or 18) P65, M66, Q68, E69, E176, D200, E562 and D583.
  • the reverse transcriptase having the mutation site or site combination has higher thermal stability than the wild-type reverse transcriptase, and can be used in reverse transcription reaction-related technologies, such as reverse transcription reaction, PCR reaction and cDNA library construction.
  • the thermostable reverse transcriptase has at least one of the following mutations: i) Q63A, Q63P or Q63G; ii) P65N, P65G, P65C, P65E, P65T, P65K, P65L, P65I or P65S; iii) M66Y or M66V; iv) Q68A, Q68E, Q68K or Q68N; v) E69K; vi) P127D, P127R or P127T; vii) P175L, P175W or P175F; viii) E176H, E176A, E176R or E176C; ix) G178N or G178T; x) G331F, G331P, G331E or G331M; xi) T332D, T332W, T332P or T332K; xii) L333A, L333K or L333N; xiii
  • thermostable reverse transcriptase further has the following mutations: Y64S, Y64V or Y64F.
  • the reverse transcriptase has the following mutations: I) Q63A, Q63P or Q63G; II) Y64S, Y64V or Y64F; III) P65N, P65G, P65C, P65E, P65T, P65K, P65L, P65I or P65S; IV) M66Y; V) Q68A, Q68E, Q68K or Q68N; VI) P127D, P127R or P127T; VII) P175L, P175W or P175F; VIII) E176H, E176A or E176C; IX) G178N or G178T; X) G331F, or XVIII) P65K, M66V, Q68K, E69K, E176R, D200N, E562Q and D583N.
  • the inventors screened multiple mutation sites and found that the thermal stability of the reverse transcriptases with the above mutations was better than that of the wild-type M-ML
  • the amino acid site positioning is based on the amino acid sequence shown in SEQ ID NO:2.
  • the above-mentioned fixed-site sequence (reference sequence) SEQ ID NO:2 of the M-MLV reverse transcriptase does not constitute a limitation on the reverse transcriptase produced by the present invention.
  • the reverse transcriptase can also be used for the improvement of other M-MLV reverse transcriptases that have already undergone mutations, as long as the mutation sites and/or site combinations described above are mutated at the corresponding positions relative to the M-MLV reverse transcriptase shown in SEQ ID NO:2, for example: at least one of the Q63, Y64, P65, M66, Q68, P127, P175, E176, G178, G331, T332, L333, F334, G105, D200, E562 and D583 sites is mutated; or the specific mutations described above are mutated at the corresponding positions relative to the M-MLV reverse transcriptase shown in SEQ ID NO:2, for example: i) Q63A, Q63P
  • the reverse transcriptase has an amino acid sequence shown in any one of SEQ ID NO: 3 to 53; or a polypeptide having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity with SEQ ID NO: 3 to 53; or a polypeptide having one or more amino acid substitutions, deletions, and/or additions compared with SEQ ID NO: 3 to 53.
  • the reverse transcriptase has an amino acid sequence shown in any one of SEQ ID NOs: 3 to 5, 9 to 17, 19 to 22, 26 to 28, 42 to 48 and 49 to 53.
  • the reverse transcriptase according to an embodiment of the present invention has a significantly better amplification efficiency in RT-qPCR reaction than wild-type and commercial M-MLV reverse transcriptase.
  • the reverse transcriptase is a mutant of M-MLV reverse transcriptase.
  • the Tm value of the reverse transcriptase is 50°C-60°C.
  • the present invention provides an isolated nucleic acid molecule.
  • the nucleic acid molecule encodes the thermostable reverse transcriptase described in the first aspect of the present invention.
  • the nucleic acid molecule can effectively obtain the reverse transcriptase, which has high thermostability and can effectively improve the reaction efficiency when applied to reverse transcription and related technologies.
  • the present application provides an expression vector carrying the isolated nucleic acid molecule described in the second aspect of the present invention.
  • the expression vector may include an optional control sequence, which is operably connected to the isolated nucleic acid molecule.
  • the control sequence is one or more control sequences that can guide the expression of the nucleic acid molecule in a host.
  • the expression vector proposed in the embodiment of the present invention can efficiently express the thermostable reverse transcriptase in a suitable host cell.
  • the nucleic acid molecule can be directly or indirectly connected to the control elements on the vector, as long as these control elements can control the translation and expression of the nucleic acid molecule.
  • these control elements can come directly from the vector itself, or they can be exogenous, that is, they are not from the vector itself.
  • "operably connected” refers to connecting the foreign gene to the vector so that the control elements in the vector, such as transcription control sequences and translation control sequences, etc., can play their expected functions of regulating the transcription and translation of the foreign gene.
  • nucleic acid molecules used to encode the thermostable reverse transcriptase can be inserted into different vectors separately and independently, and it is common to insert them into the same vector.
  • Commonly used vectors can be, for example, plasmids, bacteriophages, etc., such as Plasmid-X plasmids.
  • the isolated nucleic acid molecule is operably linked to a promoter.
  • the promoter includes one selected from the group consisting of: ⁇ -PL promoter, tac promoter, trp promoter, araBAD promoter, T7 promoter and trc promoter.
  • the present invention provides a recombinant cell, the recombinant cell comprising the nucleic acid molecule described in the second aspect of the present invention, the expression vector described in the third aspect, or expressing the thermostable reverse transcriptase described in the first aspect.
  • the host cell used to express the target gene or nucleic acid molecule can be a prokaryotic cell.
  • the thermostable reverse transcriptase of the present invention is expressed by a prokaryotic cell, such as Escherichia coli.
  • the recombinant cell is obtained by transfecting or transforming the expression vector.
  • the recombinant cell can efficiently and massively express the above-mentioned thermostable reverse transcriptase under appropriate conditions, the thermostable reverse transcriptase has high thermal stability, and the reverse transcription reaction, PCR reaction and construction of sequencing library using the reverse transcriptase all have high reaction efficiency.
  • the recombinant cells of the present invention are not particularly limited and can be prokaryotic cells, eukaryotic cells or bacteriophages.
  • the prokaryotic cells can be Escherichia coli, Bacillus subtilis, Streptomyces or Proteus mirabilis, etc.
  • the eukaryotic cells include fungi such as Pichia pastoris, Saccharomyces cerevisiae, Schizosaccharomyces or Trichoderma, insect cells such as armyworms, plant cells such as tobacco, or mammalian cells such as BHK cells, CHO cells, COS cells or myeloma cells.
  • the recombinant cells of the present invention are preferably Escherichia coli.
  • thermostable conditions refer to conditions suitable for the expression of the thermostable reverse transcriptase described in this application. It is easy for those skilled in the art to understand that the conditions suitable for the expression of the thermostable reverse transcriptase include, but are not limited to, suitable transformation or transfection methods, suitable transformation or transfection conditions, healthy host cell states, suitable host cell density, suitable cell culture environment, and suitable cell culture time. "Suitable conditions” are not particularly limited, and those skilled in the art can optimize the most suitable conditions for the expression of the reverse transcriptase according to the specific environment of the laboratory.
  • the present invention provides a method for preparing the thermostable reverse transcriptase described in the first aspect.
  • the method comprises: introducing the expression vector described in the third aspect into a host cell; culturing the host cell introduced with the expression vector under conditions suitable for protein expression to obtain the thermostable reverse transcriptase.
  • the method according to an embodiment of the present invention can effectively express and obtain the thermostable reverse transcriptase in large quantities.
  • thermostable reverse transcriptase described in the first aspect may further include at least one of the following additional technical features:
  • the host cell is Escherichia coli.
  • the expression efficiency of the thermostable reverse transcriptase is higher.
  • the host cell does not include animal germ cells, fertilized eggs or embryonic stem cells.
  • the present invention provides a kit.
  • the kit comprises the thermostable reverse transcriptase described in the first aspect of the present invention.
  • the use of a kit containing the thermostable reverse transcriptase can effectively improve the efficiency of reverse transcription and related reactions.
  • the above-mentioned kit may further include at least one of the following additional technical features:
  • the kit further comprises at least one of the following: an RNA extraction reagent, one or more DNA polymerases, one or more buffers, one or more primers, and one or more terminators.
  • the terminator comprises dideoxynucleotide.
  • the present invention provides a reverse transcription method.
  • the method comprises the following steps: reverse transcribing at least one RNA template in the presence of at least one reverse transcriptase to obtain cDNA, wherein the reverse transcriptase comprises the aforementioned thermostable reverse transcriptase.
  • the thermostable reverse transcriptase has a high thermal stability, and therefore, the efficiency of the reverse transcription method according to an embodiment of the present invention is higher than the reverse transcription reaction using a wild-type or commercial reverse transcriptase.
  • the reverse transcription method may further include at least one of the following additional technical features:
  • At least one reverse transcription primer is also present. It will be appreciated by those skilled in the art that when performing the reverse transcription reaction, it is also necessary to use a reverse transcription primer for a specific region of the nucleic acid template, and those skilled in the art can design primers according to the target region on the nucleic acid template.
  • the reverse transcription is performed at a temperature of 35°C to 65°C, preferably 37°C to 55°C, more preferably 42°C to 55°C, and even more preferably 50°C to 55°C.
  • the thermostable reverse transcriptase has a high thermal stability and can be used at a temperature of 35°C to 65°C, has a high reverse transcription reaction efficiency at a temperature of 42°C to 55°C, and when the reverse transcription temperature is 50°C to 55°C, the reverse transcription reaction efficiency is still very high.
  • the present invention provides a method for RT-PCR.
  • the method comprises: performing a reverse transcription reaction on at least one RNA template in the presence of at least one reverse transcriptase and at least one DNA polymerase to obtain cDNA, wherein the reverse transcriptase comprises the thermostable reverse transcriptase described in the first aspect.
  • the thermostable reverse transcriptase described in the first aspect has high thermal stability, and the thermostable reverse transcriptase can effectively improve the reaction efficiency of RT-PCR. Therefore, the efficiency of the RT-PCR method described in the present application is higher than that of a PCR reaction using a wild-type reverse transcriptase or a commercial reverse transcriptase.
  • the above RT-PCR method may further include at least one of the following additional technical features:
  • At least one reverse transcription primer is also present.
  • the reverse transcription reaction is carried out at a temperature of 35°C to 65°C.
  • the reverse transcription reaction is carried out at a temperature of 42°C to 55°C.
  • the reverse transcription reaction is carried out at a temperature of 50°C to 55°C.
  • the RT-PCR method further comprises: subjecting the cDNA to a first polymerase chain reaction in the presence of at least one pair of primers and at least one DNA polymerase.
  • the present invention provides a RT-PCR method.
  • the method comprises the following steps: performing a reverse transcription reaction and a second polymerase chain reaction on at least one RNA template in the presence of at least one reverse transcriptase and at least one DNA polymerase, wherein the reverse transcriptase comprises the thermostable reverse transcriptase described in the first aspect.
  • the thermostable reverse transcriptase described in the first aspect has high thermal stability, and the thermostable reverse transcriptase can effectively improve the reaction efficiency of RT-PCR. Therefore, the efficiency of the RT-PCR method described in the present application is higher than that of a PCR reaction using a wild-type reverse transcriptase or a commercial reverse transcriptase.
  • the above RT-PCR method further includes at least one of the following additional technical features:
  • At least one pair of primers is further present in the reverse transcription reaction and the second polymerase chain reaction.
  • the reverse transcription reaction includes a stage performed at a temperature of 35°C to 65°C.
  • the reverse transcription reaction includes a stage performed at a temperature of 42°C to 55°C.
  • the reverse transcription reaction is performed at a temperature of 50°C to 55°C.
  • the present invention provides a method for constructing a sequencing library.
  • the method comprises enriching nucleic acid molecules connected with a connector, wherein the nucleic acid molecules are obtained by reverse transcription reaction of the RNA sample to be tested using the reverse transcription method described above.
  • the thermostable reverse transcriptase has high thermal stability and can effectively improve the reaction efficiency of the reverse transcription reaction related technology. Therefore, the method according to the embodiment of the present invention can construct a high-quality sequencing library.
  • the method for constructing a sequencing library may further include at least one of the following additional technical features:
  • the enrichment treatment comprises subjecting the nucleic acid molecules connected with adapters to a third polymerase chain reaction in the presence of at least one pair of PCR primers and at least one DNA polymerase.
  • the enrichment process further includes a purification process.
  • the purification process is performed by magnetic bead purification.
  • the source of the RNA to be tested includes but is not limited to animals (such as rats, mice, cats, dogs, horses, cattle, pigs, chickens, ducks, geese, quails, pigeons, nematodes, zebrafish, etc.), plants (such as rice, Arabidopsis, wheat, corn, sweet potatoes, peanuts), protists, eukaryotic algae, bacteria, archaea, fungi, viroids or viruses. For example, multicellular or single cells in these biological samples can be processed to obtain RNA.
  • animals such as rats, mice, cats, dogs, horses, cattle, pigs, chickens, ducks, geese, quails, pigeons, nematodes, zebrafish, etc.
  • plants such as rice, Arabidopsis, wheat, corn, sweet potatoes, peanuts
  • protists eukaryotic algae, bacteria, archaea, fungi, viroids or viruses.
  • exemplary bacteria include but are not limited to: one or more of Staphylococcus, Streptococcus, Listeria, Erysipelothrix, Nephrobacterium, Bacillus, Clostridium, Mycobacterium, Actinomycetes, Nocardia, Corynebacterium, Coccus, Bacillus anthracis, Bacillus erysipelothrix, Bacillus tetani, Listeria, Bacillus anthracis, Mycobacterium tuberculosis, Escherichia coli, Proteus, Shigella dysenteriae, Klebsiella pneumoniae, Brucella, Clostridium perfringens, Haemophilus influenzae, Haemophilus parainfluenzae, Moraxella catarrhalis, and Acinetobacter.
  • the exemplary viruses include but are not limited to: one or more of: adenoviridae (aden), arenaviridae, astroviridae, bunyaviridae, caliciviridae, flaviviridae, hepadnaviridae, single-molecule negative-strand RNA virus order, nidovirales, picornaviridae, orthomyxoviridae, papillomaviridae, parvoviridae, polyomaviridae, poxviridae, reoviridae, and retroviridae.
  • adenoviridae aden
  • arenaviridae astroviridae
  • bunyaviridae caliciviridae
  • flaviviridae flaviviridae
  • hepadnaviridae single-molecule negative-strand RNA virus order
  • nidovirales picornaviridae
  • orthomyxoviridae papillom
  • the RNA sample to be tested includes but is not limited to samples from cells, tissues, soil, saliva, sweat, feces, urine, blood, serum or plasma.
  • the present invention provides a sequencing library.
  • the sequencing library is obtained by the method described in the tenth aspect.
  • the reverse transcriptase has high thermal stability and can effectively improve the reaction efficiency of reverse transcription reaction-related technologies. Therefore, the method according to the embodiment of the present invention can construct a high-quality sequencing library.
  • the present invention provides a sequencing method.
  • the method comprises sequencing the sequencing library described in the eleventh aspect and performing data analysis. It can be understood by those skilled in the art that sequencing the sequencing library and performing data analysis can detect and verify the sequencing library to determine whether the sequencing library is a target library.
  • the present invention proposes the use of the thermostable reverse transcriptase described in the first aspect in constructing a sequencing library.
  • the thermostable reverse transcriptase has high thermal stability and can effectively improve the reaction efficiency of technologies related to reverse transcription reactions. Therefore, all technologies applied to reverse transcription can use the thermostable reverse transcriptase.
  • the sequencing library is not particularly limited. For example, RNA-seq, exon sequencing, small RNA-seq, single-cell DNA sequencing, single-cell mRNA sequencing, methylation sequencing, Moleculo long sequencing, Ribozero and directional RNA sequencing libraries can all be constructed using the above-mentioned thermostable reverse transcriptase.
  • the present invention proposes the use of the thermostable reverse transcriptase described in the first aspect in sequencing.
  • the thermostable reverse transcriptase has high thermal stability and can effectively improve the reaction efficiency of technologies related to reverse transcription reactions. Therefore, any technology applied to reverse transcription can use the thermostable reverse transcriptase.
  • the sequencing method and sequencing platform are not particularly limited, and any sequencing that can use the thermostable reverse transcriptase is within the protection scope of this application.
  • the present invention proposes a method for detecting the gene content in a sample to be tested.
  • it includes: using the reverse transcriptase described in the first aspect to perform a reverse transcription reaction on the RNA sample to be tested to obtain cDNA; using the cDNA as a template, performing a fourth polymerase chain reaction on the cDNA under the condition of the presence of a predetermined primer, and the predetermined primer is designed with the gene fragment to be tested as the target amplification fragment; and determining the expression amount of the gene to be tested in the sample to be tested according to the content of the fourth polymerase chain reaction product.
  • the method according to the embodiment of the present invention can effectively detect the gene content in the sample to be tested, and the method can be used for diagnostic purposes and/or non-diagnostic purposes, such as scientific research, specifically, for example, for screening biological samples with specific genes or genes with specific contents for subsequent research. It can also be used to detect the gene content of the sample to be tested from an individual source, and compare the obtained gene content with the standard level of the gene content of the related disease to determine whether the individual suffers from the gene-related disease. The value of the standard level can be determined by comparing and analyzing the differences in the content of the gene in the samples to be tested of a large number of individuals with the gene-related disease and a large number of healthy individuals, and verifying it.
  • the above method for detecting the gene content in the sample to be tested may further include at least one of the following additional technical features:
  • the RNA sample to be tested includes a sample from an animal, a plant, a protist, a eukaryotic algae, a bacterium, an archaea, a fungus, a viroid or a virus.
  • the RNA sample includes a sample from cells, tissues, soil, saliva, sweat, feces, urine, blood, serum or plasma.
  • the reverse transcriptase provided by the present application has high thermal stability and can be applied to related technologies involving reverse transcription reactions to achieve amplification of complex templates and amplification of full-length cDNA, and the reaction tolerance temperature is increased, thereby improving the amplification efficiency.
  • FIG1 is a graph showing the detection results of one-step RT-qPCR of wild-type M-MLV reverse transcriptase and its mutants and commercial M-MLV reverse transcriptase according to an embodiment of the present invention.
  • first and second are used for descriptive purposes only and should not be understood as indicating or implying relative importance or implicitly indicating the number of the indicated technical features. Therefore, the features defined as “first” and “second” may explicitly or implicitly include at least one of the features. In the description of the present invention, the meaning of “plurality” is at least two, such as two, three, etc., unless otherwise clearly and specifically defined.
  • any values of the ranges disclosed in this article are not limited to the precise ranges or values, and these ranges or values should be understood to include values close to these ranges or values.
  • the endpoint values of each range, the endpoint values of each range and the individual point values, and the individual point values can be combined with each other to obtain one or more new numerical ranges, which should be regarded as specifically disclosed in this article.
  • reverse transcriptase refers to a protein, polypeptide or polypeptide fragment that exhibits reverse transcriptase activity.
  • reverse transcriptase activity or reverse transcription activity refers to the ability to synthesize a DNA chain using RNA as a template.
  • mutation refers to a protein that has one or more mutations compared to a reference sequence (such as the wild-type DNA sequence shown in SEQ ID NO: 1 or the wild-type amino acid sequence shown in SEQ ID NO: 2). Of course, this mutation can occur at the nucleic acid level or at the amino acid level.
  • identity is used to describe an amino acid sequence or nucleic acid sequence relative to a reference sequence, and the percentage of identical amino acids or nucleotides between two amino acid sequences or nucleic acid sequences is determined by conventional methods, for example, see Ausubel et al., eds. (1995), Current Protocols in Molecular Biology, Chapter 19 (Greene Publishing and Wiley-Interscience, New York); and the ALIGN program (Dayhoff (1978), Atlas of Protein Sequence and Structure 5: Suppl. 3 (National Biomedical Research Foundation, Washington, D.C.). There are many algorithms for aligning sequences and determining sequence identity, including the homology alignment algorithm of Needleman et al. (1970) J. Mol. Biol.
  • Computer programs that utilize these algorithms are also available and include, but are not limited to, ALIGN or Megalign (DNASTAR) software, or WU-B LAST-2 (Altschul et al., Meth. Enzym., 266:460-480 (1996)); or GAP, BESTFIT, BLAST Altschul et al., supra, FASTA, and TFASTA, available in the Genetics Computing Group (GCG) package, Version 8, Madison, Wisconsin, USA; and CLUSTAL in the PC/Gene program provided by Intelligenetics, Mountain View, California.
  • GCG Genetics Computing Group
  • PCR polymerase chain reaction
  • amino acid residues herein are the standard three-letter and/or one-letter codes used in the art to refer to one of the 20 common L-amino acids.
  • amino acid site when an amino acid site is indicated, it is expressed in accordance with the common expression in the art, that is, "amino acid abbreviation + site”, such as “Q63”, where "Q” represents the amino acid at the site, and "63” is the corresponding amino acid site;
  • amino acid abbreviation before mutation + site + amino acid abbreviation after mutation such as "Q63A”, where "Q” represents the amino acid before mutation, "63” is the corresponding mutation site, and "A” represents the amino acid after mutation.
  • Q and “A” are both single-letter abbreviations commonly used in the art to represent amino acids.
  • the present invention provides reverse transcriptases, kits, and compositions comprising the reverse transcriptases.
  • the reverse transcriptase provided by the present invention has at least one of the mutation sites of L333, P65, Q68, P175, E176, F334, G178, G331, T332, P127 and G105, wherein the amino acid site positioning of the reverse transcriptase is based on the amino acid sequence shown in SEQ ID NO: 2, and in addition, the reverse transcriptase may further have at least one of the mutation sites of Q63, Y64, M66, D200, E562 and D583.
  • the reverse transcriptase has the following mutation sites or site combinations: 1) Q63; 2) Y64; 3) P65; 4) M665) Q68; 6) P127; 7) P175; 8) E176; 9) G178; 10) G331; 11) T332; 12) L333; 13) F334; 14) E176, E562 and D583; 15) L333, E562 and D583; 16) M66, Q68, G105, E562 and D583; 17) M66, Q68, E176, D200, E562 and D583; or 18) P65, M66, Q68, E69, E176, D200, E562 and D583.
  • the reverse transcriptase has at least one of the following mutations: i) Q63A, Q63P or Q63G; ii) P65N, P65G, P65C, P65E, P65T, P65K, P65L, P65I or P65S; iii) M66Y or M66V; iv) Q68A, Q68E, Q68K or Q68N; v) E69K; vi) P127D, P127R or P127T; vii) P175L, P175W or P175F; viii) P175L, P175W or P175F; ) E176H, E176A, E176R or E176C; ix) G178N or G178T; x) G331F, G331P, G331E or G331M; xi) T332D, T332W, T332P or T332K; xii) L333A
  • the reverse transcriptase has the following mutations: I) Q63A, Q63P or Q63G; II) Y64S, Y64V or Y64F; III) P65N, P65G, P65C, P65E, P65T, P65K, P65L, P65I or P65S; IV) M66Y; V) Q68A, Q68E, Q68K or Q68N; VI) P127D, P127R or P127T; VII) P175L, P175W or P175F; VIII) E176H, E176A or E176C; IX) G178N or G178T; X) G331F, G331P, G331E or G33 1M; XI) T332D, T332W, T332P or T332K; XII) L333A or L333N; XIII) F334S, F334T, F334N, F334H or
  • the present invention also provides a test kit.
  • the test kit provided by the present invention can be used for generating, amplifying nucleic acid molecules (single-stranded or double-stranded) or for sequencing.
  • the test kit provided by the present application includes a loadable carrier, such as a box or a hard case, etc. These loadable carriers are equipped with one or more containers, such as vials, tubes, etc.
  • One or more reverse transcriptases provided by the present application can be installed in these containers.
  • one or more DNA polymerases, one or more buffers suitable for nucleic acid synthesis, one or more nucleotides and/or one or more nucleic acid extraction reagents can also be installed in the same or different containers.
  • composition provided by the present invention may also include one or more nucleotides, one or more nucleic acid extraction reagents, one or more buffers, and one or more DNA polymerases in addition to one or more reverse transcriptases (e.g., two, three, four, eight, ten, fifteen, twenty, thirty, forty, fifty, etc.) described in the present invention.
  • the composition of the present invention may also include one or more oligonucleoside primers. The composition is obtained by mixing the above-mentioned components.
  • the nucleic acid sequence encoding wild-type M-MLV reverse transcriptase was obtained, and then the nucleic acid sequence was introduced into the pET-22b(+) expression plasmid (purchased from GenScript Technology Co., Ltd.) between the NdeI and EcoRI restriction sites.
  • the vector has 6 His at the C-terminus of the M-MLV sequence to facilitate protein purification.
  • the expression vector was named pET-22b-M-MLV.
  • nucleotide sequence encoding wild-type M-MLV reverse transcriptase (SEQ ID NO: 1) is as follows:
  • amino acid sequence of wild-type M-MLV reverse transcriptase (SEQ ID NO: 2) is shown below:
  • the expression plasmid of the M-MLV reverse transcriptase mutant was constructed by site-directed mutagenesis, wherein the inventors designed forward and reverse mutation primer pairs, used pET-22b-M-MLV as a template, and used Pfu DNA polymerase (purchased from Promega) for site-directed mutagenesis PCR to obtain the corresponding M-MLV reverse transcriptase mutant expression vectors.
  • the specific method is as follows:
  • Table 1 PCR reaction system for constructing M-MLV reverse transcriptase mutant expression vector
  • Reaction components/final concentration 1 ⁇ Pfu buffer (containing MgSO 4 ) 0.2 mM dNTPs 0.5 ⁇ M forward primer 0.5 ⁇ M reverse primer 0.05U/ ⁇ L pfu DNA polymerase 1ng/ ⁇ L template (pET-22b-M-MLV) H2O
  • Table 2 PCR reaction conditions for constructing M-MLV reverse transcriptase mutant expression vector
  • the wild-type M-MLV reverse transcriptase and its mutants are expressed by the promoter of pET22b, and 6 His tags are fused at the C-terminus.
  • the His tags can be used for Ni column affinity purification to obtain the corresponding crude enzyme solutions. The specific methods are as follows:
  • Example 1 The wild-type and mutant plasmids obtained in Example 1 were transformed into BL21 competent cells (purchased from Tiangen Biochemical Technology (Beijing) Co., Ltd.);
  • the crude M-MLV enzyme supernatant prepared in the previous step was subjected to Ni affinity purification.
  • the main steps were as follows: the supernatant was added to the wells of the balanced His MuLtiTrap HP purification plate (purchased from GE), and the solution was removed using a vacuum pressure device after standing for 15 min; 2 mL of M-MLV reverse transcriptase Ni column A solution was added for rinsing, and the solution was removed using a vacuum pressure device; a 96-well plate was placed under the His MuLtiTrap HP purification plate and fixed, and then 100 ⁇ L of M-MLV reverse transcriptase Ni column B solution (50 mM Tris, 500 mM NaCl, 5% Glycerol, 500 mM Imidazole, pH 7.5) was added for elution, and the eluted protein was collected by centrifugation at 500 g for 2 min; 100 ⁇ L of glycerol was added to the sample wells of the
  • the wild-type M-MLV reverse transcriptase and mutants obtained in Example 2 were subjected to thermal stability assay.
  • the StepOne Plus Real-Time PCR System was used for detection.
  • the specific reaction conditions were set in full accordance with the instructions of the kit, and then the wild-type M-MLV reverse transcriptase and mutants were analyzed using Protein Thermal Shift Software and the Tm value of each protein was derived.
  • the specific detection principle is: as the temperature rises, the protein structure changes, the hydrophobic domain is exposed, and it combines with the fluorescent dye to produce fluorescence.
  • the changes between the temperature (Melt Curve) and the fluorescence value are detected in real time by the qPCR instrument, and the Tm values of the wild-type M-MLV reverse transcriptase and its mutants are compared to judge their stability, and the mutants with Tm values higher than the wild-type are screened out.
  • the specific experimental results after screening are shown in Table 3, and the specific mutation mode and sequence of each mutant are shown in Table 4.
  • M-MLV reverse transcriptase is a normothermic enzyme, and its polymerization activity decreases with increasing temperature. At the same reaction temperature, the polymerization activity of the mutant was detected by comparing the amount of polymerization products of the mutant and wild-type M-MLV RT.
  • chain extension occurs under the action of the crudely extracted wild-type M-MLV reverse transcriptase and its mutant obtained in Example 2 to obtain a hybrid chain of extended RNA and cDNA.
  • Polymerization reactions are performed under different reaction temperature conditions, fluorescent molecules are added to the reaction products, and the activity of the reverse transcriptase is calculated by detecting the RNA/cDNA amount.
  • the specific reaction system is shown in Table 5.
  • the M-MLV reverse transcriptase and its mutants prepared in Example 2 without treatment and the M-MLV reverse transcriptase and its mutants treated at 60°C for 10 minutes were tested for polymerization activity according to the polymerization activity detection method described in Example 3, and the heat resistance of the M-MLV reverse transcriptase and its mutants was verified by comparing the polymerization activity of the M-MLV reverse transcriptase and its mutants treated at 60°C for 10 minutes with that of the M-MLV reverse transcriptase and its mutants not subjected to heat treatment.
  • the residual polymerization activity of the M-MLV reverse transcriptase and its mutants after being treated at 60°C for 10 minutes compared with that of the M-MLV reverse transcriptase and its mutants not subjected to heat treatment is shown in Table 7.
  • RT-14 10.8% RT-37 38.4% RT-15 24.3% RT-38 63.9% RT-16 46.9% RT-39 52.8% RT-17 67.0% RT-40 98.6% RT-18 55.1% RT-41 60.8% RT-19 46.7% RT-42 37.2% RT-20 65.6% RT-43 43.4% RT-21 36.5% RT-44 34.6% RT-22 66.4% RT-45 47.8% RT-23 65.9% RT-46 49.0% RT-24 34.7% WT 30.7%
  • the inventor further tested the influence of template and primer on the thermal stability of the P65 site mutant of the M-MLV reverse transcriptase described in the present application.
  • the detection method adopts the method described in Example 3, and the thermal stability of the M-MLV reverse transcriptase mutant is detected in the system with template/primer (T/P).
  • the specific experimental method is as follows: the reaction system shown in Table 8 and Table 9 is prepared, and the M-MLV reverse transcriptase mutants W313F and L435G, which have been confirmed to improve the thermal stability of the enzyme in the reaction by improving the combination of T/P, are used as positive controls, and the StepOne Plus Real-Time PCR System is used for detection, and then the Protein Thermal Shift Software is used for analysis and the Tm value of each protein is derived.
  • the thermal stability of the M-MLV reverse transcriptase in the above system without T/P that is, removing oligo(dT) 16 and poly(rA) 50, is detected as a comparison.
  • the test results are shown in Table 10.
  • the Tm value of the M-MLV reverse transcriptase P65 mutant in the presence of T/P increases compared to that in the absence of T/P, while the difference between the wild-type M-MLV reverse transcriptase with and without T/P is not obvious.
  • the M-MLV reverse transcriptase P65 mutant may improve the thermal stability of the enzyme in the reaction by improving the binding with the template/primer (T/P) rather than improving the heat resistance of the enzyme itself.
  • the heat resistance of M-MLV reverse transcriptase and its P65 site mutant in the reaction system is determined by detecting the relative activity of the M-MLV reverse transcriptase and its P65 site mutant obtained in Example 2 at 55°C compared to that at 37°C.
  • the polymerization activity was detected using the polymerization activity detection method described in Example 4, wherein the reaction temperatures were 37°C and 55°C, and the relative activity of the 55°C reaction compared to the 37°C reaction is shown in Table 11.
  • the relative activity of the P65 site mutant at 55°C/37°C is higher than that of the WT, indicating that the P65 site mutant (in the presence of T/P) has higher thermal stability.
  • the M-MLV reverse transcriptase and its mutants obtained in Example 2 were used to perform one-step RT-qPCR test.
  • the reaction system is shown in Table 12, and the reaction procedure is shown in Table 13.
  • the primer and probe sequences used in the one-step RT-qPCR are:
  • GADPH-F upstream primer: 5’-CAACGGATTTGGTCGTATTGG-3’ (SEQ ID NO:54).
  • GADPH-R downstream primer: 5’-GCAACAATATCCACTTTACCAGAGTTAA-3’ (SEQ ID NO:55).
  • GADPH-P (probe): 5’-HEX-CGCCTGGTCACCAGGGCTGC-BHQ1-3’ (SEQ ID NO: 56).
  • ⁇ Ct(Mut-WT) is the difference between the Ct value of the M-MLV reverse transcriptase mutant and the Ct value of the wild-type M-MLV reverse transcriptase under the same conditions.
  • ⁇ Ct(Mut-WT) less than 0 indicates that the M-MLV reverse transcriptase mutant reaches the threshold earlier than the wild-type M-MLV reverse transcriptase, and the one-step RT-qPCR effect is better than the wild-type.
  • the results show that most mutants are better than wild-type M-MLV reverse transcriptase in one-step RT-qPCR.
  • Component/Final Concentration 1x PCR Buffer 0.27ng/ ⁇ L M-MLV reverse transcriptase or its mutants 0.5U/ ⁇ L HostStart Taq DNA polymerase 0.2U/ ⁇ L RNase Inhibitor 5mU/ ⁇ L Heat-labile UDG 0.5 ⁇ M GADPH-F 0.5 ⁇ M GADPH-R 0.25 ⁇ M GADPH-P 16.7pg/ ⁇ L HEK293T Total RNA DEPC H2O
  • This example uses the method described in Example 8 to further test the amplification effect of the M-MLV reverse transcriptase and its mutants and the commercial M-MLV RH - (purchased from Takara) in one-step RT-qPCR under different concentrations of RNA template input, and the final concentrations of RNA in the system are 16.7pg/ ⁇ L, 1.67pg/ ⁇ L, and 0.167pg/ ⁇ L, respectively.
  • the test results are shown in Figure 1.
  • the Ct of the commercial M-MLV RH - is lower than that of the wild-type M-MLV reverse transcriptase as a whole, and the amplification efficiency is better; while the Ct values of the M-MLV reverse transcriptase mutants RT-12, RT-40, RT-44, RT-47 to RT-51 are lower than those of the commercial M-MLV RH - , and their amplification effects are better than those of the commercial M-MLV RH - reverse transcriptase and the wild-type M-MLV reverse transcriptase.

Landscapes

  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Engineering & Computer Science (AREA)
  • Organic Chemistry (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Engineering & Computer Science (AREA)
  • Biotechnology (AREA)
  • Molecular Biology (AREA)
  • Biochemistry (AREA)
  • Biomedical Technology (AREA)
  • General Health & Medical Sciences (AREA)
  • Microbiology (AREA)
  • Biophysics (AREA)
  • Physics & Mathematics (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Analytical Chemistry (AREA)
  • Immunology (AREA)
  • Plant Pathology (AREA)
  • Medicinal Chemistry (AREA)
  • Enzymes And Modification Thereof (AREA)

Abstract

La présente invention concerne une transcriptase inverse thermostable et son utilisation. La transcriptase inverse thermostable présente au moins une des positions de mutation L333, P65, Q68, P127, P175, E176, G178, G331, T332, F334 et G105, les positions d'acides aminés de la transcriptase inverse thermostable étant fondées sur la séquence d'acides aminés représentée dans SEQ ID NO : 2. La transcriptase inverse présente une plus grande stabilité thermique.
PCT/CN2022/130031 2022-11-04 2022-11-04 Transcriptase inverse thermostable et son utilisation WO2024092768A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
PCT/CN2022/130031 WO2024092768A1 (fr) 2022-11-04 2022-11-04 Transcriptase inverse thermostable et son utilisation

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
PCT/CN2022/130031 WO2024092768A1 (fr) 2022-11-04 2022-11-04 Transcriptase inverse thermostable et son utilisation

Publications (1)

Publication Number Publication Date
WO2024092768A1 true WO2024092768A1 (fr) 2024-05-10

Family

ID=90929432

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2022/130031 WO2024092768A1 (fr) 2022-11-04 2022-11-04 Transcriptase inverse thermostable et son utilisation

Country Status (1)

Country Link
WO (1) WO2024092768A1 (fr)

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN103348004A (zh) * 2011-02-09 2013-10-09 株式会社百奥尼 具有改善的热稳定性的逆转录酶
CN110291196A (zh) * 2016-12-14 2019-09-27 宝生物工程株式会社 耐热逆转录酶突变体
CN112695019A (zh) * 2021-03-23 2021-04-23 翌圣生物科技（上海）有限公司 逆转录酶突变体及其应用
CN113174381A (zh) * 2021-06-08 2021-07-27 翌圣生物科技(上海)股份有限公司 增强型mmlv逆转录酶突变体及其应用

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN103348004A (zh) * 2011-02-09 2013-10-09 株式会社百奥尼 具有改善的热稳定性的逆转录酶
CN110291196A (zh) * 2016-12-14 2019-09-27 宝生物工程株式会社 耐热逆转录酶突变体
CN112695019A (zh) * 2021-03-23 2021-04-23 翌圣生物科技（上海）有限公司 逆转录酶突变体及其应用
CN113174381A (zh) * 2021-06-08 2021-07-27 翌圣生物科技(上海)股份有限公司 增强型mmlv逆转录酶突变体及其应用

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
DATABASE Protein 13 August 2018 (2018-08-13), "p80 RT [Moloney murine leukemia virus]", XP093168017, Database accession no. NP_955591 *

Similar Documents

Publication Publication Date Title
JP7375845B2 (ja) 改変された耐熱性ｄｎａポリメラーゼ
JPH06504196A (ja) モルモシポ・アフリカヌスからの純化された熱安定性核酸ポリメラーゼ
CN111996179A (zh) 一种dna聚合酶及其在pcr检测中的应用
KR20140022962A (ko) 내열성 ｄｎａ 폴리머라제를 포함하는 효소 조제물 및 그 제조 방법, 및 검출 대상생물의 검출 방법
JP6224613B2 (ja) 改善された活性を有するｄｎａポリメラーゼ
JP2015504651A (ja) 改善された活性を有するｄｎａポリメラーゼ
JP2003510052A (ja) 改良されたポリヌクレオチド合成のための方法と組成物
WO2020132966A1 (fr) Transcriptase inverse ayant une activité enzymatique accrue et application de celle-ci
EP3822349A1 (fr) Mutant d'adn polymérase adapté à l'amplification d'acide nucléique à partir d'arn
WO2005118815A1 (fr) Polypeptides ayant une activité d’adn polymérase
CA2839964A1 (fr) Adn polymerases ayant une activite amelioree
JP2018042567A (ja) 核酸増幅法
WO2024092768A1 (fr) Transcriptase inverse thermostable et son utilisation
JP7014256B2 (ja) 核酸増幅試薬
CN109943549B (zh) 一种超高速扩增型Taq DNA聚合酶
CN117487775B (zh) 一种高酶活的Taq DNA聚合酶及其应用
WO2024009873A1 (fr) Polymérase d'acide nucléique ayant une activité de transcription inverse
JP7342403B2 (ja) 改変されたｄｎａポリメラーゼ
JP6493209B2 (ja) 核酸増幅法
CN113637085B (zh) 融合dna聚合酶突变体及其在等温扩增中的应用
US20230272356A1 (en) C-terminal peptide extensions with increased activity
CN112251423B (zh) 一种m-mlv逆转录酶体及其应用
WO2024092713A1 (fr) Mutant de transcriptase inverse mmlv
JP2024008527A (ja) マンガンを使用しない逆転写方法
WO2024092712A1 (fr) Mutant de transcriptase inverse mmlv

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 22964073

Country of ref document: EP

Kind code of ref document: A1