CN114045274A - Thermostable reverse transcriptase mutants - Google Patents
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Abstract
The invention relates to the technical field of biology, and particularly relates to a thermostable reverse transcriptase mutant. The reverse transcriptase mutant has one or more of the following mutations on the basis of wild type M-MLV reverse transcriptase: K15E, D235R, E441K and K445E; and has significantly improved thermal stability.
Description
Technical Field
The invention relates to the technical field of biology, and particularly relates to a thermostable reverse transcriptase mutant.
Background
Reverse transcriptase (EC 2.7.7.49) was originally found to be the enzyme responsible for replication of the RNA virus genome. It can use RNA or single-stranded DNA (ssDNA) as a template, and has both RNase H activity and DNA polymerase activity. Moloney murine leukemia virus reverse transcriptase (M-MLV RT), because of its high catalytic activity and fidelity, is currently the most widely used reverse transcriptase in the synthesis of complementary DNA (cDNA). The M-MLV reverse transcriptase active protein is a 75kDa sized monomer with two active sites, one in the N-terminal domain for DNA polymerization and the other in the C-terminal domain for RNase H activity.
M-MLV reverse transcriptase is thermolabile and its activity drops to 50% (T (50)) when incubated for 10 min at 44 ℃ in the absence of template primer (T/P). The activity of wild-type M-MLV RT decreased to 50% even after incubation at 47 ℃ for 10 min in the presence of template and primer T/P (28. mu.M).
The thermal stability of M-MLV RT is improved to some extent by methods that eliminate RNase H activity while retaining the complete RNA-directed DNA polymerase activity. For example, RNase H-M-MLV RT (D524N) showed higher thermostability, with a 4-fold longer half-life at 50 ℃ compared to M-MLV RT. Generally, the optimal temperature for application of M-MLV RT is at 37 ℃ and RNase H-M-MLV RT is recommended to be used at 42 ℃.
However, applications in the field of molecular biology still require further increases in the reaction temperature for reverse transcription to open up RNA secondary structures, thereby reducing non-specific primer binding and improving M-MLV RT fidelity by reducing mismatch stability.
In view of the above, the present invention is particularly proposed.
Disclosure of Invention
The invention aims to provide a reverse transcriptase mutant, and the amino acid sequence of the mutant is shown in SEQ ID NO: 1, the moloney murine leukemia virus (M-MLV) reverse transcriptase has one or more of the following mutations:
K15E, D235R, E441K and K445E.
Compared with M-MLV reverse transcriptase without corresponding mutation, the reverse transcriptase mutant has remarkably improved thermal stability, so that the temperature fluctuation condition in the reverse transcription process is not required to be considered too much, nonspecific primer combination can be reduced, the fidelity of M-MLV RT is improved by reducing the mismatch stability, and the application prospect is wider.
The invention also relates to isolated nucleic acids encoding the reverse transcriptase mutants described above, and vectors comprising the nucleic acids described above.
The invention also relates to a host cell, the genome of which incorporates a nucleic acid as described above, or which is transformed with a vector as described above.
The present invention also relates to a method for producing a reverse transcriptase mutant, comprising:
a) culturing a host cell as described above;
b) expressing the reverse transcriptase mutant; and are
c) Isolating the reverse transcriptase mutant from the host cell.
The invention also relates to a kit comprising the reverse transcriptase mutant as described above.
The invention also relates to a composition obtained by mixing the components in the kit.
The present invention also relates to methods for reverse transcribing one or more nucleic acid molecules comprising:
i) using a reverse transcriptase mutant as described above, and contacting with a template RNA, and
ii) incubating under a reaction system sufficient to produce DNA complementary to said template RNA.
The present invention also relates to a method for detecting an RNA marker in a sample, comprising:
i) contacting an RNA marker with a reverse transcriptase mutant as described above;
ii) incubating under a reaction system sufficient to produce DNA complementary to said RNA marker; and
iii) detecting the presence of DNA synthesized in step ii), thereby detecting the RNA marker in the sample.
The present invention also relates to a method for producing a reverse transcriptase with enhanced thermostability, comprising:
introducing a mutation in a nucleic acid encoding an M-MLV reverse transcriptase such that the expression product of said nucleic acid has at least one of the following mutations:
K15E, D235R, E441K and K445E;
the position of the mutation is shown as SEQ ID NO: 1 is referred to as amino acid of M-MLV reverse transcriptase.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, and it is obvious that the drawings in the following description are some embodiments of the present invention, and other drawings can be obtained by those skilled in the art without creative efforts.
FIG. 1 is a schematic representation of a reverse transcription reaction employed in one embodiment of the present invention;
FIG. 2 shows the comparison of the thermostability of a wild-type reverse transcriptase provided in one embodiment of the present invention with K15E, D235R, E441K and K445E; panel A: the reaction is carried out for 35 cycles of 5min at 42 ℃, 3min at 95 ℃, 30s at 95 ℃ and 1min at 60 ℃; lane M: GeneRuler ultra-low range DNA ladder (Thermo Scientific), lanes 1-2: K15E, lanes 3-4: D235R, lanes 5-6: E441K, lanes 7-8: K445E, lanes 9-10: wild-type reverse transcriptase, lanes 11-12: no template control; panel B: the reaction is carried out for 35 cycles of 5min at 55 ℃, 3min at 95 ℃, 30s at 95 ℃ and 1min at 60 ℃; sample set-up was the same as Panel a; panel C: the reaction is carried out for 35 cycles of 5min at 65 ℃, 3min at 95 ℃, 30s at 95 ℃ and 1min at 60 ℃; sample set-up was the same as Panel a; panel D: the reaction is carried out for 35 cycles of 5min at 70 ℃, 3min at 95 ℃, 30s at 95 ℃ and 1min at 60 ℃; sample set-up was the same as Panel a.
Detailed Description
Reference will now be made in detail to embodiments of the invention, one or more examples of which are described below. Each example is provided by way of explanation, not limitation, of the invention. In fact, it will be apparent to those skilled in the art that various modifications and variations can be made in the present invention without departing from the scope or spirit of the invention. For instance, features illustrated or described as part of one embodiment, can be used on another embodiment to yield a still further embodiment.
Unless otherwise defined, all terms (including technical and scientific terms) used in disclosing the invention are to be interpreted as commonly understood by one of ordinary skill in the art to which this invention belongs. The following definitions serve to better understand the teachings of the present invention by way of further guidance. The terminology used in the description of the invention herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention.
As used herein, the terms "comprising," "including," and "comprising" are synonymous, inclusive or open-ended, and do not exclude additional, unrecited members, elements, or method steps.
The recitation of numerical ranges by endpoints herein includes all numbers and fractions subsumed within that range, as well as the recited endpoints.
The present invention relates to concentration values, which include fluctuations within a certain range. For example, it may fluctuate within a corresponding accuracy range. For example, 2%, may be allowed to fluctuate within 0.1%. For values that are larger or do not require more fine control, the meaning is also allowed to include greater fluctuations. For example, 100mM, may allow fluctuations within the range of. + -. 1%, + -2%, + -5%, etc. The molecular weight is referred to, allowing the meaning to include fluctuations of ± 10%.
In the present invention, the terms "plurality", and the like mean, unless otherwise specified, 2 or more in number.
In the present invention, the technical features described in the open type include a closed technical solution composed of the listed features, and also include an open technical solution including the listed features.
In the present invention, "preferably", "better" and "preferable" are only embodiments or examples with better description, and it should be understood that the scope of the present invention is not limited by them.
All documents referred to herein are incorporated by reference into this application as if each were individually incorporated by reference. The citation referred to herein is incorporated by reference in its entirety for all purposes unless otherwise in conflict with the present disclosure's objectives and/or technical solutions. Where a citation is referred to herein, the definition of a reference in the document, including features, terms, nouns, phrases, etc., that is relevant, is also incorporated by reference. In the present invention, when the citation is referred to, the cited examples and preferred embodiments of the related art features are also incorporated by reference into the present application, but the present invention is not limited to the embodiments. It should be understood that where the citation conflicts with the description herein, the application will control or be adapted in accordance with the description herein.
In the present invention, "thermostable" or "thermostable" refers to the ability of an M-MLV reverse transcriptase to still have reverse transcription activity under conditions suitable for reverse transcription reactions to occur, but at higher reaction temperatures. The reaction temperature is about 30 ℃ to 70 ℃, for example 35 ℃, 37 ℃, 39 ℃, 40 ℃, 42 ℃, 45 ℃, 50 ℃, 55 ℃, 60 ℃, 65 ℃, preferably 65 ℃ to 70 ℃.
The invention relates to a reverse transcriptase mutant, the amino acid sequence of which is shown in SEQ ID NO: 1, has one or more of the following mutations:
K15E, D235R, E441K and K445E.
The amino acid sequence of the reverse transcriptase mutant corresponding to the single mutation site is exemplified by the amino acid sequence shown in sequence as SEQ ID NO: 2 to 5.
Compared with M-MLV reverse transcriptase without corresponding mutation, the reverse transcriptase mutant has obviously improved thermal stability, and has no obvious adverse effect on the reverse transcription efficiency, the reverse transcription template sensitivity, the fidelity and the like of the M-MLV reverse transcriptase.
Reverse transcriptase mutants are not excluded in addition to SEQ ID NO: the possibility of mutating M-MLV reverse transcriptase other than 1, i.e., the reverse transcriptase mutants claimed in the present application, may be enzymes substantially similar to the reverse transcriptase mutants (particularly, the reverse transcriptase mutants shown in SEQ ID NO: 2-5).
By "substantially similar" is meant an enzyme in which a given nucleic acid or amino acid sequence shares a sequence of at least about 80% identity, at least about 90% identity, at least about 95% identity, at least about 96% identity, at least about 97% identity, at least about 98% identity, at least about 99% identity, or at least about 99.5% identity with a reference sequence, and retains at least one of the sites of mutation in K15E, D235R, E441K, and K445E, retains reverse transcriptase activity, and retains greater thermostability (its thermostability resembles, e.g., is not less than, at least one of the enzymes set forth in SEQ ID NOS: 2-5).
Substantially similar enzymes typically undergo conservative amino acid substitutions as compared to a reference sequence, substitutions which are generally regarded as conservative substitutions are those in the aliphatic amino acids Ala, Val, Leu and Ile which are substituted for one another, the exchange of the hydroxyl residues Ser and Thr, the exchange of the acidic residues Asp and Glu, the exchange of the amide residues Asn and Gln, the exchange of the basic residues Lys and Arg and the substitution of the aromatic residues Phe, Tyr.
In addition, substantially similar enzymes also include derivatives obtained by natural processes (such as processing and other post-translational modifications), or by chemical modification techniques, such as by the addition of one or more polyethylene glycol molecules, sugars, phosphates, and/or other such molecules, wherein the one or more molecules are not naturally attached to the protein. Derivatives include salts. Such chemical modifications are described in detail in basic texts and in more detailed monographs, as well as in a large number of research documents, and they are well known to those skilled in the art. It is understood that the same type of modification may be present to the same or different degrees at several sites in a given protein or polypeptide. In addition, a given protein or polypeptide may contain many types of modifications. Modifications can occur anywhere in a protein or polypeptide, including the peptide backbone, the amino acid side chains, and the amino or carboxyl termini. Modifications include, for example, acetylation, acylation, ADP-ribosylation, amidation, covalent attachment of flavin, covalent attachment of a heme moiety, covalent attachment of a nucleotide or nucleotide derivative, covalent attachment of a lipid or lipid derivative, covalent attachment of phosphatidylinositol, cross-linking, cyclization, disulfide bond formation, demethylation, formation of covalent cross-links, formation of cysteine, formation of pyroglutamic acid, methylation, gamma-carboxylation, glycosylation, GPI-anchoring, hydroxylation, iodination, methylation, myristoylation, oxidation, proteolytic processing, phosphorylation, prenylation, racemization, glycosylation, lipid attachment, sulfation, gamma-carboxylation of glutamic acid residues, alkylation and ADP-ribosylation, selenization, sulfation, transfer RNA-mediated addition of amino acids to proteins (such as arginylation), and ubiquitination. They may also be bound to vitamins such as biotin, folic acid or vitamin B12. See, e.g., Proteins-Structure And Molecular Properties,2nd Ed., T.E.Creighton, W.H.Freeman And Company, New York (1993) And world, F., "Postrelational Protein Modifications: perspectra and Prospectra, "pgs.1-12 in Posttranslation equivalent Modification Of Proteins, B.C. Johnson, Ed., Academic Press, New York (1983); seifter et al, meth. enzymol.182: 626 + 646(1990) and Rattan et al, "Protein Synthesis: posttranslation Modifications and Aging, "Ann.N.Y.Acad.Sci.663: 48-621992). The term "derivative" includes chemical modifications that result in a protein or polypeptide becoming branched or cyclic, with or without branching. Cyclic, branched and branched circular proteins or polypeptides can be naturally processed post-translationally and can also be made entirely by synthetic methods. In some embodiments, the compound may be covalently linked to a carrier protein, such as serum albumin or other plasma proteins.
Substantially similar enzymes may have no RNase H activity or have attenuated RNase H activity.
Substantially similar enzymes may comprise other mutations known in the art to facilitate functional performance of M-MLV reverse transcriptase, e.g., they may have a mutation comprising at least one amino acid position selected from the group consisting of:
Q19，Y64，R116，D124，H126，Y133，K152，T197，V223，L435，D524；
amino acids that may be used to replace Tyr include Lys, Arg, His, Asp, Glu, Ala, Val, Leu, Ile, Pro, Met, Trp, Gly, Ser, Thr, Cys, Asn, or Gln. Amino acids that may be used in place of Arg include Tyr, His, Asp, Glu, Ala, Val, Leu, ILe, Pro, Met, Trp, Gly, Ser, Thr, Cys, Phe, Asn, or Gln. Amino acids that may be used in place of Lys include Tyr, Arg, His, Asp, Glu, Ala, Val, Leu, Ile, Pro, Met, Trp, Gly, Ser, Thr, Cys, Asn, or Gln. Amino acids that may be used in place of Glu include Lys, Arg, His, Asp, Tyr, Ala, Val, Leu, Ile, Pro, Met, Trp, Gly, Ser, Thr, Cys, Asn, or Gln. Amino acids that may be used in place of Thr include Lys, Arg, His, Asp, Glu, Ala, Val, Leu, Ile, Pro, Met, Trp, Gly, Ser, Tyr, Cys, Asn, or Gln. Amino acids that may be used to replace Val include Lys, Arg, His, Asp, Glu, Ala, Tyr, Leu, Ile, Pro, Met, Trp, Gly, Ser, Thr, Cys, Asn, or Gln. Amino acids that may be used in place of Leu include Lys, Arg, His, Asp, Glu, Ala, Val, Tyr, Ile, Pro, Met, Trp, Gly, Ser, Thr, Cys, Asn, or Gln. Amino acids that may be used in place of Asp include Lys, Arg, His, Leu, Glu, Ala, Val, Tyr, Ile, Pro, Met, Trp, Gly, Ser, Thr, Cys, Asn, or Gln.
These mutants can be prepared by well-known methods such as site-directed mutagenesis.
The above effects can be easily verified by those skilled in the art on the basis of the present application without any inventive effort, and thus a reverse transcriptase mutant obtained by mutation on the basis of the above M-MLV reverse transcriptase is also within the scope of the present application.
Preferably, the reverse transcriptase mutant comprises a tag. Further preferably, a tag is fused to the C-terminus of the reverse transcriptase mutant. Tags are attached to proteins for various purposes, for example, to facilitate ease of purification, to aid in proper folding of the protein, to prevent precipitation of the protein, to alter chromatographic properties, to modify or label the protein or to tag the protein. Examples of tags include Arg-tag, His-tag, Strep-tag, Flag-tag, T7-tag, V5-peptide-tag, GST-tag and c-Myc-tag. The preferred tag in the present invention is a His-tag consisting of six histidine residues.
According to a further aspect of the invention, it also relates to an isolated nucleic acid encoding a reverse transcriptase mutant as described above.
The nucleic acid is typically RNA or DNA. The nucleic acid may be single-stranded or double-stranded. A nucleic acid is "operably linked" when it is placed into a functional relationship with another nucleic acid sequence. For example, a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the coding sequence. It is preferable to use DNA nucleic acid when it is ligated to a vector. The nucleic acid may be codon optimized for more efficient expression in a desired host cell.
According to a further aspect of the invention, it also relates to a vector comprising a nucleic acid as described above.
The term "vector" refers to a nucleic acid delivery vehicle into which a polynucleotide can be inserted. When a vector is capable of expressing a protein encoded by an inserted polynucleotide, the vector is referred to as an expression vector. The vector may be introduced into a host cell by transformation, transduction, or transfection, and the genetic material elements carried thereby are expressed in the host cell. Vectors are well known to those skilled in the art and include, but are not limited to: a plasmid; phagemid; a cosmid; artificial chromosomes such as Yeast Artificial Chromosomes (YACs), Bacterial Artificial Chromosomes (BACs), or artificial chromosomes (PACs) derived from P1; bacteriophage such as lambda phage or M13 phage, animal virus, etc. Animal viruses that may be used as vectors include, but are not limited to, retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpes viruses (e.g., herpes simplex virus), poxviruses, baculoviruses, papilloma viruses, papilloma polyoma vacuolatum viruses (e.g., SV 40). In some embodiments, regulatory elements commonly used in genetic engineering, such as enhancers, promoters, Internal Ribosome Entry Sites (IRES), and other expression control elements (e.g., transcription termination signals, or polyadenylation signals and poly-U sequences, etc.) are included in the vectors of the present invention.
The invention also relates to a host cell in the genome of which a nucleic acid as described above, or transformed with a vector as described above, is present.
The term "host cell" refers to a cell which can be used for introducing a vector, and includes, but is not limited to, prokaryotic cells such as Escherichia coli or Bacillus subtilis, fungal cells such as yeast cells or Aspergillus, insect cells such as S2 Drosophila cells or Sf9, or animal cells such as fibroblast, CHO cells, COS cells, NSO cells, HeLa cells, BHK cells, HEK 293 cells or human cells. The host cell is preferably a eukaryotic cell,
according to still another aspect of the present invention, there is also provided a method of producing a reverse transcriptase mutant, comprising:
a) culturing a host cell as described above;
b) expressing the reverse transcriptase mutant; and are
c) Isolating the reverse transcriptase mutant from the host cell.
Isolation of the reverse transcriptase mutant is understood to mean the isolation of the crude product and may also be understood to include purification and/or concentration procedures, e.g.the product of interest may be purified from the cell culture contents according to standard procedures in the art, including ammonium sulphate precipitation, affinity columns, column chromatography, gel electrophoresis, etc. Such techniques are within the skill of the art and do not limit the invention. The expressed protein can be filtered and concentrated by a conventional method. Soluble mixtures and polymers can also be removed by conventional methods, such as molecular sieves, ion exchange. The obtained product is immediately frozen, such as-70 deg.C to-80 deg.C, or lyophilized.
According to a further aspect of the invention, it also relates to a kit comprising a reverse transcriptase mutant as described above.
The kit is preferably used to perform reverse transcription.
In some embodiments, it further comprises one or more of RNA extraction reagents, reverse transcription reaction solution, dNTPs, water, dideoxynucleotides, and reverse transcription primers.
The reverse transcription primer may be an Oligo dT primer, a random primer or a gene-specific primer, as required.
The term "kit" refers to any article of manufacture (e.g., a package or container) comprising at least one device, which may further comprise instructions for use, supplemental reagents, and/or components or assemblies for use in the methods described herein or steps thereof.
Preferably, the nucleic acid components and protein components of the kit, such as primers and reverse transcriptase mutants, are stored in the kit in dry powder form. The components can also be realized in lyophilized form, for example in the form of one or more so-called lyophilized beads. Lyophilized beads are generally understood to mean lyophilizates which are compressed into spherical form after production (after which the substance is generally present as a powder).
According to a further aspect of the invention, it also relates to a composition comprising a reverse transcriptase mutant as described above.
In some embodiments, the composition is obtained by mixing the components of the kit as described above.
According to yet another aspect of the invention, there is also provided a method for reverse transcription of one or more nucleic acid molecules comprising:
i) using a reverse transcriptase mutant as described above, and contacting with a template RNA, and
ii) incubating under a reaction system sufficient to produce DNA complementary to said template RNA.
According to a further aspect of the invention, it also relates to a method for detecting an RNA marker in a sample, comprising:
i) contacting an RNA marker with a reverse transcriptase mutant as described above;
ii) incubating under a reaction system sufficient to produce DNA complementary to said RNA marker; and
iii) detecting the presence of DNA synthesized in step ii), thereby detecting the RNA marker in the sample.
In some embodiments, the sample is selected from the group consisting of blood, plasma, serum, urine, bile, cerebrospinal fluid, swab, clinical specimen, organ sample, and tissue sample.
In some embodiments, the sample is obtained from a cell culture, a source suspected of being contaminated, or a subject.
In some embodiments, the subject is selected from the group consisting of humans, animals (e.g., rat, mouse, cat, dog, horse, cow, sheep, pig, chicken, duck, goose, quail, pigeon, nematode, zebrafish) and plants (e.g., rice, arabidopsis, wheat, corn).
It is readily understood that the above-described method is a universal method applicable to different species and different purposes. In some preferred embodiments, however, the above methods are used for the detection of disease.
The RNA marker may be used to indicate a microorganism, cell, virus, bacterium, fungus, parasite, mammalian species, genetic condition or disease. RNA can be used as a marker in various applications. The detected RNA may be indicative of itself, or it may be indicative of the presence of DNA or expression of a target gene, which in turn is indicative of a disease, the presence of a pathogen, or the like. The RNA itself may indicate the presence of viral RNA, in particular retroviral RNA. Retroviruses cause a variety of diseases such as cancer, AIDS, autoimmunity, and diseases of the central nervous system, bone and joints, such as myeloid leukemia, erythroid leukemia, lymphoid leukemia, lymphoma, sarcoma, breast cancer, renal cancer, aplastic anemia, hemolytic anemia, autoimmune diseases, immunodeficiency, osteopetrosis, arthritis, peripheral neuropathy, encephalopathy, neurodegeneration, dementia, pneumonia, and adenomatosis. Viruses that induce such diseases include Human Immunodeficiency Virus (HIV), human T-lympho virus (HTLV), Rous Sarcoma Virus (RSV), and Murine Mammary Tumor Virus (MMTV). However, RNA markers may indicate gene expression. Many genes are expressed only under specific conditions (including disease conditions) or by specific species. Accordingly, the presence of a protein (or corresponding mRNA) may be indicative of a disease state, cell, or pathogen. For example, cancer cells are characterized by specific markers whose nucleic acids can be used for their detection and quantification. Examples which may be mentioned (in particular oncogenes and tumor suppressor genes) are: such as p53, ras family genes erb-B2, c-myc, mdm2, c-fos, DPC4, FAP, nm23, RET, WT1, etc., LOH such as for p53, DCC, APC, Rb, etc., and microsatellite instability of BRCA1 and BRCA2, MSH2, MLH1, WT1, etc., in hereditary tumors; and tumor RNA, such as CEA, cytokeratins such as CK20, BCL-2, MUC1, particularly tumor-specific splice variants thereof, MAGE3, Muc18, tyrosinase, PSA, PSM, BA46, Mage-1, and the like, or other morphogenic RNA, such as mammary silk inhibin, hCG, GIP, motilin, hTG, SCCA-1, AR, ER, PR, various hormones, and the like; furthermore, especially the expression of RNAs and proteins which influence the metastatic profile, i.e.molecules involved in angiogenesis, motility, adhesion and matrix degradation, such as bFGF, bFGF-R, VEGF-Rs, for example VEGF-R1 or VEGF-R2, E-cadherin, integrins, selectins, MMPs, TIMPs, SF-R etc., cell cycle profiles or proliferation profiles such as cyclins (e.g.the expression ratio of cyclins D, E and B), Ki67, p120, p21, PCNA etc., or apoptosis profiles such as FAS (receptor and/or ligand), TNF (receptor and/or ligand), perforin, granzyme B, BAX, bcl-2, caspase 3 etc. Alternatively, the RNA may be indicative of DNA of a pathogen other than a retrovirus.
Exemplary viruses include: one or more of adenoviridae (adenoviridae), arenaviridae (arenaviridae), astroviridae (astroviridae), orthoviridae (bunyaviridae), caliciviridae (caliciviridae), flaviviridae (flaviviridae), hepaviridae (hepeviridae), mononegavirales (mononegavirales), reticuloviridae (nidovirales), picornaviridae (picornaviridae), orthomyxoviridae (orthomyxoviridae), papilloma virus (papioviridae), parvovirus (paraviridae), polyomaviridae (polyomaviridae), poxviridae (poxviridae), reoviridae (reoviridae), retroviridae (retroviridae), and togaviridae (togaviridae).
Exemplary bacteria include: one or more of staphylococcus, streptococcus, listeria, erysipelothrix, nephrobacter, bacillus, clostridium, mycobacterium, actinomyces, nocardia, corynebacterium, rhodococcus, and/or one or more of anthrax, erysipelothrix, tetanus, listeria, mycobacterium aerobicum, bacillus tubercle, bacillus extracolicus, proteus, dysentery, pneumobacillus, brucella, aerobacter, haemophilus influenzae, haemophilus parainfluenza, moraxella catarrhalis, acinetobacter, yersinia, legionella pneumophila, pertussis, bordetella parapertussis, shigella, pasteurella, vibrio cholerae, and haemophilus parahaemolyticus.
Exemplary fungi include: one or more of coccidioidomycosis immitis, pediococcus pluvialis, histoplasma capsulatum, histoplasma donae, lobelomyces lobbiensis, paracoccidioides brasiliensis, blastomyces dermatitidis, sporothrix schenckii, penicillium marneffei, candida albicans, candida glabrata, candida tropicalis, candida vitis vinifera, aspergillus oryzae, Exophiala jeansi, chromocor pellicii, chromocor verruckeri, chromocor dermatitidis, geotrichum candidum, pediococcus borealis, cryptococcus neoformans, myceliophthora sp, rhizopus oryzae, mucor india, Absidia, Coptomyces racemosus, Chaetomium fortunei, coniothrix gmelini, coniothrix guanidium, Isosporotrichum islandicum, nosema sibirici, Sporotrichomonas well as cryptosporium venenatum.
Exemplary parasites include: one or more of an alimentary canal endoparasite, a liver endoparasite, a lung endoparasite, a brain tissue parasite, a blood vessel endoparasite, a lymphatic endoparasite, a muscle tissue parasite, a cell endoparasite, a bone tissue parasite, and an intraocular parasite.
According to a further aspect of the invention, there is also provided a method for producing a reverse transcriptase having enhanced thermostability (or referred to as a method for improving thermostability of a reverse transcriptase), comprising:
introducing a mutation in a nucleic acid encoding an M-MLV reverse transcriptase such that the expression product of said nucleic acid has at least one of the following mutations:
K15E, D235R, E441K and K445E;
the position of the mutation is shown as SEQ ID NO: 1 is referred to as amino acid of M-MLV reverse transcriptase.
Since it is known that mutations at the positions K15E, D235R, E441K and K445E can be used to increase the thermostability, any method suitable for generating mutations or for generating the desired recombinant protein sequence can be applied.
It should be noted that the reference sequence of the M-MLV reverse transcriptase described above does not constitute a limitation on the reverse transcriptase produced by the present invention, except that the sequence shown in SEQ ID NO: 1, it can also be used for the improvement of other M-MLV reverse transcriptases that have undergone other mutations, only the mutations of K15E, D235R, E441K and K445E need to occur in the same way as compared to the variants of SEQ ID NO: 1, and the corresponding site can be found by a person skilled in the art through a conventional sequence analysis method.
Embodiments of the present invention will be described in detail with reference to examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. The experimental procedures for the conditions not specified in the following examples, preferably with reference to the guidelines given in the present invention, may also be performed according to the experimental manual or the conventional conditions in the art, and may also be performed according to other experimental procedures known in the art, or according to the conditions suggested by the manufacturer.
In the following specific examples, the measurement parameters relating to the components of the raw materials, if not specified otherwise, may be subject to slight deviations within the accuracy of the weighing. Temperature and time parameters are involved to allow for acceptable deviation due to instrument test accuracy or operational accuracy.
Examples
M-MLV reverse transcriptase with a C-terminal His tag (sequence shown below) was expressed in C3019(New England Biolabs, MA) and plasmid construction used pBAD vector with kanamycin resistance.
MLNIEDEHRLHETSKEPDVSLGSTWLSDFPQAWAETGGMGLAVRQAPLIIPLKATSTPVSIKQYPMSQEARLGIKPHIQRLLDQGILVPCQSPWNTPLLPVKKPGTNDYRPVQDLREVNKRVEDIHPTVPNPYNLLSGLPPSHQWYTVLDLKDAFFCLRLHPTSQPLFAFEWRDPEMGISGQLTWTRLPQGFKNSPTLFDEALHRDLADFRIQHPDLILLQYVDDLLLAATSELDCQQGTRALLQTLGNLGYRASAKKAQICQKQVKYLGYLLKEGQRWLTEARKETVMGQPTPKTPRQLREFLGTAGFCRLWIPGFAEMAAPLYPLTKTGTLFNWGPDQQKAYQEIKQALLTAPALGLPDLTKPFELFVDEKQGYAKGVLTQKLGPWRRPVAYLSKKLDPVAAGWPPCLRMVAAIAVLTKDAGKLTMGQPLVILAPHAVEALVKQPPDRWLSNARMTHYQALLLDTDRVQFGPVVALNPATLLPLPEEGLQHNCLDILAEAHGTRPDLTDQPLPDADHTWYTNGSSLLQEGQRKAGAAVTTETEVIWAKALPAGTSAQRAELIALTQALKMAEGKKLNVYTDSRYAFATAHIHGEIYRRRGLLTSEGKEIKNKDEILALLKALFLPKRLSIIHCPGHQKGHSAEARGNRMADQAARKAAITETPDTSTLLGSGSSG(SEQ ID NO：1)-HHHHHH(His tag)
The bacteria after vector transformation were first cultured at 37 ℃ for 5 hours to reach an OD600nm of 0.5, after which 0.2% arabinose was added to the bacterial culture to induce protein expression at 25 ℃. After overnight protein induction, bacteria were collected by centrifugation, and the supernatant was incubated with His-agarose beads after sonication to purify M-MLV reverse transcriptase. Finally, the protein was eluted from His-agarose beads using a 200mM Imidazole buffer and normalized to 10 ng/. mu.l for subsequent assay of reverse transcriptase enzyme activity.
The activity of the reverse transcriptase was tested by one-step RT-PCR as shown in FIG. 1.
RT-PCR reactions were prepared as 10. mu.l mixtures containing 10ng reverse transcriptase, 1ng human 293RNA, 1 Xmod buffer (20mM Tris-HCl, 10mM (NH)4)2SO4)、10mM KCl、2mM MgSO40.1% Triton-X100, 80mM Tris-Acetate, 3mM Mg-Acetate, pH 8.8@25 ℃), 10mM DTT, 0.2. mu.M forward primer (CCCATGTTCGTCATGGGTGT), 0.2. mu.M reverse primer (TGGTCATGAGTCCTTCCACGATA), 0.2. mu.l 10mM dNTP, 25ng Taq polymerase D578R (ref: taq polymerase mutants for faster amplification, U.S. patent No.: #10,865,441). The thermostability was determined by first performing a reverse transcription reaction of the reverse transcriptase mutants at different temperatures (42 ℃, 55 ℃, 65 ℃, 70 ℃ for 5 minutes) and then testing the RT-PCR activity by a PCR step programmed for 35 cycles of 95 ℃ for 3 minutes (95 ℃ for 30 seconds, 60 ℃ for 1 minute).
Compared with the wild type reverse transcriptase, the four RT mutants K15E, D235R, E441K and K445E show higher activity than the wild type M-MLV RT when reverse transcription is carried out at 65 ℃/70 ℃ for 5 minutes. A comparison of RT-PCR activities at 42 ℃, 55 ℃, 65 ℃ and 70 ℃ is shown in FIG. 1. In the same buffer and RT-PCR reaction system, the wild-type reverse transcriptase lost detectable RT-PCR activity at 65 ℃ and 70 ℃, but K15E, D235R, E441K and K445E still retained its activity, with K15E having significantly stronger activity at 65 ℃ relative to other mutants, and also having stronger specificity at 65 ℃ and 70 ℃.
The above-mentioned embodiments only express several embodiments of the present invention, and the description thereof is more specific and detailed, but not construed as limiting the scope of the invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention. Therefore, the protection scope of the patent of the present invention should be subject to the appended claims, and the description and the drawings can be used for explaining the contents of the claims.
SEQUENCE LISTING
<110> Wuhan Ebola Biotech Co., Ltd
<120> thermostable reverse transcriptase mutant
<160> 5
<170> PatentIn version 3.5
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Ser Gly Ser Ser Gly
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<223> K445E mutant
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Pro Val Ala Ala Gly Trp Pro Pro Cys Leu Arg Met Val Ala Ala Ile
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420 425 430
Val Ile Leu Ala Pro His Ala Val Glu Ala Leu Val Glu Gln Pro Pro
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Ala Thr Leu Leu Pro Leu Pro Glu Glu Gly Leu Gln His Asn Cys Leu
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Asp Ile Leu Ala Glu Ala His Gly Thr Arg Pro Asp Leu Thr Asp Gln
500 505 510
Pro Leu Pro Asp Ala Asp His Thr Trp Tyr Thr Asn Gly Ser Ser Leu
515 520 525
Leu Gln Glu Gly Gln Arg Lys Ala Gly Ala Ala Val Thr Thr Glu Thr
530 535 540
Glu Val Ile Trp Ala Lys Ala Leu Pro Ala Gly Thr Ser Ala Gln Arg
545 550 555 560
Ala Glu Leu Ile Ala Leu Thr Gln Ala Leu Lys Met Ala Glu Gly Lys
565 570 575
Lys Leu Asn Val Tyr Thr Asp Ser Arg Tyr Ala Phe Ala Thr Ala His
580 585 590
Ile His Gly Glu Ile Tyr Arg Arg Arg Gly Leu Leu Thr Ser Glu Gly
595 600 605
Lys Glu Ile Lys Asn Lys Asp Glu Ile Leu Ala Leu Leu Lys Ala Leu
610 615 620
Phe Leu Pro Lys Arg Leu Ser Ile Ile His Cys Pro Gly His Gln Lys
625 630 635 640
Gly His Ser Ala Glu Ala Arg Gly Asn Arg Met Ala Asp Gln Ala Ala
645 650 655
Arg Lys Ala Ala Ile Thr Glu Thr Pro Asp Thr Ser Thr Leu Leu Gly
660 665 670
Ser Gly Ser Ser Gly
675
Claims (16)
1. A reverse transcriptase mutant, the amino acid sequence of which is set forth in SEQ ID NO: 1, has one or more of the following mutations:
K15E, D235R, E441K and K445E.
2. The reverse transcriptase mutant of claim 1, wherein the amino acid sequence is selected from the group consisting of SEQ ID NO: 2 to 5.
3. The mutant reverse transcriptase of claim 1 or 2, comprising a tag, preferably a tag fused to the C-terminus of said mutant reverse transcriptase.
4. The reverse transcriptase mutant of claim 3, wherein said tag is any one of an Arg tag, a His tag, a Strep tag, a Flag tag, a T7 tag, a V5-peptide tag, a GST tag and a c-Myc tag.
5. An isolated nucleic acid encoding the reverse transcriptase mutant of any one of claims 1 to 4.
6. A vector comprising the nucleic acid of claim 5.
7. A host cell having incorporated in its genome the nucleic acid of claim 5 or transformed with the vector of claim 6.
8. A method of producing a mutant reverse transcriptase comprising:
a) culturing the host cell of claim 7;
b) expressing the reverse transcriptase mutant; and are
c) Isolating the reverse transcriptase mutant from the host cell.
9. A kit comprising the reverse transcriptase mutant of any one of claims 1 to 4.
10. The kit of claim 9, further comprising one or more of RNA extraction reagents, reverse transcription reaction solution, dNTPs, water, dideoxynucleotides, and reverse transcription primers.
11. A composition obtained by mixing the components of the kit of claim 9 or 10.
12. A method for reverse transcription of one or more nucleic acid molecules, comprising:
i) contacting a template RNA with the reverse transcriptase mutant of any one of claims 1 to 4, and
ii) incubating under a reaction system sufficient to produce DNA complementary to said template RNA.
13. A method for detecting an RNA marker in a sample, comprising:
i) contacting the reverse transcriptase mutant of any one of claims 1 to 4 with an RNA marker;
ii) incubating under a reaction system sufficient to produce DNA complementary to said RNA marker; and
iii) detecting the presence of DNA synthesized in step ii), thereby detecting the RNA marker in the sample.
14. The method according to claim 13, wherein the sample is selected from the group consisting of blood, plasma, serum, urine, bile, cerebrospinal fluid, a swab, a clinical specimen, an organ sample and a tissue sample, and/or wherein the sample is obtained from a cell culture, a source suspected of being contaminated or a subject, in particular wherein the subject is selected from the group consisting of a human, an animal and a plant.
15. The method according to any one of claims 12 to 14, wherein the temperature of the incubation is between 30 ℃ and 70 ℃, preferably between 65 ℃ and 70 ℃.
16. A method for producing a reverse transcriptase having enhanced thermostability, comprising:
introducing a mutation in a nucleic acid encoding an M-MLV reverse transcriptase such that the expression product of said nucleic acid has at least one of the following mutations:
K15E, D235R, E441K and K445E;
the position of the mutation is shown as SEQ ID NO: 1 is referred to as amino acid of M-MLV reverse transcriptase.
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| CN115011578A (en) * | 2022-04-24 | 2022-09-06 | 江南大学 | Enhanced M-MLV reverse transcriptase mutant and application thereof |
| CN116694598A (en) * | 2023-06-30 | 2023-09-05 | 翌圣生物科技(上海)股份有限公司 | Murine leukemia virus reverse transcriptase mutant and application thereof |
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