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WO2013097760A1 - Reagent for improving performance of reverse transcriptase - Google Patents

Reagent for improving performance of reverse transcriptase Download PDF

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Publication number
WO2013097760A1
WO2013097760A1 PCT/CN2012/087800 CN2012087800W WO2013097760A1 WO 2013097760 A1 WO2013097760 A1 WO 2013097760A1 CN 2012087800 W CN2012087800 W CN 2012087800W WO 2013097760 A1 WO2013097760 A1 WO 2013097760A1
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Prior art keywords
reverse transcriptase
reagent
reagent according
agent
reducing agent
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PCT/CN2012/087800
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French (fr)
Chinese (zh)
Inventor
杨彩兰
王维
文春描
尹珊珊
万强
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思洛生物技术股份有限公司
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Publication of WO2013097760A1 publication Critical patent/WO2013097760A1/en

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/26—Preparation of nitrogen-containing carbohydrates
    • C12P19/28—N-glycosides
    • C12P19/30—Nucleotides
    • C12P19/34—Polynucleotides, e.g. nucleic acids, oligoribonucleotides
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/12—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
    • C12N9/1241—Nucleotidyltransferases (2.7.7)
    • C12N9/1276—RNA-directed DNA polymerase (2.7.7.49), i.e. reverse transcriptase or telomerase
    • 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
    • C12N9/96—Stabilising an enzyme by forming an adduct or a composition; Forming enzyme conjugates
    • 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

Definitions

  • the present invention is in the field of biotechnology and, in particular, relates to an agent for improving the performance of a reverse transcriptase. Background technology
  • Reverse transcripatase is an enzyme that directs the synthesis of complementary DNA (cDNA) by deoxynucleotide triphosphate by using RNA as a template. It mainly performs the following functions: DNA polymerization relying on RNA or DNA as a template; chain conversion; degradation RNase H function of RNA in RNA-DNA hybrids. Therefore, reverse transcriptase is often used in the construction of cDNA libraries.
  • Both AMV and M-MLV reverse transcriptase have two major activities: RNA-dependent DNA synthetase activity and RNase H activity.
  • RNase H activity leads to a decrease in catalytic efficiency of reverse transcriptase, which is undesirable in the synthesis of full-length cDNA.
  • RNase H and DNA synthetase compete with each other for the hybridization of the RNA template with the DNA primer or the cDNA extension strand, and degrade the RNA strand in the RNA:DNA complex, thereby reducing the yield of cDNA synthesis and increasing the RNA template. The minimum amount.
  • RNA templates that are degraded by RNase H activity can no longer serve as an effective substrate for the synthesis of cDNA, reducing the yield and length of cDNA synthesis.
  • Wild-type M-MLV is generally not suitable for synthesizing full-length cDNAs greater than 5 kb. Attenuating or eliminating the RNase H activity of reverse transcriptase can solve the above problems.
  • M-MLV reverse transcriptase which removes or weakens RNase H activity by genetic recombination technology is most used in the construction of cDNA libraries and synthesis of full-length cDNAs. mutant. It has also been reported in the literature that M-MLV reverse transcriptase with RNase H activity removed can be obtained by PEG modification technique. Non-genetically recombined, non-chemically modified techniques that attenuate or remove the RNase H activity of reverse transcriptase without affecting its RNA-dependent DNA synthetase activity, such as additive technology, lack extensive research results and technical support, Research work still needs to be further developed.
  • the secondary structure of the mRNA molecule itself may cause partial mRNA to be difficult to synthesize the first strand cDNA, thereby affecting the reverse transcription efficiency.
  • these secondary structures can be formed.
  • the formation of RNA secondary structures can be reduced by increasing the temperature of the solution in which the RNA molecules are placed. Therefore, in many cases, it is desirable to perform reverse transcription of RNA under reaction conditions exceeding 37 °C.
  • 37 °C e.g., 50 V
  • a reverse transcription enzyme that increases thermal stability, reduces terminal deoxynucleotidyl transferase activity, and/or increases fidelity by mutation or modification techniques is disclosed in CN01810163.1. The enzyme retained at least 95% of reverse transcriptase activity after heating to 50 °C for 5 min.
  • WO2009125006 discloses a technique for screening proteins, and the reverse transcriptase (Maxima Reverse Transcriptase) obtained by this method retains more than 90% of reverse transcriptase activity after heating to 50 ° C for 60 minutes. Additive technology is not covered in either of the above two patents.
  • a reagent for improving the properties of reverse transcriptase of the present invention which is a thiol blocking agent or a disulfide bond reducing agent or a combination of the two.
  • the sulfhydryl blocking agent is iodoacetic acid, sodium iodoacetate, iodoacetamide, bromoacetic acid, sodium bromoacetate, methyl methylthiosulfonate (Methyl)
  • methanethiosulfonate MMTS
  • N-ethylmaleimide NEM
  • the thiol blocking agent is iodoacetic acid, and the final use concentration is 1-10 mM, more preferably 1-5 mM, and most preferably 2-4 mM.
  • the thiol blocking agent is iodoacetamide in a final concentration of from 0.5 to 50 mM, more preferably from 0.5 to 10 mM, most preferably from 1 to 5 mM.
  • the disulfide bond reducing agent is 2-mercaptoethanol, cysteine hydrochloride, dithiothreitol (DTT), tris(2-carboxyethyl)phosphine.
  • DTT dithiothreitol
  • the disulfide bond reducing agent is dithiothreitol, and the final use concentration is from 1 to 50 mM, more preferably from 1 to 30 mM, and most preferably from 1 to 20 mM.
  • the disulfide bond reducing agent is 2-mercaptoethanol, and the final use concentration is 5-300 mM, more preferably 5-200 mM, and most preferably 30-100 mM.
  • the disulfide bond reducing agent is tris(2-carboxyethyl)phosphine hydrochloride, and the final use concentration is from 0.1 to 20 mM, more preferably from 0.1 to 1 OmM, most preferably from 0.5 to 10 mM.
  • the sulfhydryl blocking agent is iodoacetic acid, sodium iodoacetate, iodoacetamide, bromoacetic acid, sodium bromoacetate, methyl thiosulfonate One or more of N-ethylmaleimide;
  • the disulfide bond reducing agent is 2-mercaptoethanol, cysteine hydrochloride, dithiothreitol, tris(2-carboxyethyl) One or more of phosphonate hydrochloride, mercaptoethylamine hydrochloride, dithioerythritol, and the like.
  • the aforementioned reagent for improving the performance of the reverse transcriptase optionally includes one or more of the following: a buffer, a nonionic detergent, a metal ion, a metal ion chelating agent, a polyol, a polysaccharide, and the like.
  • the polyol is selected from one or more selected from the group consisting of glycerin, mannitol, sorbitol, inositol, mercaptan, polyethylene glycol, xylitol and the like.
  • the polysaccharide is selected from one or more of the group consisting of glucose, dextran, mannose, sucrose, lactose, galactose, fructose, sucrose, trehalose, maltose, inulin, and the like.
  • the present invention also provides a method for improving the performance of reverse transcriptase, which comprises formulating the aforementioned reagent for improving the performance of reverse transcriptase into a mother liquor of a certain concentration, and then mixing the reverse transcriptase solution with the mother liquor to bring the reagent to the final stage. Use concentration.
  • the formulated mother liquor of a certain concentration may be 2-100 times, preferably 2-20 times, more preferably 2-10 times of the final use concentration.
  • the reverse transcriptase is M-MLV (Moloney murine leukemia virus reverse transcriptase), RSV (Lloyd's sarcoma virus reverse transcriptase), RAV (Lloyd's virus reverse transcriptase), AMV (bird amyloplast)
  • M-MLV Moloney murine leukemia virus reverse transcriptase
  • RSV Lioyd's sarcoma virus reverse transcriptase
  • RAV Lloyd's virus reverse transcriptase
  • AMV bird amyloplast
  • leukemia virus HIV (human immunodeficiency virus reverse transcriptase) and the like, preferably M-MLV.
  • the invention further provides a kit comprising the reagent for improving the performance of a reverse transcriptase.
  • RNA or DNA-dependent DNA synthetase activity is increased or significantly improved
  • thiol-blocking agents can inhibit the activity of RNase H enzymes.
  • thiol-blocking agents for inhibiting reverse transcriptase RNase H activity to obtain reverse transcriptase which removes or weakens RNase H activity.
  • the low concentration of reducing agent acts as a protective protein, -SH, which prevents the formation of intramolecular or intermolecular disulfide bonds between the cysteines in the protein.
  • High concentrations of reducing agents can destroy already formed disulfide bonds and even reduce the cysteine residues of most proteins.
  • the reagents and methods of the invention may be used to block the partial thiol group of the reverse transcriptase (eg, iodoacetamide can be thiolated histidine and cysteine), or a portion thereof.
  • the re-formation and arrangement of disulfide bonds may result in a change in the native conformation of the reverse transcriptase, which ultimately results in a change in the properties of the reverse transcriptase.
  • the conformation of the RNase H domain and the synthetase domain of the reverse transcriptase, the conformation of the functional subunit and the structural subunit, and the conformation of the RNase can be achieved by techniques such as genetic recombination and amino acid modification.
  • the conformation of the primer grip structure, the ⁇ -helix and the a-helix, loop structure of the H-active center changes, making the RNase H active center insensitive to the substrate or unable to accurately conform to the substrate, resulting in Reverse transcriptase RNase 11 is down or lost in life (Sharon J. Schultz, James J. Champoux, Virus Research: 134 (2008) 86-103).
  • Optional polyols or (and) polysaccharides increase the stability of the reverse transcriptase.
  • the polyhydroxy compound is capable of forming hydrogen bonds with protein molecules, which facilitates preferential hydration of the protein to form a solvent layer.
  • Sugar is a non-specific stabilizer for proteins.
  • the presence of polysaccharides, especially disaccharides, can affect the refolding process of the protein, thereby increasing the stability of the protein.
  • the protective effect of the polyhydroxy compound and polysaccharide on the protein is related to the selected reagent itself and the type of protein.
  • Optional buffers, nonionic detergents, metal salts (metal ions), metal ion chelators are necessary to maintain reverse transcriptase activity and increase its stability.
  • the reagents of the present invention may be formulated in the form of a mother liquor, diluted at the time of use, or may be prepared in various forms which can be directly mixed with reverse transcriptase.
  • the method for improving the performance of reverse transcriptase provided by the present invention is an additive technique different from genetic recombination technology and chemical modification technology, and the purpose of improving reverse transcriptase performance is mainly achieved by adding a specific chemical reagent.
  • the reagents and methods provided by the invention can effectively improve the performance of reverse transcriptase (such as M-MLV, AMV, HIV, etc.), so that it has stronger extension ability, higher reaction sensitivity, and can be used higher.
  • the reverse transcription temperature overcomes the difficulty of reverse transcription due to the secondary structure of the template RNA and is suitable for constructing a high proportion of full-length cDNA libraries and synthesizing longer cDNAs.
  • Figure ⁇ is the result of 50 °C thermal stability test of M-MLV reverse transcriptase treated with 50 mM DTT in Example 4 of the present invention; wherein, A: M-MLV after 50 mM DTT treatment; B: control, M-MLV (RNase H- ) (other company products); C: Control, M-MLV (Slo Bio Company products); D: Control, M-MLV (other company products).
  • Example 2 is a result of performing first strand cDNA synthesis of M-MLV reverse transcriptase treated with 30 mM DTT in Example 5 of the present invention; wherein, the first strand cDNA corresponding to A, B, and C in the left panel is 9 kb in length, respectively. , 12kb, 14kb. M is a marker; the right panel is a schematic diagram of the length of the synthesized first strand cDNA, and A, B, and C are detection points corresponding to cDNA lengths of 9 kb, 12 kb, and 14 kb, respectively.
  • Fig. 3A and Fig. 3B show the results of an experiment for amplifying HCV genomic RNA by M-MLV reverse transcriptase treated with 30 mM DTT in Example 5; wherein, Fig. 3A is an amplification curve, and Fig. 3B is a linear relationship.
  • Figure 4 is a graph showing the amplification of MS2 genomic RNA by M-MLV reverse transcriptase treated with 60 mM DTT in Example 6 of the present invention at 42 ° C, 45 ° C, 50 ° C, 55 ° C, and 60 ° C, respectively. Results; wherein: A: control, M-MLV (RNase H-) (other company products), B: 60-mM DTT-treated M-MLV. detailed description
  • Wild type M-MLV reverse transcriptase (Silo biological, containing ImM DTT) was added to the following reagent A in a volume ratio of 9:1. After blowing and mixing for a few minutes, the resulting mixture was added to the following reagent B in a volume ratio of 9:1, and mixed by blowing for several minutes.
  • Wild type M-MLV reverse transcriptase (Silo biological, containing 1 mM DTT) was added to the following reagent A in a volume ratio of 9:1. After blowing and mixing for a few minutes, press the volume ratio of 9:1. The mixed solution is added to the following reagent B, and the mixture is blown and mixed for several minutes.
  • Wild type M-MLV reverse transcriptase (Silo biological, containing ImM DTT) was added to the following reagent A in a volume ratio of 4:1, and mixed by pipetting for several minutes.
  • Wild type M-MLV reverse transcriptase (Silo biological, containing ImM DTT) was added to the following reagent at a volume ratio of 9:1, and mixed by pipetting for several minutes.
  • the 50 mM DTT-treated M-MLV sample solution was incubated with wild type M-MLV (Slo Bio and other company products), mutant M-MLV (RNaseH-) (other company products) enzyme solution at 50 ° C for 5 min, The reverse transcriptase RT activity was measured at 30 min, 60 min, 120 min, and 240 min, and the remaining viability was calculated. Among them, two wild-type M-MLV samples and one mutant M-MLV (RNaseH-) sample were used as controls. The results are shown in Figure 1.
  • Example 4 can reduce its RNase H activity and greatly increase its heat resistance at 50 °C without substantially affecting the wild-type M-MLV reverse transcriptase RT activity.
  • Example 5 Reagents for improving reverse transcriptase performance DTT was added to the following reagents and mixed by pipetting for several minutes.
  • the obtained M-MLV enzyme solution was used for two-step RT-PCR, and the first strand cDNA synthesis was carried out according to the experimental protocol provided in the product specification. The test results are shown in Figure 2.
  • the obtained M-MLV enzyme solution was used for two-step RT-qPCR, and the HCV genomic RNA was amplified according to the experimental protocol provided in the product manual. The test results are shown in Figure 3.
  • the M-MLV treated with the reagent of Example 5 was used to amplify HCV genomic RNA, and the amplification efficiency was 95.123%, and R 2 was 1.
  • the results showed that the M-MLV treated by the reagent of Example 5 has high amplification sensitivity and can be used for accurate quantification of RNA molecules.
  • Example 5 can reduce its RNase H activity and increase the length and amplification sensitivity of the synthesized cDNA fragment without affecting the activity of M-MLV reverse transcriptase RT.
  • Wild type M-MLV reverse transcriptase (Silo biological, containing ImM DTT) was added to the following reagent at a volume ratio of 14:1, and mixed by pipetting for several minutes.
  • the M-MLV sample solution treated with the above reagents was subjected to two-step RT-PCR amplification of MS2 genomic RNA at 42 ° C, 45 ° C, 50 ° C, 55 ° C, and 60 ° C, respectively.
  • the amount of template RNA is lpg, and specific primers are used.
  • the control is M-MLV (RNase H-) (other company products). The results are shown in Figure 4.
  • the M-MLV enzyme solution treated with the reagents of the example was amplified by two-step RT-PCR at 42 ° C, 45 ° C, 50 ° C, 55 ° C, and 60 ° C.
  • the MS2 genomic RNA can obtain an amplification product of 2 kb in size.
  • the cDNA yield is much higher than M-MLV (RNase H - ).
  • the reagent for improving the performance of reverse transcriptase has the property of improving reverse transcriptase M-MLV, which has stronger elongation ability and higher reaction sensitivity characteristics, and further tests, results Indicates that the above is used to improve reverse transcriptase performance
  • the reagents are suitable for all reverse transcriptase performance including M-MLV, such as RSV, RAV, AMV, HIV.

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Abstract

Provided in the present invention is a reagent for improving the performance of a reverse transcriptase, and the major constituent of the reagent is a sulfhydryl blocking agent or a disulfide bond-reducing agent or a mixture thereof. Also provided in the present invention is a method for improving the performance of the reverse transcriptase, comprising adding a sulfhydryl blocking agent or a disulfide bond-reducing agent or a mixture thereof to the reverse transcriptase.

Description

一种用于改进逆转录酶性能的试剂  A reagent for improving the performance of reverse transcriptase
技术领域 Technical field
本发明属于生物技术领域， 具体地说， 涉及一种用于改进逆转录 酶性能的试剂。 说 背景技术  The present invention is in the field of biotechnology and, in particular, relates to an agent for improving the performance of a reverse transcriptase. Background technology
逆转录酶（ Reverse transcripatase )是以 RNA为模板， 指导三磷酸 脱氧核苷酸合成互补 DNA ( cDNA ) 的酶， 主要行使以下功能： 依赖 RNA或 DNA为模板的 DNA聚合作用； 链转换作用； 降解 RNA-DNA 杂合体中 RNA的 RNase H功能。 因此逆转录酶常用于 cDNA文库的构 书  Reverse transcripatase is an enzyme that directs the synthesis of complementary DNA (cDNA) by deoxynucleotide triphosphate by using RNA as a template. It mainly performs the following functions: DNA polymerization relying on RNA or DNA as a template; chain conversion; degradation RNase H function of RNA in RNA-DNA hybrids. Therefore, reverse transcriptase is often used in the construction of cDNA libraries.
建。 目前从鸟类成髓细胞白血病病毒（Avian Myeloblastosis Virus , 简 称 AMV )中获得的 AMV逆转录酶以及通过在大肠杆菌 ( E.coli 中重 组表达获得的莫洛尼氏鼠白血病病毒 （ Moloney Murine Leukemia Virus , 简称 M-MLV ) 的 M-MLV逆转录酶是两种最常用的逆转录酶。 build. AMV reverse transcriptase currently obtained from Avian Myeloblastosis Virus (AMV) and Moloney Murine Leukemia Virus obtained by recombinant expression in E. coli M-MLV reverse transcriptase, referred to as M-MLV, is the two most commonly used reverse transcriptases.
无论 AMV还是 M-MLV逆转录酶， 都具有两种主要的活性： RNA 依赖的 DNA合成酶活性和 RNase H活性。 其中， RNase H活性会导致 逆转录酶催化效率降低， 是在合成全长 cDNA中所不希望具有的。 一 方面， RNase H同 DNA合成酶相互竟争 RNA模板与 DNA引物或 cDNA 延伸链间形成的杂合链， 并降解 RNA:DNA复合物中的 RNA链， 从而 降低 cDNA合成的产量， 增加 RNA模板的最小用量。 另一方面， 因 RNase H活性导致降解的 RNA模板不能再作为合成 cDNA的有效底 物， 降低了 cDNA合成的产量和长度。 通常野生型 M-MLV不适用于合 成大于 5kb的全长 cDNA。 减弱或消除逆转录酶的 RNase H活性能够解 决上述问题。  Both AMV and M-MLV reverse transcriptase have two major activities: RNA-dependent DNA synthetase activity and RNase H activity. Among them, RNase H activity leads to a decrease in catalytic efficiency of reverse transcriptase, which is undesirable in the synthesis of full-length cDNA. On the one hand, RNase H and DNA synthetase compete with each other for the hybridization of the RNA template with the DNA primer or the cDNA extension strand, and degrade the RNA strand in the RNA:DNA complex, thereby reducing the yield of cDNA synthesis and increasing the RNA template. The minimum amount. On the other hand, RNA templates that are degraded by RNase H activity can no longer serve as an effective substrate for the synthesis of cDNA, reducing the yield and length of cDNA synthesis. Wild-type M-MLV is generally not suitable for synthesizing full-length cDNAs greater than 5 kb. Attenuating or eliminating the RNase H activity of reverse transcriptase can solve the above problems.
目前， 在构建 cDNA文库和合成全长 cDNA时使用最多的是通过 基因重组技术得到的去除或削弱了 RNase H活性的 M-MLV逆转录酶 突变体。 也有文献报道通过 PEG修饰技术可得到 RNase H活性去除了 的 M-MLV逆转录酶。对于既能减弱或去除逆转录酶的 RNase H活性同 时又不影响其 RNA依赖的 DNA合成酶活性的非基因重组、 非化学修 饰技术， 如添加剂技术还缺乏广泛的研究成果和技术支持， 相关的研 究工作仍需进一步展开。 At present, M-MLV reverse transcriptase which removes or weakens RNase H activity by genetic recombination technology is most used in the construction of cDNA libraries and synthesis of full-length cDNAs. mutant. It has also been reported in the literature that M-MLV reverse transcriptase with RNase H activity removed can be obtained by PEG modification technique. Non-genetically recombined, non-chemically modified techniques that attenuate or remove the RNase H activity of reverse transcriptase without affecting its RNA-dependent DNA synthetase activity, such as additive technology, lack extensive research results and technical support, Research work still needs to be further developed.
此外， 由于 mRNA分子自身的二级结构可能导致部分 mRNA难以 合成第一链 cDNA, 进而影响逆转录效率。 例如， 当 RNA分子的某些 区域具有足够的互补性而发生杂交并形成双链 RNA时，就能形成这些 二级结构。通常，可以通过提高 RNA分子所处溶液的温度来降低 RNA 二级结构的形成。 因此， 在许多情况下， 希望在超过 37 °C的反应条件 下来完成 RNA的逆转录。然而，本领域已知当逆转录酶在超过 37 °C (如 50 V ) 的条件下孵育时通常导致活性丧失。  In addition, the secondary structure of the mRNA molecule itself may cause partial mRNA to be difficult to synthesize the first strand cDNA, thereby affecting the reverse transcription efficiency. For example, when certain regions of the RNA molecule are sufficiently complementary to hybridize and form a double-stranded RNA, these secondary structures can be formed. In general, the formation of RNA secondary structures can be reduced by increasing the temperature of the solution in which the RNA molecules are placed. Therefore, in many cases, it is desirable to perform reverse transcription of RNA under reaction conditions exceeding 37 °C. However, it is known in the art that when reverse transcriptase is incubated at conditions above 37 °C (e.g., 50 V), loss of activity is typically caused.
CN01810163.1中公开了一种通过突变或修饰技术增加了热稳定 性、 降低了末端脱氧核苷酸转移酶活性， 和 /或增加了保真度的逆转 录酶。 该酶在加热至 50 °C , 维持 5min后保留了至少 95%的逆转录酶活 性。 WO2009125006中公开了一种筛选蛋白的技术， 通过该方法得到 的逆转录酶 ( Fermentas产品 Maxima Reverse Transcriptase )在加热至 50 °C , 维持 60min后保留了 90%以上的逆转录酶活性。 以上两篇专利 中均未涉及到添加剂技术。  A reverse transcription enzyme that increases thermal stability, reduces terminal deoxynucleotidyl transferase activity, and/or increases fidelity by mutation or modification techniques is disclosed in CN01810163.1. The enzyme retained at least 95% of reverse transcriptase activity after heating to 50 °C for 5 min. WO2009125006 discloses a technique for screening proteins, and the reverse transcriptase (Maxima Reverse Transcriptase) obtained by this method retains more than 90% of reverse transcriptase activity after heating to 50 ° C for 60 minutes. Additive technology is not covered in either of the above two patents.
由此可见，寻求除常用的基因重组技术之外的可用于改进逆转录 酶性能的方法成为亟待解决的问题。 发明内容  Thus, it has been found that methods other than the commonly used gene recombination techniques for improving the performance of reverse transcriptase have become an urgent problem to be solved. Summary of the invention
本发明的目的是提供一种用于改进逆转录酶性能的试剂。  It is an object of the present invention to provide an agent for improving the performance of reverse transcriptase.
为了实现本发明目的，本发明的一种用于改进逆转录酶性能的试 剂， 所述试剂为巯基封闭剂或二硫键还原剂或二者的组合物。  For the purpose of the present invention, a reagent for improving the properties of reverse transcriptase of the present invention, which is a thiol blocking agent or a disulfide bond reducing agent or a combination of the two.
当所述试剂仅含巯基封闭剂时， 所述巯基封闭剂为碘乙酸、碘乙 酸纳、 碘乙酰胺、 溴乙酸、 溴乙酸纳、 甲基硫代磺酸甲酯 （Methyl methanethiosulfonate , MMTS )、 N-乙基马来酸亚胺( N-ethylmaleimide , NEM ) 等中的一种或多种。 When the reagent contains only a sulfhydryl blocking agent, the sulfhydryl blocking agent is iodoacetic acid, sodium iodoacetate, iodoacetamide, bromoacetic acid, sodium bromoacetate, methyl methylthiosulfonate (Methyl) One or more of methanethiosulfonate, MMTS), N-ethylmaleimide (NEM), and the like.
优选地， 所述巯基封闭剂为碘乙酸， 最终使用浓度为 1-lOmM, 更优选 l-5mM, 最优选 2-4mM。  Preferably, the thiol blocking agent is iodoacetic acid, and the final use concentration is 1-10 mM, more preferably 1-5 mM, and most preferably 2-4 mM.
优选地， 所述巯基封闭剂为碘乙酰胺， 最终使用浓度为 0.5-50mM, 更优选 0.5-10mM, 最优选 l-5mM。  Preferably, the thiol blocking agent is iodoacetamide in a final concentration of from 0.5 to 50 mM, more preferably from 0.5 to 10 mM, most preferably from 1 to 5 mM.
当所述试剂仅含二硫键还原剂时， 所述二硫键还原剂为 2-巯基乙 醇、 盐酸半胱氨酸、 二硫苏糖醇（DTT )、 三 (2-羧乙基)膦盐酸盐、 盐 酸巯基乙胺、 二硫赤藓糖醇等中的一种或多种。  When the reagent contains only a disulfide bond reducing agent, the disulfide bond reducing agent is 2-mercaptoethanol, cysteine hydrochloride, dithiothreitol (DTT), tris(2-carboxyethyl)phosphine. One or more of hydrochloride, mercaptoethylamine, dithioerythritol, and the like.
优选地， 所述二硫键还原剂为二硫苏糖醇， 最终使用浓度为 l-50mM, 更优选 l-30mM, 最优选 l-20mM。  Preferably, the disulfide bond reducing agent is dithiothreitol, and the final use concentration is from 1 to 50 mM, more preferably from 1 to 30 mM, and most preferably from 1 to 20 mM.
优选地， 所述二硫键还原剂为 2-巯基乙醇， 最终使用浓度为 5-300mM, 更优选 5-200mM, 最优选 30-100mM。  Preferably, the disulfide bond reducing agent is 2-mercaptoethanol, and the final use concentration is 5-300 mM, more preferably 5-200 mM, and most preferably 30-100 mM.
优选地， 所述二硫键还原剂为三 (2-羧乙基)膦盐酸盐， 最终使用 浓度为 0.1-20mM, 更优选 0.1-1 OmM, 最优选 0.5-10mM。  Preferably, the disulfide bond reducing agent is tris(2-carboxyethyl)phosphine hydrochloride, and the final use concentration is from 0.1 to 20 mM, more preferably from 0.1 to 1 OmM, most preferably from 0.5 to 10 mM.
当所述试剂为巯基封闭剂和二硫键还原剂的混合物时，所述巯基 封闭剂为碘乙酸、 碘乙酸纳、 碘乙酰胺、 溴乙酸、 溴乙酸纳、 甲基硫 代磺酸甲酯、 N-乙基马来酰亚胺中的一种或多种； 所述二硫键还原剂 为 2-巯基乙醇、 盐酸半胱氨酸、 二硫苏糖醇、 三 (2-羧乙基)膦盐酸盐、 盐酸巯基乙胺、 二硫赤藓糖醇等中的一种或多种。  When the reagent is a mixture of a sulfhydryl blocking agent and a disulfide bond reducing agent, the sulfhydryl blocking agent is iodoacetic acid, sodium iodoacetate, iodoacetamide, bromoacetic acid, sodium bromoacetate, methyl thiosulfonate One or more of N-ethylmaleimide; the disulfide bond reducing agent is 2-mercaptoethanol, cysteine hydrochloride, dithiothreitol, tris(2-carboxyethyl) One or more of phosphonate hydrochloride, mercaptoethylamine hydrochloride, dithioerythritol, and the like.
前述的用于改进逆转录酶性能的试剂中，任选包括以下物质中的 一种或多种： 缓冲剂、非离子型去污剂、金属离子、金属离子螯合剂、 多元醇及多糖等。  The aforementioned reagent for improving the performance of the reverse transcriptase optionally includes one or more of the following: a buffer, a nonionic detergent, a metal ion, a metal ion chelating agent, a polyol, a polysaccharide, and the like.
其中， 所述多元醇选自甘油、 甘露醇、 山梨醇、 肌醇、 硫醇、 聚 乙二醇、 木糖醇等中的一种或多种。  Wherein the polyol is selected from one or more selected from the group consisting of glycerin, mannitol, sorbitol, inositol, mercaptan, polyethylene glycol, xylitol and the like.
所述多糖选自葡萄糖、 葡聚糖、 甘露糖、 蔗糖、 乳糖、 半乳糖、 果糖、 蔗糖、 海藻糖、 麦芽糖、 菊糖等中的一种或多种。 本发明还提供一种改进逆转录酶性能的方法，将前述的用于改进 逆转录酶性能的试剂配制成一定浓度的母液，然后将逆转录酶溶液与 该母液混合， 使所述试剂达到最终使用浓度。 The polysaccharide is selected from one or more of the group consisting of glucose, dextran, mannose, sucrose, lactose, galactose, fructose, sucrose, trehalose, maltose, inulin, and the like. The present invention also provides a method for improving the performance of reverse transcriptase, which comprises formulating the aforementioned reagent for improving the performance of reverse transcriptase into a mother liquor of a certain concentration, and then mixing the reverse transcriptase solution with the mother liquor to bring the reagent to the final stage. Use concentration.
其中， 所配制的一定浓度的母液可以是最终使用浓度的 2-100倍， 优选 2-20倍， 更优选 2- 10倍。  Wherein, the formulated mother liquor of a certain concentration may be 2-100 times, preferably 2-20 times, more preferably 2-10 times of the final use concentration.
所述逆转录酶为 M-MLV (莫洛尼鼠白血病病毒逆转录酶）、 RSV (劳氏肉瘤病毒逆转录酶）、 RAV (劳氏伴随病毒逆转录酶）、 AMV (鸟类成髓细胞白血病病毒）、 HIV (人免疫缺陷病毒逆转录酶） 等 中的一种或多种， 优选 M-MLV。  The reverse transcriptase is M-MLV (Moloney murine leukemia virus reverse transcriptase), RSV (Lloyd's sarcoma virus reverse transcriptase), RAV (Lloyd's virus reverse transcriptase), AMV (bird amyloplast) One or more of leukemia virus, HIV (human immunodeficiency virus reverse transcriptase) and the like, preferably M-MLV.
本发明进一步提供含有所述用于改进逆转录酶性能的试剂的试 剂盒。  The invention further provides a kit comprising the reagent for improving the performance of a reverse transcriptase.
本发明人经过长期的摸索和研究试验，发现将一定浓度的巯基封 闭剂或二硫键还原剂或二者的混合物以适当的比例与逆转录酶溶液 混合后， 可以部分或全面改进逆转录酶的以下各项性能：  After long-term exploration and research experiments, the inventors have found that a certain concentration of sulfhydryl blocking agent or disulfide bond reducing agent or a mixture of the two can be partially or completely improved by reverse transcriptase after mixing with a reverse transcriptase solution in an appropriate ratio. The following properties:
( a ) RNase H活性降低或显著降低；  (a) a decrease or a significant decrease in RNase H activity;
( b ) RNA或 DNA依赖的 DNA合成酶活性耐热性能提高或显著提 高；  (b) RNA or DNA-dependent DNA synthetase activity is increased or significantly improved;
( c )合成的 cDNA更长、 产量更高；  (c) The synthesized cDNA is longer and the yield is higher;
( d ) 灵敏度提高。  (d) Increased sensitivity.
本领域知晓，巯基封闭剂可以抑制 RNase H 酶的活性， 然而未见 将巯基封闭剂用于抑制逆转录酶 RNase H活性以得到去除或削弱了 RNase H活性的逆转录酶的相关报道。  It is known in the art that thiol-blocking agents can inhibit the activity of RNase H enzymes. However, there is no report on the use of thiol-blocking agents for inhibiting reverse transcriptase RNase H activity to obtain reverse transcriptase which removes or weakens RNase H activity.
低浓度的还原剂起保护蛋白 -SH的作用， 可阻止蛋白质中的半胱 氨酸之间形成蛋白质分子内或分子间的二硫键。而高浓度的还原剂可 以破坏已经形成的二硫键甚至可以还原大多数蛋白的半胱氨酸残基。  The low concentration of reducing agent acts as a protective protein, -SH, which prevents the formation of intramolecular or intermolecular disulfide bonds between the cysteines in the protein. High concentrations of reducing agents can destroy already formed disulfide bonds and even reduce the cysteine residues of most proteins.
不希望受理论的约束，釆用本发明的试剂和方法可使逆转录酶部 分巯基被封闭 （如碘乙酰胺可垸基化组氨酸和半胱氨酸）， 或者部分 二硫键重新形成和排布， 可能导致逆转录酶的天然构象被改变， 最终 表现为逆转录酶部分性能被改变。 Without wishing to be bound by theory, the reagents and methods of the invention may be used to block the partial thiol group of the reverse transcriptase (eg, iodoacetamide can be thiolated histidine and cysteine), or a portion thereof. The re-formation and arrangement of disulfide bonds may result in a change in the native conformation of the reverse transcriptase, which ultimately results in a change in the properties of the reverse transcriptase.
已知在现有技术中， 通过基因重组、 氨基酸修饰等技术可使逆转 录酶的 RNase H结构域和合成酶结构域的链接处的构象、 功能亚基与 结构亚基接触面的构象、 RNase H活性中心引物捕获（ primer grip )结 构、 α-螺旋和颈环（a-helix、 loop )结构的构象发生改变， 促使 RNase H活性中心对底物不敏感或无法准确与底物契合， 从而导致逆转录酶 RNase 11活个生下降或丢失 ( Sharon J.Schultz, James J.Champoux, Virus Research: 134(2008)86-103 )。  It is known in the prior art that the conformation of the RNase H domain and the synthetase domain of the reverse transcriptase, the conformation of the functional subunit and the structural subunit, and the conformation of the RNase can be achieved by techniques such as genetic recombination and amino acid modification. The conformation of the primer grip structure, the α-helix and the a-helix, loop structure of the H-active center changes, making the RNase H active center insensitive to the substrate or unable to accurately conform to the substrate, resulting in Reverse transcriptase RNase 11 is down or lost in life (Sharon J. Schultz, James J. Champoux, Virus Research: 134 (2008) 86-103).
任选多元醇或（和）多糖可提高逆转录酶的稳定性。 多羟基化合 物能够与蛋白质分子形成氢键，有助于蛋白质优先水化，形成溶剂层。 糖是蛋白质的非特异性稳定剂。 多糖， 尤其是二糖的存在能够影响蛋 白质的重折叠过程， 从而提高蛋白质的稳定性。 同时， 多羟基化合物 和多糖对蛋白质的保护作用与所选试剂本身及蛋白质的种类有关。  Optional polyols or (and) polysaccharides increase the stability of the reverse transcriptase. The polyhydroxy compound is capable of forming hydrogen bonds with protein molecules, which facilitates preferential hydration of the protein to form a solvent layer. Sugar is a non-specific stabilizer for proteins. The presence of polysaccharides, especially disaccharides, can affect the refolding process of the protein, thereby increasing the stability of the protein. At the same time, the protective effect of the polyhydroxy compound and polysaccharide on the protein is related to the selected reagent itself and the type of protein.
任选缓冲剂、 非离子型去污剂、 金属盐 （金属离子）、 金属离子 螯合剂对于维持逆转录酶活性、 提高其稳定性是必需的。  Optional buffers, nonionic detergents, metal salts (metal ions), metal ion chelators are necessary to maintain reverse transcriptase activity and increase its stability.
根据不同的需要， 本发明涉及的试剂可以配制成母液形式， 在使 用时进行稀释， 也可制备成能够直接与逆转录酶混合的各种形式。  Depending on the needs, the reagents of the present invention may be formulated in the form of a mother liquor, diluted at the time of use, or may be prepared in various forms which can be directly mixed with reverse transcriptase.
本发明的优点在于：  The advantages of the invention are:
(一）本发明提供的改进逆转录酶性能的方法是有别于基因重组 技术和化学修饰技术的一种添加剂技术，主要通过添加特定的化学试 剂来达到改进逆转录酶性能的目的。  (I) The method for improving the performance of reverse transcriptase provided by the present invention is an additive technique different from genetic recombination technology and chemical modification technology, and the purpose of improving reverse transcriptase performance is mainly achieved by adding a specific chemical reagent.
(二） 本发明提供的试剂和方法能有效的改进逆转录酶 （如 M-MLV、 AMV、 HIV等） 的性能， 使之具有更强的延伸能力， 更高 的反应灵敏度， 可以使用较高的反转录温度， 克服了因模板 RNA的二 级结构引起的反转录困难， 适合用于构建高比例的全长 cDNA文库和 合成较长 cDNA。 附图说明 (2) The reagents and methods provided by the invention can effectively improve the performance of reverse transcriptase (such as M-MLV, AMV, HIV, etc.), so that it has stronger extension ability, higher reaction sensitivity, and can be used higher. The reverse transcription temperature overcomes the difficulty of reverse transcription due to the secondary structure of the template RNA and is suitable for constructing a high proportion of full-length cDNA libraries and synthesizing longer cDNAs. DRAWINGS
图丄为本发明实施例 4中以 50mM DTT处理后的 M-MLV逆转录酶 50°C热稳定性试验结果； 其中， A: 50mM DTT处理后的 M-MLV; B: 对照， M-MLV ( RNase H- ) (其他公司产品）； C: 对照， M-MLV (思 洛生物公司产品）； D: 对照， M-MLV (其他公司产品）。  Figure 丄 is the result of 50 °C thermal stability test of M-MLV reverse transcriptase treated with 50 mM DTT in Example 4 of the present invention; wherein, A: M-MLV after 50 mM DTT treatment; B: control, M-MLV (RNase H- ) (other company products); C: Control, M-MLV (Slo Bio Company products); D: Control, M-MLV (other company products).
图 2为本发明实施例 5中用 30mM DTT处理后的 M-MLV逆转录酶 进行第一链 cDNA合成的结果； 其中， 左图中 A、 B、 C对应的第一链 cDNA长度分别为 9kb、 12kb、 14kb。 M为 marker; 右图为合成的第一 链 cDNA长度检测示意图， A、 B、 C为对应 cDNA长度分别为 9kb、 12kb、 14kb的检测点。  2 is a result of performing first strand cDNA synthesis of M-MLV reverse transcriptase treated with 30 mM DTT in Example 5 of the present invention; wherein, the first strand cDNA corresponding to A, B, and C in the left panel is 9 kb in length, respectively. , 12kb, 14kb. M is a marker; the right panel is a schematic diagram of the length of the synthesized first strand cDNA, and A, B, and C are detection points corresponding to cDNA lengths of 9 kb, 12 kb, and 14 kb, respectively.
图 3A和图 3B为本发明实施例 5中用 30mM DTT处理后的 M-MLV 逆转录酶扩增 HCV基因组 RNA的试验结果； 其中， 图 3A为扩增曲线， 图 3B为线性关系。  Fig. 3A and Fig. 3B show the results of an experiment for amplifying HCV genomic RNA by M-MLV reverse transcriptase treated with 30 mM DTT in Example 5; wherein, Fig. 3A is an amplification curve, and Fig. 3B is a linear relationship.
图 4为本发明实施例 6中用 60mM DTT处理后的 M-MLV逆转录酶 分别在 42°C、 45°C、 50°C、 55°C、 60°C下扩增 MS2基因组 RNA的试验 结果； 其中， A: 对照， M-MLV ( RNase H- ) (其他公司产品）， B: 60mM DTT处理后的 M-MLV。 具体实施方式  Figure 4 is a graph showing the amplification of MS2 genomic RNA by M-MLV reverse transcriptase treated with 60 mM DTT in Example 6 of the present invention at 42 ° C, 45 ° C, 50 ° C, 55 ° C, and 60 ° C, respectively. Results; wherein: A: control, M-MLV (RNase H-) (other company products), B: 60-mM DTT-treated M-MLV. detailed description
以下实施例用于说明本发明， 但不用来限制本发明的范围。 若未 特别指明，实施例中所用的技术手段为本领域技术人员所熟知的常规 手段， 所用原料均为巿售商品。  The following examples are intended to illustrate the invention but are not intended to limit the scope of the invention. Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art, and the raw materials used are all commercially available products.
实施例 1 用于改进逆转录酶性能的试剂 Example 1 Reagents for improving reverse transcriptase performance
按 9:1的体积比将野生型 M-MLV逆转录酶 （思洛生物， 含 ImM DTT )加入到下述试剂 A中。 吹打混匀数分钟后， 按 9:1的体积比将所 得混合液加入到下述试剂 B中， 吹打混匀数分钟。  Wild type M-MLV reverse transcriptase (Silo biological, containing ImM DTT) was added to the following reagent A in a volume ratio of 9:1. After blowing and mixing for a few minutes, the resulting mixture was added to the following reagent B in a volume ratio of 9:1, and mixed by blowing for several minutes.
试剂 A的配方：  Reagent A formula:
碘乙酸 50mM Tris-HCl 20mM (pH7.5, 4°C ) Iodoacetic acid 50mM Tris-HCl 20mM (pH 7.5, 4°C)
NaCl lOOmM  NaCl lOOmM
EDTA O.lmM  EDTA O.lmM
Triton X® -100 0.1% (V/V)  Triton X® -100 0.1% (V/V)
甘油 50% (V/V)  Glycerin 50% (V/V)
试剂 B的配方  Reagent B formula
DTT 25mM  DTT 25mM
Tris-HCl 20mM (pH7.5, 4°C )  Tris-HCl 20mM (pH 7.5, 4°C)
NaCl lOOmM  NaCl lOOmM
EDTA O.lmM  EDTA O.lmM
Triton X® -100 0.1% (V/V)  Triton X® -100 0.1% (V/V)
甘油 50% (V/V)  Glycerin 50% (V/V)
以所得酶溶液为样本， 利用荧光实时定量分析系统 （安捷伦， Mx3000p )、实时定量 PCR ( ABI, StepOne™ Real-Time PCR System )、 PCR仪、 电泳仪等仪器， 参照使用说明书， 测定样本溶液的逆转录酶 RT活性和 RNaseH活性， 并计算剩余活力。 结果见表 1。  Using the obtained enzyme solution as a sample, using a real-time quantitative quantitative analysis system (Agilent, Mx3000p), real-time quantitative PCR (ABI, StepOneTM Real-Time PCR System), PCR instrument, electrophoresis instrument, etc., according to the instruction manual, the sample solution is determined. Reverse transcriptase RT activity and RNaseH activity were calculated and residual viability was calculated. The results are shown in Table 1.
由表 1结果可知， M-MLV经过母液浓度为 50mM的碘乙酸处理后， RT活性损失了 20%, RNaseH活性损失了 91%。 该结果表明， 实施例 1 的试剂可以在基本不影响 M-MLV逆转录酶 RT活性的情况下， 显著降 低其 RNaseH活性。  From the results of Table 1, it was found that M-MLV was treated with iodoacetic acid having a mother liquor concentration of 50 mM, and the RT activity was lost by 20%, and the RNaseH activity was lost by 91%. This result indicates that the reagent of Example 1 can significantly reduce its RNaseH activity without substantially affecting the M-MLV reverse transcriptase RT activity.
M-MLVRT, RNaseH剩余活力 碘乙酸终浓度 （mM) 0 5  M-MLVRT, residual activity of RNaseH Final concentration of iodoacetic acid (mM) 0 5
RT剩余活力 （％) 100 80  RT residual energy (%) 100 80
RNaseH剩余活力 （％) 100 9 实施例 2 用于改进逆转录酶性能的试剂  RNaseH Residual Activity (%) 100 9 Example 2 Reagents for improving reverse transcriptase performance
按 9:1的体积比将野生型 M-MLV逆转录酶 （思洛生物， 含 ImM DTT)加入到下述试剂 A中。 吹打混匀数分钟后， 按 9:1的体积比将所 得混合液加入到下述试剂 B中， 吹打混匀数分钟 Wild type M-MLV reverse transcriptase (Silo biological, containing 1 mM DTT) was added to the following reagent A in a volume ratio of 9:1. After blowing and mixing for a few minutes, press the volume ratio of 9:1. The mixed solution is added to the following reagent B, and the mixture is blown and mixed for several minutes.
试剂 A的配方：  Reagent A formula:
碘乙酰胺 40mM  Iodoacetamide 40mM
Tris-HCl 20mM (pH7.5, 4°C )  Tris-HCl 20mM (pH 7.5, 4°C)
NaCl lOOmM  NaCl lOOmM
EDTA O.lmM  EDTA O.lmM
Triton X® -100 0.1% (V/V)  Triton X® -100 0.1% (V/V)
甘油 50% (V/V)  Glycerin 50% (V/V)
试剂 B的配方:  Reagent B formula:
DTT 20mM  DTT 20mM
Tris-HCl 20mM (pH7.5, 4°C )  Tris-HCl 20mM (pH 7.5, 4°C)
NaCl lOOmM  NaCl lOOmM
EDTA O.lmM  EDTA O.lmM
Triton X® -100 0.1% (V/V)  Triton X® -100 0.1% (V/V)
甘油 50% (V/V)  Glycerin 50% (V/V)
以所得酶溶液为样本， 利用荧光实时定量分析系统 （安捷伦， Mx3000p )、实时定量 PCR ( ABI, StepOne™ Real-Time PCR System )、 PCR仪、 电泳仪等仪器， 参照使用说明书， 测定样本溶液的 RT活性和 RNaseH活性， 并计算剩余活力。 结果见表 2。  Using the obtained enzyme solution as a sample, using a real-time quantitative quantitative analysis system (Agilent, Mx3000p), real-time quantitative PCR (ABI, StepOneTM Real-Time PCR System), PCR instrument, electrophoresis instrument, etc., according to the instruction manual, the sample solution is determined. RT activity and RNaseH activity, and calculate residual viability. The results are shown in Table 2.
由表 2结果可知， M-MLV经过母液浓度为 40mM的碘乙酰胺处理 后， RT活性损失了 17%, RNaseH活性全部损失。 该结果表明， 实施 例 2的试剂可以在基本不影响 M-MLV逆转录酶 RT活性的情况下，去除 其 RNaseH活性。  From the results of Table 2, it was found that M-MLV was treated with iodoacetamide having a mother liquor concentration of 40 mM, and the RT activity was lost by 17%, and the RNaseH activity was completely lost. This result indicates that the reagent of Example 2 can remove its RNaseH activity without substantially affecting the M-MLV reverse transcriptase RT activity.
表 2 M-MLVRT, RNaseH剩余活力 碘乙酰胺终浓度 （mM) 0 4  Table 2 M-MLVRT, residual activity of RNaseH Iodine acetamide final concentration (mM) 0 4
RT剩余活力 （％) 100 83  RT residual energy (%) 100 83
RNaseH剩余活力 （％) 100 0 实施例 3 用于改进逆转录酶性能的试剂 RNaseH residual energy (%) 100 0 Example 3 Reagents for improving reverse transcriptase performance
按 4:1的体积比将野生型 M-MLV逆转录酶 （思洛生物， 含 ImM DTT)加入到下述试剂 A中， 吹打混匀数分钟。  Wild type M-MLV reverse transcriptase (Silo biological, containing ImM DTT) was added to the following reagent A in a volume ratio of 4:1, and mixed by pipetting for several minutes.
试剂 A的配方：  Reagent A formula:
碘乙酰胺 25mM  Iodoacetamide 25mM
Tris-HCl 20mM (pH7.5, 4°C )  Tris-HCl 20mM (pH 7.5, 4°C)
NaCl lOOmM  NaCl lOOmM
EDTA O.lmM  EDTA O.lmM
Triton X® -100 0.1% (V/V)  Triton X® -100 0.1% (V/V)
甘油 50% (V/V)  Glycerin 50% (V/V)
以所得酶溶液为样本， 利用荧光实时定量分析系统 （安捷伦， Mx3000p )、实时定量 PCR ( ABI, StepOne™ Real-Time PCR System )、 PCR仪、 电泳仪等仪器， 参照使用说明书， 测定样本溶液的 RT活性和 RNase H活性， 并计算剩余活力。 结果见表 3。  Using the obtained enzyme solution as a sample, using a real-time quantitative quantitative analysis system (Agilent, Mx3000p), real-time quantitative PCR (ABI, StepOneTM Real-Time PCR System), PCR instrument, electrophoresis instrument, etc., according to the instruction manual, the sample solution is determined. RT activity and RNase H activity, and calculate residual viability. The results are shown in Table 3.
由表 3结果可知， M-MLV经过母液浓度为 25mM的碘乙酰胺处理 后， RT活性损失了 48%, RNaseH活性全部损失。 该结果表明， 实施 例 3的试剂可以在损失 M-MLV部分 RT活性的情况下， 去除其 RNase H 活性。  From the results in Table 3, it was found that M-MLV was treated with iodoacetamide having a mother liquor concentration of 25 mM, and the RT activity was lost by 48%, and the RNaseH activity was completely lost. This result indicates that the reagent of Example 3 can remove its RNase H activity while losing the partial RT activity of M-MLV.
M-MLVRT, RNaseH剩余活力 碘乙酰胺终浓度 （mM) 0 5  M-MLVRT, RNaseH Remaining Activity Iodoacetamide Final Concentration (mM) 0 5
RT剩余活力 （％) 100 52  RT Remaining Vitality (%) 100 52
RNaseH剩余活力 （％) 100 0  RNaseH residual energy (%) 100 0
实施例 4 用于改进逆转录酶性能的试剂 Example 4 Reagents for improving reverse transcriptase performance
按 9:1的体积比将野生型 M-MLV逆转录酶 （思洛生物， 含 ImM DTT)加入到下述试剂中， 吹打混匀数分钟。  Wild type M-MLV reverse transcriptase (Silo biological, containing ImM DTT) was added to the following reagent at a volume ratio of 9:1, and mixed by pipetting for several minutes.
按以下配方配制试剂：  Formulate the reagents as follows:
DTT 50mM Tris-HCl 20mM (pH7.5, 4°C ) DTT 50mM Tris-HCl 20mM (pH 7.5, 4°C)
NaCl 150mM  NaCl 150mM
EDTA O.lmM  EDTA O.lmM
Triton X® -100 0.1% (V/V)  Triton X® -100 0.1% (V/V)
甘油 50% (V/V)  Glycerin 50% (V/V)
以所得酶溶液为样本， 利用荧光实时定量分析系统 （安捷伦， Mx3000p )、实时定量 PCR ( ABI, StepOne™ Real-Time PCR System )、 PCR仪、 电泳仪等仪器， 参照使用说明书， 测定样本溶液的 RT活性和 RNaseH活性， 并计算剩余活力。 结果见表 4。  Using the obtained enzyme solution as a sample, using a real-time quantitative quantitative analysis system (Agilent, Mx3000p), real-time quantitative PCR (ABI, StepOneTM Real-Time PCR System), PCR instrument, electrophoresis instrument, etc., according to the instruction manual, the sample solution is determined. RT activity and RNaseH activity, and calculate residual viability. The results are shown in Table 4.
将 50mM DTT处理过的 M-MLV样本溶液与野生型 M-MLV (思洛 生物和其他公司产品）、 突变型 M-MLV (RNaseH-) (其他公司产品 ) 酶溶液 50°C共同孵育 5min、 30min、 60min、 120min、 240min, 检测 逆转录酶 RT活性， 并计算剩余活力。 其中， 以两个野生型 M-MLV样 本和一个突变型 M-MLV (RNaseH-)样本为对照。 结果见图 1。  The 50 mM DTT-treated M-MLV sample solution was incubated with wild type M-MLV (Slo Bio and other company products), mutant M-MLV (RNaseH-) (other company products) enzyme solution at 50 ° C for 5 min, The reverse transcriptase RT activity was measured at 30 min, 60 min, 120 min, and 240 min, and the remaining viability was calculated. Among them, two wild-type M-MLV samples and one mutant M-MLV (RNaseH-) sample were used as controls. The results are shown in Figure 1.
由表 4结果可知， M-MLV经过母液浓度为 50mM的 DTT处理后， RT活性仅损失了 6%, RNase H活性则损失了 41%。  From the results in Table 4, it was found that M-MLV was only treated with DTT at a mother liquor concentration of 50 mM, and the RT activity was only lost by 6%, and the RNase H activity was lost by 41%.
由图 1可知， M-MLV经过母液浓度为 50mM的 DTT处理后， 其耐 热性能大幅提高， 50°C孵育 240min后， RT活性仅损失了 28%, 优于野 生型 M-MLV和突变型 M-MLV ( RNase H- )。  It can be seen from Fig. 1 that the heat resistance of M-MLV after treatment with DTT having a mother liquor concentration of 50 mM is greatly improved. After incubation at 50 ° C for 240 min, the RT activity is only lost by 28%, which is superior to wild type M-MLV and mutant. M-MLV (RNase H- ).
以上该结果表明， 实施例 4的试剂可以在基本不影响野生型 M-MLV逆转录酶 RT活性的前提下， 降低其 RNase H活性， 同时大幅 提高其 50°C耐热性。  The above results indicate that the reagent of Example 4 can reduce its RNase H activity and greatly increase its heat resistance at 50 °C without substantially affecting the wild-type M-MLV reverse transcriptase RT activity.
表 4 M-MLVRT, RNaseH剩余活力  Table 4 M-MLVRT, RNaseH residual vitality
DTT终浓度 （mM) 0 5 DTT final concentration (mM) 0 5
RT剩余活力 （％) 100 94 RT Remaining Vitality (%) 100 94
RNaseH剩余活 （％) 100 59 RNaseH residual activity (%) 100 59
实施例 5 用于改进逆转录酶性能的试剂 DTT )加入到下述试剂中， 吹打混匀数分钟。 Example 5 Reagents for improving reverse transcriptase performance DTT) was added to the following reagents and mixed by pipetting for several minutes.
按以下配方配制试剂：  Formulate the reagents as follows:
DTT 30mM  DTT 30mM
Tris-HCl 20mM ( pH 7.5 , 4°C )  Tris-HCl 20mM (pH 7.5, 4°C)
NaCl lOOmM  NaCl lOOmM
EDTA O.lmM  EDTA O.lmM
Triton X® - 100 0.1% ( v/v )  Triton X® - 100 0.1% ( v/v )
甘油 50% ( V/V )  Glycerol 50% (V/V)
以所得酶溶液为样本， 利用荧光实时定量分析系统 （安捷伦， Mx3000p )、实时定量 PCR ( ABI, StepOne™ Real-Time PCR System )、 PCR仪、 电泳仪等仪器， 按照使用说明书， 测定样本溶液的 RT活性和 RNase H活性， 并计算剩余活力。 结果见表 5。  Using the obtained enzyme solution as a sample, using a real-time quantitative quantitative analysis system (Agilent, Mx3000p), real-time quantitative PCR (ABI, StepOneTM Real-Time PCR System), PCR instrument, electrophoresis instrument, etc., according to the instruction manual, the sample solution is determined. RT activity and RNase H activity, and calculate residual viability. The results are shown in Table 5.
将所得 M-MLV酶液用于两步法 RT-PCR , 按照产品说明书提供的 实验方案进行第一链 cDNA合成。 试验结果见图 2。  The obtained M-MLV enzyme solution was used for two-step RT-PCR, and the first strand cDNA synthesis was carried out according to the experimental protocol provided in the product specification. The test results are shown in Figure 2.
将所得 M-MLV酶液用于两步法 RT-qPCR, 按照产品说明书提供 的实验方案， 扩增 HCV基因组 RNA。 试验结果见图 3。  The obtained M-MLV enzyme solution was used for two-step RT-qPCR, and the HCV genomic RNA was amplified according to the experimental protocol provided in the product manual. The test results are shown in Figure 3.
由表 5结果可知， M-MLV经过实施例试剂处理后， RT活性仅损失 了 11%, RNase H活性则损失了 63%。  From the results in Table 5, it was found that M-MLV lost only 11% of the RT activity and 63% of the RNase H activity after the treatment with the example reagent.
由图 2结果可知， M-MLV经过实施例 5的试剂处理后， 可用于合 成长达 14kb的第一链 cDNA, 且逆转录完全。  As is apparent from the results of Fig. 2, after the M-MLV was treated with the reagent of Example 5, it was used to synthesize the first-strand cDNA of 14 kb, and the reverse transcription was complete.
由图 3结果可知， 将实施例 5的试剂处理过的 M-MLV用于扩增 HCV基因组 RNA, 扩增效率为 95.123%, R2为 1。 结果表明， 经过实 施例 5的试剂处理后的 M-MLV具有高的扩增灵敏度，可以用于 RNA分 子的精确定量。 As is clear from the results of Fig. 3, the M-MLV treated with the reagent of Example 5 was used to amplify HCV genomic RNA, and the amplification efficiency was 95.123%, and R 2 was 1. The results showed that the M-MLV treated by the reagent of Example 5 has high amplification sensitivity and can be used for accurate quantification of RNA molecules.
以上该结果表明， 实施例 5的试剂可以在基本不影响 M-MLV逆转 录酶 RT活性的前提下， 降低其 RNase H活性， 提高其合成 cDNA片段 的长度和扩增灵敏度。 M-MLV RT、 RNase H剩余活力 The above results indicate that the reagent of Example 5 can reduce its RNase H activity and increase the length and amplification sensitivity of the synthesized cDNA fragment without affecting the activity of M-MLV reverse transcriptase RT. M-MLV RT, RNase H residual vitality
DTT终浓度 （ mM ) 0 3DTT final concentration ( mM ) 0 3
RT剩余活力 （％) 100 89RT residual energy (%) 100 89
RNase H剩余活 （％) 100 37 RNase H Remaining Activity (%) 100 37
实施例 6 用于改进逆转录酶性能的试剂 Example 6 Reagents for improving reverse transcriptase performance
按 14:1的体积比将野生型 M-MLV逆转录酶 （思洛生物， 含 ImM DTT )加入到下述试剂中， 吹打混匀数分钟。  Wild type M-MLV reverse transcriptase (Silo biological, containing ImM DTT) was added to the following reagent at a volume ratio of 14:1, and mixed by pipetting for several minutes.
按以下配方配制试剂：  Formulate the reagents as follows:
DTT 60mM  DTT 60mM
Tris-HCl 20mM ( pH 7.5 , 4°C )  Tris-HCl 20mM (pH 7.5, 4°C)
NaCl lOOmM  NaCl lOOmM
EDTA O. lmM  EDTA O. lmM
Triton X® -100 0.1% ( V/V )  Triton X® -100 0.1% ( V/V )
甘油 50% ( V/V )  Glycerol 50% (V/V)
蔗糖 1 % ( W/V )  Sucrose 1 % ( W/V )
将上述试剂处理过的 M-MLV样本溶液分别在 42°C、 45 °C、 50°C、 55°C、 60°C下， 用两步法 RT-PCR扩增 MS2基因组 RNA。 其中， 模板 RNA量为 lpg, 使用特异性引物。 对照为 M-MLV ( RNase H- ) (其他 公司产品）。 结果见图 4。  The M-MLV sample solution treated with the above reagents was subjected to two-step RT-PCR amplification of MS2 genomic RNA at 42 ° C, 45 ° C, 50 ° C, 55 ° C, and 60 ° C, respectively. Among them, the amount of template RNA is lpg, and specific primers are used. The control is M-MLV (RNase H-) (other company products). The results are shown in Figure 4.
如图 4所示， 经过实施例试剂处理后的 M-MLV酶液， 在 42 °C、 45°C、 50°C、 55°C、 60°C下，通过两步法 RT-PCR扩增 MS2基因组 RNA, 均能得到 2kb大小的扩增产物。 且 cDNA产量远高于 M-MLV ( RNase H - )。  As shown in Figure 4, the M-MLV enzyme solution treated with the reagents of the example was amplified by two-step RT-PCR at 42 ° C, 45 ° C, 50 ° C, 55 ° C, and 60 ° C. The MS2 genomic RNA can obtain an amplification product of 2 kb in size. And the cDNA yield is much higher than M-MLV (RNase H - ).
以上实施例仅验证了所述用于改进逆转录酶性能的试剂具有改 进逆转录酶 M-MLV的性能， 使之具有更强的延伸能力， 更高的反应 灵敏度的特性， 经进一步试验， 结果表明所述用于改进逆转录酶性能 的试剂适用于所有包括 M-MLV在内的， 如 RSV、 RAV、 AMV、 HIV 等逆转录酶性能的改进。 The above examples only verified that the reagent for improving the performance of reverse transcriptase has the property of improving reverse transcriptase M-MLV, which has stronger elongation ability and higher reaction sensitivity characteristics, and further tests, results Indicates that the above is used to improve reverse transcriptase performance The reagents are suitable for all reverse transcriptase performance including M-MLV, such as RSV, RAV, AMV, HIV.
虽然，上文中已经用一般性说明及具体实施方案对本发明作了详 尽的描述， 但在本发明基础上， 可以对之作一些修改或改进， 这对本 领域技术人员而言是显而易见的。 因此， 在不偏离本发明精神的基础 上所做的这些修改或改进， 均属于本发明要求保护的范围。  Although the present invention has been described in detail with reference to the preferred embodiments of the present invention, it will be apparent to those skilled in the art. Therefore, such modifications or improvements made without departing from the spirit of the invention are intended to be within the scope of the invention.

Claims

权 利 要 求 书 Claim
1、 一种用于改进逆转录酶性能的试剂， 其特征在于， 所述试剂 为巯基封闭剂或二硫键还原剂或二者的混合物。 An agent for improving the performance of a reverse transcriptase, characterized in that the agent is a sulfhydryl blocking agent or a disulfide bond reducing agent or a mixture of the two.
2、 如权利要求 1所述的试剂， 其特征在于， 所述试剂为巯基封闭 剂。  The reagent according to claim 1, wherein the reagent is a sulfhydryl blocking agent.
3、 如权利要求 2所述的试剂， 其特征在于， 所述巯基封闭剂为碘 乙酸、碘乙酸纳、碘乙酰胺、 溴乙酸、 溴乙酸纳、 甲基硫代磺酸甲酯、 N-乙基马来酰亚胺中的一种或多种。  The reagent according to claim 2, wherein the sulfhydryl blocking agent is iodoacetic acid, sodium iodoacetate, iodoacetamide, bromoacetic acid, sodium bromoacetate, methyl methylthiosulfonate, N- One or more of ethyl maleimide.
4、 如权利要求 3所述的试剂， 其特征在于， 所述巯基封闭剂为碘 乙酸， 最终使用浓度为 l-10mM。  The reagent according to claim 3, wherein the thiol blocking agent is iodoacetic acid, and the final use concentration is l-10 mM.
5、 如权利要求 3所述的试剂， 其特征在于， 所述巯基封闭剂为碘 乙酰胺， 最终使用浓度为 0.5-50mM。  The reagent according to claim 3, wherein the thiol blocking agent is iodoacetamide, and the final use concentration is 0.5-50 mM.
6、 如权利要求 1所述的试剂， 其特征在于， 所述试剂为二硫键还 原剂。  The reagent according to claim 1, wherein the reagent is a disulfide bond reducing agent.
7、 如权利要求 6所述的试剂， 其特征在于， 所述二硫键还原剂为 2-巯基乙醇、 盐酸半胱氨酸、 二硫苏糖醇、 三 (2-羧乙基)膦盐酸盐、 盐酸巯基乙胺、 二硫赤藓糖醇中的一种或多种。  The reagent according to claim 6, wherein the disulfide bond reducing agent is 2-mercaptoethanol, cysteine hydrochloride, dithiothreitol, and tris(2-carboxyethyl)phosphine salt. One or more of an acid salt, mercaptoethylamine hydrochloride, or dithioerythritol.
8、 如权利要求 7所述的试剂， 其特征在于， 所述二硫键还原剂为 二硫苏糖醇， 最终使用浓度为 l-50mM。  The reagent according to claim 7, wherein the disulfide bond reducing agent is dithiothreitol, and the final use concentration is from 1 to 50 mM.
9、 如权利要求 7所述的试剂， 其特征在于， 所述二硫键还原剂为 2-巯基乙醇， 最终使用浓度为 5-300mM。  The reagent according to claim 7, wherein the disulfide bond reducing agent is 2-mercaptoethanol, and the final use concentration is 5-300 mM.
10、 如权利要求 7所述的试剂， 其特征在于， 所述二硫键还原剂 为三 (2-羧乙基)膦盐酸盐， 最终使用浓度为 0.1 -20mM。  The reagent according to claim 7, wherein the disulfide bond reducing agent is tris(2-carboxyethyl)phosphine hydrochloride, and the final use concentration is from 0.1 to 20 mM.
11、 如权利要求 1所述的试剂， 其特征在于， 所述试剂为巯基封 闭剂和二硫键还原剂的混合物。  The reagent according to claim 1, wherein the reagent is a mixture of a sulfhydryl group-blocking agent and a disulfide-bonding reducing agent.
12、 如权利要求 11所述的试剂， 其特征在于， 所述巯基封闭剂为 碘乙酸、 碘乙酸纳、 碘乙酰胺、 溴乙酸、 溴乙酸纳、 甲基硫代磺酸甲 酯、 N-乙基马来酰亚胺中的一种或多种； 所述二硫键还原剂为 2-巯基 乙醇、 盐酸半胱氨酸、 二硫苏糖醇、 三 (2-羧乙基)膦盐酸盐、 盐酸巯 基乙胺、 二硫赤藓糖醇中的一种或多种。 The reagent according to claim 11, wherein the sulfhydryl blocking agent is iodoacetic acid, sodium iodoacetate, iodoacetamide, bromoacetic acid, sodium bromoacetate, methylthiosulfonate One or more of an ester, N-ethylmaleimide; the disulfide bond reducing agent is 2-mercaptoethanol, cysteine hydrochloride, dithiothreitol, and tris(2-carboxyl One or more of phosphonium hydrochloride, mercaptoethylamine hydrochloride, dithioerythritol.
13、 如权利要求 1-12任一项所述的试剂， 其特征在于， 所述试剂 中任选包括以下物质中的一种或多种： 缓冲剂、 非离子型去污剂、 金 属离子、 金属离子螯合剂、 多元醇及多糖。  The reagent according to any one of claims 1 to 12, wherein the reagent optionally comprises one or more of the following: a buffer, a non-ionic detergent, a metal ion, Metal ion chelating agents, polyols and polysaccharides.
14、 如权利要求 13所述的试剂， 其特征在于， 所述多元醇选自甘 油、 甘露醇、 山梨醇、 肌醇、 硫醇、 聚乙二醇、 木糖醇中的一种或多 种。  The reagent according to claim 13, wherein the polyol is one or more selected from the group consisting of glycerin, mannitol, sorbitol, inositol, thiol, polyethylene glycol, and xylitol. .
15、 如权利要求 13所述的试剂， 其特征在于， 所述多糖选自葡萄 糖、 葡聚糖、 甘露糖、 蔗糖、 乳糖、 半乳糖、 果糖、 蔗糖、 海藻糖、 麦芽糖、 菊糖中的一种或多种。  The reagent according to claim 13, wherein the polysaccharide is selected from the group consisting of glucose, dextran, mannose, sucrose, lactose, galactose, fructose, sucrose, trehalose, maltose, and inulin. Kind or more.
16、一种改进逆转录酶性能的方法，其特征在于，将权利要求 1-15 任一项所述的试剂配制成一定浓度的母液，然后将逆转录酶溶液与该 母液混合， 使所述试剂达到最终使用浓度。  A method for improving the performance of a reverse transcriptase, characterized in that the reagent according to any one of claims 1 to 15 is formulated into a mother liquor of a certain concentration, and then the reverse transcriptase solution is mixed with the mother liquor to cause the The reagent reaches the final use concentration.
17、 如权利要求 16所述的方法， 其特征在于， 所述逆转录酶为 M-MLV、 RSV、 RAV、 AMV、 HIV中的一种或多种。  17. The method according to claim 16, wherein the reverse transcriptase is one or more of M-MLV, RSV, RAV, AMV, and HIV.
18、 如权利要求 17所述的方法， 其特征在于， 所述逆转录酶为  18. The method of claim 17, wherein the reverse transcriptase is
19、 含有权利要求 1-15任一项所述用于改进逆转录酶性能的试剂 的试剂盒。 19. A kit comprising an agent for improving reverse transcriptase properties according to any one of claims 1-15.
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