CN115354036A - Stabilizer of reverse transcriptase - Google Patents
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Abstract
The invention discloses a stabilizer of reverse transcriptase, which comprises acetylated bovine serum albumin and one or more substances selected from monosaccharide, disaccharide or ATP. The stabilizer of the reverse transcriptase can enable the structure of the reverse transcriptase to be more stable at normal temperature, keeps good biological activity, is low in storage cost, can meet the requirement of transportation of the reverse transcriptase at normal temperature, and greatly reduces the risk cost of the reverse transcriptase in the aspect of transportation.
Description
Technical Field
The invention relates to the technical field of molecular diagnosis, in particular to a novel stabilizer of reverse transcriptase.
Background
In 1970 Temin et al discovered reverse transcriptase in oncogenic RNA viruses and suggested that this enzyme is involved in the oncogenic properties of the virus. Reverse transcriptase was later found to be prevalent not only in RNA viruses but also in mammalian embryonic cells and dividing lymphocytes. The discovery of reverse transcriptase indicates that genetic information can be transferred from RNA to DNA. It has facilitated the study of molecular biology, biochemistry and virology and has become a powerful tool for these disciplines.
Reverse transcriptase mainly comprises the following three activities: (1) reverse transcription Activity: and catalyzing dNTP polymerization by taking the RNA as a template to generate a DNA-RNA heteroduplex. (2) RNase H activity: the RNA in the heteroduplex is hydrolyzed to give a single DNA strand (cDNA) complementary to the RNA. (3) DNA-directed DNA polymerase activity: double-stranded DNA is synthesized by using the cDNA single strand as a template and the dNTP as a substrate.
The reverse transcriptases that have been studied at present include reverse transcription of human immunodeficiency virus type 1 (PDB: 1 HMV); reverse transcriptase of Moloney Murine Leukemia Virus (MMLV); avian myeloblastosis virus reverse transcriptase (AMV); telomerase reverse transcriptase enzyme which maintains the presence of chromosomal telomeres in eukaryotic cells. Of these, MMLV and AMV are two reverse transcriptases commonly used in molecular PCR assays, and MMLV is the most common, a recombinant DNA polymerase that synthesizes complementary DNA strands from single-stranded RNA, DNA or RNA-DNA hybridization. Compared to AMV, MMLV lacks endoDNA enzyme activity and RNase H activity is lower.
In recent years, with the development of molecular biology and the expansion of the scientific field, reverse transcriptase has been more remarkably demanded. Because the preservation condition of reverse transcriptase is strict, the preservation temperature of the reverse transcriptase is generally 0-4 ℃, ice crystallization of solute below zero degree can cause concentration of salt, the pH value of the solution is changed, and thereby, the connection between enzyme sulfydryl can be caused to become dimercapto bond, the active center of the enzyme is damaged, and the enzyme is denatured. Therefore, the preservation and transport of reverse transcriptase become more of a concern.
In the prior art, the reverse transcriptase is modified by BSA acetylation, and after the modification, the reaction temperature of the reverse transcriptase can be increased, but the half-life period of the reverse transcriptase is still short, and the thermal stability of the reverse transcriptase cannot meet the use requirement. Therefore, a new treatment method of reverse transcriptase is required to solve the above problems.
Disclosure of Invention
In one aspect of the present invention, a stabilizer for reverse transcriptase is provided to overcome the disadvantage of poor thermostability of reverse transcriptase in the prior art.
The technical scheme provided by the invention is as follows:
a stabilizer of reverse transcriptase contains acetylated bovine serum albumin and one or more substances selected from monosaccharide, disaccharide or ATP.
In the present invention, the inventors have found that the interaction between acetylated bovine serum albumin, one or more substances selected from monosaccharides, disaccharides and ATP, and the reverse transcriptase makes the structure of the reverse transcriptase more stable at room temperature and maintains good biological activity. Preferably, in certain embodiments of the present invention, the monosaccharide is one or more selected from glucose, ribose, fructose, deoxyribose, and galactose, and the disaccharide is one or more selected from maltose, lactose, and sucrose. More preferably, in one embodiment of the present invention, the monosaccharide is dextran, ribose or deoxyribose.
In the present invention, the acetylated bovine serum albumin and the one or more selected from monosaccharides, disaccharides and ATP may be mixed in any suitable ratio. However, in order to achieve better effects, in certain embodiments of the present invention, the mass ratio of the acetylated bovine serum albumin to the one or more selected from monosaccharides, disaccharides and ATP is 10:1 to 100:1.
in the present invention, the acetylated bovine serum albumin may be in the form of a solid or a liquid. When the acetylated bovine serum albumin is a solid powder, it may be dissolved in a suitable solvent in use. Preferably, the solvent is water. More preferably, in a embodiment of the invention, the solvent is water free from nuclease.
To achieve better results, in certain embodiments of the invention, the stabilizing agent for reverse transcriptase may further comprise, for example, but not limited to, buffers, rnase inhibitors, dnase inhibitors, preservatives, pigments, cosolvents, primers, terminators, and the like.
In another aspect of the present invention, there is provided a method for treating a reverse transcriptase, comprising mixing the reverse transcriptase with acetylated bovine serum albumin and one or more of said monosaccharide, disaccharide or ATP in a ratio of 10:1 to 100:1, and mixing and uniformly mixing. The mixing can be achieved by repeated inversion, or by vortex oscillation. Preferably, in an embodiment of the present invention, the acetylated bovine serum albumin is first mixed with the one or more selected from monosaccharides, disaccharides and ATP, and then the mixture is added to the reverse transcriptase and mixed.
Preferably, the reverse transcriptase is human immunodeficiency virus type 1 reverse transcriptase (PDB: 1 HMV), moloney murine leukemia virus reverse transcriptase (MMLV), avian myeloblastosis virus reverse transcriptase (AMV), or telomerase reverse transcriptase which maintains the presence of chromosomal telomeres in eukaryotic cells.
In the present invention, the acetylated bovine serum albumin may be obtained by purchasing commercially available products, or may be prepared by an appropriate method. In some embodiments of the present invention, the preparation method of the acetylated bovine serum albumin comprises: and reacting the bovine serum albumin solution with an acetylation reagent in an alkaline environment, and drying and crushing the product to obtain the powder of the acetylated bovine serum albumin.
Acetylation of proteins refers to a reaction in which an acetyl group of a donor is transferred to a terminal amino acid residue (α amino group) or a lysine residue(s) in a chain of an acceptor protein. The above reaction can be carried out in an alkaline environment. For better results, the alkaline environment may be at a pH of 8.0 to 8.5, which is adjusted with an alkaline agent. Preferably, in certain embodiments of the invention, the alkaline agent is sodium carbonate, sodium bicarbonate, sodium sulfite, sodium hydroxide, potassium hydroxide, sodium acetate, sodium phosphate, sodium hypochlorite, ammonium bicarbonate or calcium carbonate. More preferably, in certain embodiments of the present invention, the alkaline agent is sodium carbonate, sodium bicarbonate or sodium sulfite.
Commonly used acetylating agents include carboxylic acylating agents, anhydride acylating agents and acyl chloride acylating agents. Preferably, in certain embodiments of the invention, the acetylating reagent is acetyl chloride, acetic anhydride, glacial acetic acid, N-methoxydiacetamide or trifluoroacetyl triflate. More preferably, in certain embodiments of the invention, the acetylating agent is acetyl chloride, acetic anhydride or glacial acetic acid.
In the present invention, the inventors also investigated the effect of the addition of different acetylation agents on the degree of acetylation of bovine serum albumin and on the effectiveness of the reverse transcriptase stabilizer of the present invention. The inventor creatively finds that the acetylation degree is improved along with the increase of the dosage of the acetylation reagent, and the comprehensive evaluation value of the functionality of the acetylated bovine serum albumin is increased rapidly and then decreased slowly. Therefore, in order to obtain better effect, in certain embodiments of the invention, when the acetylation reagent is acetic anhydride, the addition amount of acetic anhydride is 5-10% of the mass of the bovine serum albumin; when the acetylation reagent is acetyl chloride, the addition amount of the acetyl chloride is 2-7% of the mass of the bovine serum albumin; when the acetylation reagent is glacial acetic acid, the addition amount of the glacial acetic acid is 5-13% of the mass of the bovine serum albumin.
In the present invention, the reaction temperature of the acetylation reaction may be in the range of-10 ℃ to 100 ℃. Preferably, in certain embodiments of the present invention, the reaction temperature of the acetylation reaction may be 0 ℃ to 40 ℃.
In some embodiments of the present invention, the obtained acetylated bovine serum albumin may be in a solution state or a solid powder state.
In another aspect of the present invention, a kit is provided, which comprises a reverse transcriptase and the above-mentioned stabilizer. In addition, in certain embodiments of the invention, the kit may further comprise one or more nucleotides, one or more DNA polymerases, one or more buffers, one or more primers, or one or more terminators. In certain embodiments of the invention, the kit may further comprise a lysis solution.
In another aspect, the invention provides a use of the reverse transcriptase stabilizer or the kit in preparation of a novel coronavirus (COVID-19) detection product.
The invention has the beneficial effects that:
the stabilizer of the reverse transcriptase can enable the structure of the reverse transcriptase to be more stable at normal temperature, keeps good biological activity, is low in storage cost, can meet the requirement of transportation of the reverse transcriptase at normal temperature, and greatly reduces the risk cost of the reverse transcriptase in the aspect of transportation.
Drawings
FIG. 1 is a graph showing the sensitivity test curve of a novel coronavirus (2019-nCoV) nucleic acid detection by an MMLV enzyme stored at-20 ℃ in an example of the present invention, wherein the threshold is set according to the principle that the threshold line just exceeds the highest point of a fluorescence curve for negative quality control quality detection;
FIG. 2 is a graph showing the sensitivity test curve of the MMLV enzyme stored at normal temperature for detecting the nucleic acid of the novel coronavirus (2019-nCoV) in the example of the present invention;
FIG. 3 is a graph showing the sensitivity test curve of the MMLV enzyme for detecting novel coronavirus (2019-nCoV) nucleic acid after being stored for two weeks at normal temperature in the example of the present invention;
fig. 4A, fig. 4B and fig. 4C are graphs showing the results of the comprehensive evaluation of the effect of different amounts of acetylation reagents on the functionality of acetylated BSA, where the acetylation reagent in fig. 4A is acetic anhydride, the acetylation reagent in fig. 4B is acetyl chloride, and the acetylation reagent in fig. 4C is glacial acetic acid.
Detailed Description
The invention discloses a stabilizer of reverse transcriptase, which can be realized by appropriately improving process parameters by a person skilled in the art by referring to the content. It is expressly intended that all such alterations and modifications which are obvious to those skilled in the art are deemed to be incorporated herein by reference, and that the techniques of the invention may be practiced and applied by those skilled in the art without departing from the spirit, scope and range of equivalents of the invention.
In the present invention, unless otherwise specified, scientific and technical terms used herein have the meanings that are commonly understood by those skilled in the art. Throughout the specification and claims, unless explicitly stated otherwise, the word "comprise", or variations such as "comprises" or "comprising", will be understood to imply the inclusion of a stated element or component but not the exclusion of any other element or component. The terms "such as," "e.g.," and the like are intended to refer to exemplary embodiments and are not intended to limit the scope of the present disclosure.
Some terms appearing in the present invention are explained below.
The term "stabilizer" refers to a material that is added to prevent a change in state or chemical change when the material is produced, placed separately, or stored. Enzyme stability is the ability to retain enzyme activity after storage. This can be determined by measuring the enzyme activity before and after storage to determine how much activity is lost. For practical purposes, the residual activity can be determined by comparing the activity of the stored sample and the frozen reference sample, both samples being analyzed simultaneously to eliminate the daily variations of the analysis.
The term "acetylated bovine serum albumin" (BSA) is a globulin in bovine serum, and BSA is generally used as a stabilizer in a preservation solution and a reaction solution of a restriction enzyme or a modification enzyme because some enzymes are unstable or have low activity at a low concentration. After BSA is added, it may play a role of "protection" or "carrier", and after many enzymes are added, the activity of BSA can be greatly improved. Enzymes that do not require the addition of BSA generally do not suffer. For most substrate DNAs, BSA allows more complete digestion and allows for repeated cleavage. BSA can stabilize the enzyme at 37 ℃ for more than 1h, since many restriction enzymes survive only 10-20min or less at 37 ℃ in BSA-free reaction buffer. While BSA can bind metal ions and other chemicals in the buffer or substrate DNA that inhibit restriction endonuclease activity. BSA is therefore extremely versatile, for example for biochemical, genetic and medical research; can be used as health food and flavoring agent; maintaining osmotic pressure, pH buffering and carrier effect; the stability and the activity of Taq enzyme are facilitated in a PCR system, and the PCR efficiency can be improved. BSA contains 581 amino acid residues containing 35 cysteines constituting 17 disulfide bonds with a free thiol group at position 34 of the peptide chain. These features allow BSA to bind to a wide variety of cations, anions, and other small molecule species. Serum protein in blood plays a role in maintaining osmotic pressure, pH buffering, carrier and the like, and because serum protein is a substance in an organism, the hydrogel prepared from BSA also has good biocompatibility and has good physiological and mechanical protection effects when used as a reverse transcriptase carrier.
The term "reverse transcription" refers to a process of synthesizing DNA using RNA as a template in biology, that is, RNA-directed DNA synthesis. In this process, the process of nucleic acid synthesis and transcription (DNA to RNA) is opposite to the flow direction of genetic information (RNA to DNA), and is called reverse transcription. The reverse transcription process is one of the replicative forms of RNA viruses, and requires catalysis by reverse transcriptase during reverse transcription. Therefore, reverse transcriptase plays an essential role in reverse transcription of RNA. Reverse transcription has been widely used in molecular biology, such as reverse transcription, which mainly extracts total RNA in tissues or cells, reverse transcribes the total RNA into cDNA using reverse transcriptase as a template, and performs PCR amplification using cDNA strands as a template to obtain a large number of copies. The advent of reverse transcription PCR has increased the sensitivity of RNA detection by several orders of magnitude, making possible the analysis of some extremely minute quantities of RNA samples. This greatly improves the efficiency of disease detection and diagnosis in the field of in vitro diagnosis.
The term "protein acetylation" refers to the transfer of acetyl groups of acetyl-CoA to lysine residues of proteins, both histones and other proteins, which are modified by acetylation. Histone acetylation is a dynamic, reversible process, regulated by Histone Acetyltransferase (HAT) and Histone Deacetylase (HDAC). HATs catalyze acetyl groups to be covalently connected with lysine, neutralize positive charge of histone and weaken electrostatic interaction of histone and DNA, so that chromatin is in an open state, transcription factors and RNA polymerase are favorably combined with specific DNA sequences, and gene transcription is activated; in contrast, HDACs catalyze the removal of acetyl groups, resulting in a compact chromosomal structure, thereby inhibiting gene transcription. In mammals, the HATs mainly include the GNAT family, CBP/p300 family and MYST family, and HDACs are classified as Zn 2+ HDAC (histone deacetylase) family and NAD + The Sirtuin (SIRT) -dependent family, the former including type i (HDAC 1/2/3/8), type iia (HDAC 4/5/7/9), type iib (HDAC 6/10), type iv (HDAC 11), the latter including SIRT 1-7 proteins, also known as type iii HDACs. Histone acetylation is one of important apparent regulatory mechanisms, and cells up-regulate gene transcription activity by maintaining histone acetylation state, thereby activating expression of sugar and lipid metabolism genes. In addition to histones, other proteins can be acetylated. Acetylation can regulate metabolism-related transcription factors such as peroxisome proliferator-activated receptor gamma coactivator 1 alpha (PGC-1 alpha), and specifically regulate enzymes in energy metabolism pathways such as glycolysis, gluconeogenesis, tricarboxylic acid cycle and fatty acid oxidation, thereby regulating and controlling cell metabolic activity. In addition, HATs and HDACs can alter metabolic-related signaling pathways, such as insulin signaling pathways, autophagy-related pathways, and thereby regulate nutrient sensing and autophagy. In conclusion, protein acetylation regulates the metabolic activity of intracellular glucose, lipids, amino acids and maintains metabolic homeostasis, so regulating protein acetylation levels is therapeuticMetabolic diseases are of great significance.
The term "polysaccharide" refers to a molecule composed of long chains of monosaccharide units joined together by glycosidic bonds. The structure of the polysaccharide can be linear to strongly branched. Examples include storage polysaccharides such as starch and glycogen; and structural polysaccharides such as cellulose and chitin. Polysaccharides include natural polysaccharides or polysaccharides that have been chemically modified by cross-linking, oxidation, acetylation, partial hydrolysis, and the like.
The term "ambient temperature" refers to the natural temperature without warming or cooling, which may mean, for example, any temperature in the range of about 10 ℃ to 30 ℃ or a temperature around about 25 ℃ or 23 ℃.
In order to make those skilled in the art better understand the technical solutions of the present invention, the present invention is further described in detail with reference to specific embodiments.
Example 1:
acetylation of BSA protein: weighing a certain amount of BSA (10 g) and dissolving in a certain amount of water, adjusting the pH value (8.0) with 1mol/L NaOH, magnetically stirring or electrically stirring, adding acetic anhydride in batches, adjusting the pH value (8.0) with 2mol/L NaOH during the reaction process, centrifuging (3000 r/min,10 min), drying by air blowing (45 ℃), crushing and storing.
Determination of the degree of acylation of BSA: the ninhydrin method Moore and Stein. The degree of acylation is reflected in the absorbance value, with a greater absorbance value indicating a lesser degree of acylation, and the amount is adjusted appropriately to ensure complete acetylation of BSA.
The other reaction conditions were fixed as follows: the reaction temperature is 20 ℃, and the dosage of the acylation reagent is 10 percent of the mass of BSA.
And (3) taking 1g of the acetylated powder obtained in the last step, adding 10mL of glucan, and performing vortex oscillation or full inversion to ensure uniform mixing to obtain a reverse transcriptase stabilizer, and adding a proper amount of reverse transcriptase, and performing full vortex oscillation to obtain the reverse transcriptase mixed enzyme.
With the increase of the using amount of the acetic anhydride, the acetylation degree is improved, and the comprehensive evaluation value of the functionality of the acetylated BSA is increased rapidly and then decreased slowly (when the adding amount of the acetic anhydride is between 5 and 10 percent, the comprehensive evaluation value of the functionality of the product is higher).
Example 2:
acetylation of BSA protein: a defined amount of BSA (10 g) was weighed out and dissolved in a defined amount of water with 1mol/L Na 2 CO 3 Adjusting pH value to 8.0-8.5, magnetically stirring or electrically stirring, adding acetyl chloride in batches, and reacting with 2mol/L Na 2 CO 3 Adjusting pH (8.0-8.5), centrifuging (3000 r/min,10 min), air drying (45 deg.C), pulverizing, and storing.
Determination of the degree of acylation of BSA: the ninhydrin method Moore and Stein. The degree of acylation is reflected in the absorbance value, the greater the absorbance value, the lesser the degree of acylation, and the amount is suitably adjusted to ensure complete acetylation of BSA.
The other reaction conditions were fixed as follows: the reaction temperature was 10 ℃ and the amount of acylating agent was 18% of that of BSA.
And (3) taking 1g of the acetylated powder obtained in the last step, adding 10mL of deoxyribose, performing vortex oscillation or fully inverting to ensure uniform mixing to obtain a reverse transcriptase stabilizer, and adding a proper amount of reverse transcriptase, and performing full vortex oscillation to obtain the reverse transcription mixed enzyme.
With the increase of the dosage of the acetyl chloride, the degree of acetylation is improved, and the comprehensive evaluation value of the functionality of the acetylated BSA is increased rapidly and then decreased slowly (when the dosage of the acetyl chloride is between 2% and 7%, the comprehensive evaluation value of the functionality of the product is higher).
Example 3:
acetylation of BSA protein: weighing a certain amount of BSA (10 g) and dissolving in a certain amount of water, adjusting the pH value (8.0-8.5) by using 1mol/L KOH, stirring by magnetic force or electric force, adding glacial acetic acid in batches, adjusting the pH value (8.0-8.5) by using 2mol/L KOH in the reaction process, centrifuging (3000 r/min,10 min), drying by air blowing (45 ℃), crushing and storing.
Determination of the degree of acylation of BSA: the ninhydrin method Moore and Stein. The degree of acylation is reflected in the absorbance value, the greater the absorbance value, the lesser the degree of acylation, and the amount is suitably adjusted to ensure complete acetylation of BSA.
The other reaction conditions were fixed as follows: the reaction temperature was 37 ℃ and the amount of acylating agent was 15% of that of BSA.
And taking 1g of the acetylated powder obtained in the last step, adding 10mL of lactose, performing vortex oscillation or full inversion to ensure uniform mixing to obtain a reverse transcriptase stabilizer, and adding a proper amount of reverse transcriptase, and performing full vortex oscillation to obtain the reverse transcriptase mixed enzyme.
With the increase of the dosage of the glacial acetic acid, the acetylation degree is improved, and the comprehensive evaluation value of the functionality of the acetylated BSA is increased rapidly and then decreased slowly (when the dosage of the glacial acetic acid is between 5% and 13%, the comprehensive evaluation value of the functionality of the product is higher).
Example 4: influence of different amounts of acetylation reagent on comprehensive evaluation value of functionality of acetylated BSA
1. 1 mg/mL BSA standard protein solution was prepared: BSA 100 mg was weighed, dissolved with ultra pure water and diluted to 100 mL. The amount of acetic anhydride added is as follows:
the results are shown in FIG. 4A, demonstrating that the overall evaluation value of functionality of the product is higher when the amount of acetic anhydride added is between 5% and 10%.
2. 1 mg/mL BSA standard protein solution was prepared: BSA 100 mg was weighed, dissolved with ultra pure water and diluted to 100 mL. The amount of acetyl chloride added is as follows:
as a result, as shown in FIG. 4B, it was confirmed that when acetyl chloride was added in an amount of 2% to 7%, the overall evaluation value of the functionality of the product was high.
3. 1 mg/mL BSA standard protein solution was prepared: BSA 100 mg was weighed, dissolved with ultra pure water and diluted to 100 mL. The amounts of glacial acetic acid added are as follows:
the results are shown in fig. 4C, which demonstrates that the overall evaluation value of functionality of the product is higher when the amount of glacial acetic acid added is between 5% and 13%.
Experimental example 1: reverse transcriptase cocktail Activity detection
The reverse transcriptase used in the experimental group in this experimental example was the reverse transcriptase cocktail prepared in example 1, and the reverse transcriptase used in the control group was the reverse transcriptase without stabilizer treatment in example 1 (M-MuLV reverse transcriptase, specification: 200U/. Mu.L Guangdong Fengcong biological Co., ltd.). The nucleic acid detection kit used in this experimental example was a novel coronavirus nucleic acid detection kit (novel coronavirus (2019-nCoV) nucleic acid detection kit (fluorescence PCR method)) from beijingtangjie diagnostic reagent ltd.
The RT-qPCR system used in this example is shown in Table 1.
TABLE 1 reaction system (30. Mu.l)
The experimental results are as follows:
the results of the control group are shown in table 2 and fig. 1, the Ct values of the RT enzymes are all below 38 when the RT enzymes are stored in the environment of-20 ℃, which indicates that the experimental verification method is effective and the RT enzymes maintain better activity.
TABLE 2-sensitivity test results for novel coronavirus nucleic acid detection with MMLV enzyme stored at 20 deg.C
When the stabilizer of the embodiment 1 of the invention is used for treating RT enzyme, the Ct value can still be controlled within an effective range when the RT enzyme is stored at normal temperature and a novel coronavirus (2019-nCoV) nucleic acid detection sensitivity test is carried out. The results are shown in table 3 and fig. 2.
TABLE 3 results of sensitivity test of novel coronavirus nucleic acid detection by MMLV enzyme stored at Normal temperature
In order to further verify whether the preparation method meets the requirement of normal-temperature transportation or not, the treated reverse transcription mixed enzyme is placed at room temperature in a laboratory for two weeks to be subjected to nucleic acid detection sensitivity test again, and the result shows that the mixed enzyme can still keep good sensitivity, so that the reverse transcription mixed enzyme synthesized by the method improves the thermal stability of the reverse transcription enzyme at room temperature, and provides possibility for long-distance transportation of the reverse transcription enzyme at room temperature. The results are shown in table 4 and fig. 3.
TABLE 4 MMLV enzyme after two weeks storage at ambient temperature
Results of sensitivity test for novel coronavirus nucleic acid detection
The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and amendments can be made without departing from the principle of the present invention, and these modifications and amendments should also be considered as the protection scope of the present invention.
Claims (14)
1. A stabilizer of reverse transcriptase, which is characterized by comprising acetylated bovine serum albumin and one or more substances selected from monosaccharide, disaccharide or ATP.
2. The stabilizer according to claim 1, wherein the monosaccharide is one or more selected from glucose, ribose, fructose, deoxyribose and galactose, and the disaccharide is one or more selected from maltose, lactose and sucrose.
3. The stabilizer of claim 2, wherein the monosaccharide is dextran, ribose, or deoxyribose.
4. The stabilizer according to claim 1, 2 or 3, wherein the mass ratio of the acetylated bovine serum albumin to the one or more selected from monosaccharides, disaccharides or ATP is 10:1 to 100:1.
5. a method for treating a reverse transcriptase, which comprises mixing and homogenizing a reverse transcriptase with acetylated bovine serum albumin and one or more substances selected from monosaccharides, disaccharides and ATP according to the mass ratio as defined in claim 1, 2 or 3.
6. The treatment method according to claim 5, wherein the acetylated bovine serum albumin is mixed with one or more of monosaccharide, disaccharide or ATP, and then added with the reverse transcriptase to be mixed uniformly; the reverse transcriptase is human immunodeficiency virus type 1 reverse transcriptase, moloney murine leukemia virus reverse transcriptase, avian myeloblastosis virus reverse transcriptase or telomerase reverse transcriptase for maintaining existence of chromosome telomere of eukaryotic cell.
7. The process according to claim 5, wherein the acetylated bovine serum albumin is prepared by: and reacting the bovine serum albumin solution with an acetylation reagent in an alkaline environment, and drying and crushing the product to obtain the powder of the acetylated bovine serum albumin.
8. The treatment method according to claim 7, wherein the alkaline environment is at a pH of 8.0 to 8.5, and the pH is adjusted by an alkaline agent.
9. The treatment process according to claim 8, wherein the alkaline agent is sodium carbonate, sodium bicarbonate, sodium sulfite, sodium hydroxide, potassium hydroxide, sodium acetate, sodium phosphate, sodium hypochlorite, ammonium bicarbonate or calcium carbonate.
10. The treatment process according to claim 9, characterized in that the alkaline agent is sodium carbonate, sodium bicarbonate or sodium sulfite.
11. The process according to claim 7, characterized in that the acetylating agent is acetyl chloride, acetic anhydride, glacial acetic acid, N-methoxydiacetamide or trifluoroacetyl triflate.
12. The process according to claim 11, characterized in that the acetylating agent is acetyl chloride, acetic anhydride or glacial acetic acid.
13. The process according to claim 12, wherein when the acetylating agent is acetic anhydride, the amount of the acetic anhydride is 5 to 10% by mass of the bovine serum albumin; when the acetylation reagent is acetyl chloride, the addition amount of the acetyl chloride is 2-7% of the mass of the bovine serum albumin; when the acetylation reagent is glacial acetic acid, the addition amount of the glacial acetic acid is 5-13% of the mass of the bovine serum albumin.
14. A kit comprising a reverse transcriptase and a stabilizer as defined in any one of claims 1 to 4.
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Citations (14)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1996015235A1 (en) * | 1994-11-11 | 1996-05-23 | Asahi Kasei Kogyo Kabushiki Kaisha | Stably preservable reverse transcriptase composition |
| CN1177638A (en) * | 1996-07-25 | 1998-04-01 | 理化学研究所 | Method for increasing activity of enzyme under high temperature |
| CN101591703A (en) * | 2008-11-22 | 2009-12-02 | 中国水产科学研究院黄海水产研究所 | The store method of loop-mediated isothermal amplification reaction reagent mixture |
| CN102242188A (en) * | 2010-05-13 | 2011-11-16 | 上海星汉生物科技有限公司 | Gene diagnosis and detection core reagent transportable at normal temperature and of high sensitivity and high specificity |
| US20120020958A1 (en) * | 2008-12-30 | 2012-01-26 | Dansk Universitet | Fibcd1 for the prevention and treatment of diseases |
| CN103184198A (en) * | 2011-12-30 | 2013-07-03 | 思洛生物技术股份有限公司 | Reagent and method for improving reverse transcriptase performance |
| CN103380770A (en) * | 2013-06-27 | 2013-11-06 | 中国农业科学院特产研究所 | Dilute solution used for preserving racoon dog seminal fluid at normal temperature, and semen collection method |
| RU2012122619A (en) * | 2012-06-01 | 2013-12-10 | Общество с ограниченной ответственностью "ИнтерЛабСервис" | METHOD FOR PRODUCING DRY MIXTURE FOR AMPLICATION OF NUCLEIC ACID AND DRY MIXTURE FOR AMPLIFICATION OF NUCLEIC ACID |
| WO2016033116A1 (en) * | 2014-08-25 | 2016-03-03 | Life Technologies Corporation | Reversibly inactivated thermostable reverse transcriptases, compositions and methods for use |
| CN106906237A (en) * | 2017-04-18 | 2017-06-30 | 淮海工学院 | A kind of preparation method of high-performance M MLV reverse transcriptases |
| CN107365754A (en) * | 2017-09-04 | 2017-11-21 | 广州和实生物技术有限公司 | A kind of lyophilized technique of M MLV reverse transcriptases |
| WO2019030118A1 (en) * | 2017-08-08 | 2019-02-14 | Thermo Fisher Scientific Baltics Uab | Glycerol-free formulations for reverse transcriptases |
| CN110885326A (en) * | 2019-11-18 | 2020-03-17 | 中粮营养健康研究院有限公司 | High water-solubility phenyl acetate compound and carboxylesterase detection kit containing same |
| US20220243180A1 (en) * | 2021-02-04 | 2022-08-04 | Longhorn Vaccines And Diagnostics, Llc | Multipurpose Compositions for Collecting and Transporting Biological Material |
-
2022
- 2022-10-24 CN CN202211298726.9A patent/CN115354036B/en active Active
Patent Citations (14)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1996015235A1 (en) * | 1994-11-11 | 1996-05-23 | Asahi Kasei Kogyo Kabushiki Kaisha | Stably preservable reverse transcriptase composition |
| CN1177638A (en) * | 1996-07-25 | 1998-04-01 | 理化学研究所 | Method for increasing activity of enzyme under high temperature |
| CN101591703A (en) * | 2008-11-22 | 2009-12-02 | 中国水产科学研究院黄海水产研究所 | The store method of loop-mediated isothermal amplification reaction reagent mixture |
| US20120020958A1 (en) * | 2008-12-30 | 2012-01-26 | Dansk Universitet | Fibcd1 for the prevention and treatment of diseases |
| CN102242188A (en) * | 2010-05-13 | 2011-11-16 | 上海星汉生物科技有限公司 | Gene diagnosis and detection core reagent transportable at normal temperature and of high sensitivity and high specificity |
| CN103184198A (en) * | 2011-12-30 | 2013-07-03 | 思洛生物技术股份有限公司 | Reagent and method for improving reverse transcriptase performance |
| RU2012122619A (en) * | 2012-06-01 | 2013-12-10 | Общество с ограниченной ответственностью "ИнтерЛабСервис" | METHOD FOR PRODUCING DRY MIXTURE FOR AMPLICATION OF NUCLEIC ACID AND DRY MIXTURE FOR AMPLIFICATION OF NUCLEIC ACID |
| CN103380770A (en) * | 2013-06-27 | 2013-11-06 | 中国农业科学院特产研究所 | Dilute solution used for preserving racoon dog seminal fluid at normal temperature, and semen collection method |
| WO2016033116A1 (en) * | 2014-08-25 | 2016-03-03 | Life Technologies Corporation | Reversibly inactivated thermostable reverse transcriptases, compositions and methods for use |
| CN106906237A (en) * | 2017-04-18 | 2017-06-30 | 淮海工学院 | A kind of preparation method of high-performance M MLV reverse transcriptases |
| WO2019030118A1 (en) * | 2017-08-08 | 2019-02-14 | Thermo Fisher Scientific Baltics Uab | Glycerol-free formulations for reverse transcriptases |
| CN107365754A (en) * | 2017-09-04 | 2017-11-21 | 广州和实生物技术有限公司 | A kind of lyophilized technique of M MLV reverse transcriptases |
| CN110885326A (en) * | 2019-11-18 | 2020-03-17 | 中粮营养健康研究院有限公司 | High water-solubility phenyl acetate compound and carboxylesterase detection kit containing same |
| US20220243180A1 (en) * | 2021-02-04 | 2022-08-04 | Longhorn Vaccines And Diagnostics, Llc | Multipurpose Compositions for Collecting and Transporting Biological Material |
Non-Patent Citations (4)
| Title |
|---|
| RAMALINGAM N: "Acetylated bovine serum albumin differentially inhibits polymerase chain reaction in microdevices", 《BIOMICROFLUIDICS》 * |
| 姚伟等: "质粒DNA小量提取法的改进", 《应用与环境生物学报》 * |
| 武瑶 等: "UNcle高通量蛋白稳定系统分析3种反转录酶", 《分析仪器》 * |
| 王春等: "一种新的蛋白释放载体――水溶性壳聚糖纳米粒子", 《科学通报》 * |
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