阅读说明：本技术 一种高效热启动Taq酶的优化及制备方法 (Optimization and preparation method of high-efficiency hot start Taq enzyme ) 是由 王河清 王晓敏 张蓉 邱欢欢 李瑞雪 刘中华 王国强 于 2021-07-19 设计创作，主要内容包括：本发明公开了一种高效Taq酶的冻干保护剂,由如下的一种或几种组合形成：10mM～50mM Tris-HCl(Ph 8.0)、5%～30%海藻糖、1%～10%甘露醇、1mg/ml～10mg/ml牛血清白蛋白、5mM～30mM叠氮化钠、0.01%～0.1%吐温20、5%～50%甘油、0.05%～0.5%SE-15、焦磷酸二乙酯处理过的水。本发明对冻干所需的保护剂进行了优化,使冻干后的酶制剂成品性能更加稳定,存储时间更长。(The invention discloses a freeze-drying protective agent for high-efficiency Taq enzyme, which is formed by one or more of the following combinations: 10 mM-50 mM Tris-HCl (Ph 8.0), 5% to 30% trehalose, 1% to 10% mannitol, 1mg/ml to 10mg/ml bovine serum albumin, 5 mM-30 mM sodium azide, 0.01% to 0.1% Tween 20, 5% to 50% glycerol, 0.05% to 0.5% SE-15 and diethyl pyrophosphate treated water. The invention optimizes the protective agent required by freeze-drying, so that the performance of the freeze-dried enzyme preparation finished product is more stable, and the storage time is longer.)

1. A freeze-drying protective agent of high-efficiency Taq enzyme is characterized in that: is formed by one or a combination of the following components: 10 mM-50 mM Tris-HCl (Ph 8.0), 5% to 30% trehalose, 1% to 10% mannitol, 1mg/ml to 10mg/ml bovine serum albumin, 5 mM-30 mM sodium azide, 0.01% to 0.1% Tween 20, 5% to 50% glycerol, 0.05% to 0.5% SE-15 and diethyl pyrophosphate treated water.

2. An enzyme storage buffer comprising: is formed by one or a combination of the following components: 10 mM-50 mM Tris-HCl (Ph 8.0), 0.01 mM-0.5 mM EDTA, 0.1 mM-10 mM DTT, 50 mM-500 mM KCl, 1-3% ammonium sulfate, 1 mM-5 mM sodium azide, 20 mg/ml-100 mg/ml hydroxypropyl-beta-cyclodextrin, 1-5M betaine, 0.1-1% BSA, 50-110 mM PVP, 1-5% potassium citrate, 10% -70% glycerol.

3. A freeze-drying method suitable for high-efficiency hot start Taq enzyme is characterized by comprising the following steps: pre-freezing, sublimation drying and analysis drying;

the pre-freezing setting is as follows: the pre-freezing temperature of the first stage reaches 0-20 ℃ within 10-40 min, and the temperature is kept for 0.5-2 h; the pre-freezing temperature of the second stage reaches-20 ℃ to-50 ℃ within 10min to 50min, and the pre-freezing temperature is kept for 1h to 5 h;

the sublimation drying is set as follows: in the first stage, the sublimation drying temperature reaches minus 45 ℃ to minus 30 ℃ within 5min to 40min, the temperature is maintained for 1 to 3 hours, and the vacuum degree is 0Pa to 30 Pa; the sublimation drying temperature of the second stage reaches-35 ℃ to-20 ℃ within 5min to 40min, the temperature is kept for 5 to 20 hours, and the vacuum degree is 0Pa to 30 Pa;

the analysis drying is set as follows: in the first stage, the resolving and drying temperature reaches 10-20 ℃ within 10-50 min, the temperature is maintained for 1-3 h, and the vacuum degree is 0 Pa; the second stage analysis drying temperature reaches 15-30 ℃ within 10-50 min, the temperature is kept for 2-7 h, and the vacuum degree is 0 Pa.

4. The freeze-drying method suitable for the high-efficiency hot start Taq enzyme according to claim 3, wherein the pre-freezing stage is provided with an annealing operation, and the annealing temperature is up to-30 ℃ within 15min and is kept for 1.5 h.

5. The freeze-drying method for Taq enzyme with high efficiency according to claim 3, wherein inert gas is injected under vacuum state after freeze-drying is finished, and the Taq enzyme is taken out of the box after the inert gas is pressed in the box.

6. The freeze-drying method for high-efficiency hot-start Taq enzyme according to claim 3, wherein the inert gas is one of nitrogen, argon, helium and carbon dioxide.

7. The freeze-drying method suitable for the high-efficiency hot start Taq enzyme according to claim 3, wherein the water content of a freeze-dried preparation finished product obtained by vacuum freeze-drying of the high-efficiency Taq enzyme preparation is 0.1% -5%.

8. The freeze-drying method for high-efficiency hot-start Taq enzyme according to claim 3, wherein 1/3 volumes of freeze-drying protective agent are added in the vacuum freeze-drying process of the high-efficiency hot-start Taq enzyme.

9. An optimized preparation method of hot start Taq enzyme is characterized in that a monoclonal antibody is used for sealing an active center of the Taq enzyme.

10. The optimized preparation method of hot start Taq enzyme according to claim 8, comprising the following steps:

step 1, obtaining Taq enzyme;

and 2, mixing the monoclonal antibody and the obtained Taq enzyme according to a certain molar ratio, and incubating at a preset temperature to form an antigen-antibody complex, thereby obtaining the hot start Taq enzyme.

11. An optimized preparation method of hot start Taq enzyme is characterized in that a chemical group and a chemical group of an active center of the enzyme are utilized to carry out chemical reaction, so that the activity of the enzyme is closed, and the hot start Taq enzyme is obtained; when the temperature rises to a preset value, the chemical bond between the chemical group and the active center of the enzyme is broken, the activity of the enzyme is released, and the aim of hot start is fulfilled.

12. The optimized preparation method of hot-start Taq enzyme according to claim 11, wherein the chemical reagent is one of acetic anhydride, benzoic anhydride, dimethylmaleic anhydride, phthalic anhydride, succinic anhydride, dimethylmaleic anhydride, citraconic anhydride or other reagents having equivalent effects.

13. An optimized preparation method of hot start Taq enzyme is characterized in that a single-chain binding protein gene sequence is connected to the 5' end of a Taq enzyme sequence, and fusion expression is carried out by utilizing escherichia coli to prepare and obtain single-chain binding protein-Taq enzyme recombinant protein; the recombined Taq enzyme can obviously improve the binding capacity between the Taq enzyme and an amplification template, can detect and clone a target gene from a sample with low copy number in a lower cycle number, and has high sensitivity and strong specificity in multiplex PCR detection.

14. A method for preparing a liquid reagent of high-efficiency hot start Taq enzyme, which comprises adding the enzyme storage buffer solution according to claim 2 to the liquid reagent of hot start Taq enzyme.

The technical field is as follows:

the invention belongs to the technical field of biological preparations, and particularly relates to an optimization and preparation method of high-efficiency hot start Taq enzyme.

Background art:

PCR (polymerase chain reaction) is a molecular biology technique for amplifying and amplifying specific DNA fragments, and utilizes the principle that DNA is changed into a single strand at high temperature of 95 ℃ in vitro, a primer is combined with the single strand at low temperature (usually about 60 ℃) according to the principle of base complementary pairing, then the temperature is adjusted to the optimal reaction temperature (about 72 ℃) of DNA polymerase, and the DNA polymerase synthesizes a complementary strand along the direction from phosphoric acid to pentose (5 '-3'). PCR is widely used in medical and biological laboratories, for example, to determine whether a physical examination shows a genetic disease, to diagnose infectious diseases, to diagnose genes, to copy genes, and to identify relatives.

DNA polymerase necessary for PCR reaction is currently widely used as a thermostable DNA polymerase isolated from Thermus Aquaticus (Taq), and thus is called Taq enzyme. Functions to polymerize deoxynucleotides to form a deoxynucleotide chain by constructing a phosphodiester bond, thereby forming a DNA molecule. Taq enzyme has milestone significance for PCR application, common Taq enzyme can be divided into rTaq enzyme and LTaq enzyme, the LTaq enzyme has stronger fidelity and better heat resistance than the rTaq enzyme. Most enzymes are denatured and inactivated at high temperature, however, Taq enzyme can endure high temperature of more than 90 ℃ without inactivation, so that enzyme addition in each cycle is not required, and the PCR technology becomes very simple. Meanwhile, the cost is greatly reduced, and the PCR technology is applied in a large quantity and is gradually applied to clinic. In widely used PCR procedures, Taq enzyme has the disadvantage of lacking 3 to 5 proofreading exonuclease activity, and thus sometimes makes mistakes when replicating DNA, resulting in errors in DNA sequence. In order to avoid the amplification of non-specific sequences in the operation process, researchers have improved the PCR technology and invented Hot start polymerase chain reaction (Hot start PCR). The technology isolates polymerase from a reaction system or uses the polymerase (hot start Taq enzyme) which can be activated under a high-temperature state, and the polymerase can play a good catalytic effect at the temperature of more than 90 ℃ so as to avoid starting reaction before the set temperature is reached.

The current hot start PCR protocol includes: (1) wax isolation method: adding reactants except polymerase into a PCR reaction tube, and adding molten paraffin; after the paraffin is solidified, polymerase is added. After the PCR reaction system is put into a PCR instrument, paraffin melts and floats to the uppermost layer in the temperature rise stage, the polymerase is contacted and mixed with other reactants in the PCR reaction system, and the paraffin also has the effect of preventing evaporation. (2) Chemically modified hot start polymerases: the polymerase is modified by chemical molecules such as formaldehyde and acid anhydride, and is started by the structural change of the polymerase under high heat. (3) Antibody-modified hot start polymerase: the active center is blocked with an immune antibody against a hot-start polymerase, which is initiated by separation of the polymerase from the antibody at high heat. (4) Covalent bond binding modification of hot start polymerase: the active center of the enzyme is blocked with a small molecule compound and the polymerase is initiated by separation from the antibody at high heat.

The information disclosed in this background section is only for enhancement of understanding of the general background of the invention and should not be taken as an acknowledgement or any form of suggestion that this information forms the prior art already known to a person skilled in the art.

The invention content is as follows:

the invention aims to provide an optimization and preparation method of high-efficiency hot start Taq enzyme, thereby overcoming the defects in the prior art.

In order to achieve the aim, the invention provides a freeze-drying protective agent for high-efficiency Taq enzyme, which is formed by one or a combination of several of the following components: 10 mM-50 mM Tris-HCl (Ph 8.0), 5% to 30% trehalose, 1% to 10% mannitol, 1mg/ml to 10mg/ml bovine serum albumin, 5 mM-30 mM sodium azide, 0.01% to 0.1% Tween 20, 5% to 50% glycerol, 0.05% to 0.5% SE-15 and diethyl pyrophosphate treated water. The protective agent required by freeze-drying is optimized, so that the performance of the freeze-dried enzyme preparation finished product is more stable, and the storage time is longer.

An enzyme storage buffer formed from one or a combination of: 10 mM-50 mM Tris-HCl (Ph 8.0), 0.01 mM-0.5 mM EDTA, 0.1 mM-10 mM DTT, 50 mM-500 mM KCl, 1-3% ammonium sulfate, 1 mM-5 mM sodium azide, 20 mg/ml-100 mg/ml hydroxypropyl-beta-cyclodextrin, 1-5M betaine, 0.1-1% BSA, 50-110 mM PVP, 1-5% potassium citrate, 10% -70% glycerol. The enzyme storage buffer solution contains optimized components, so that the performance of the enzyme preparation in a liquid state can be more stable, and the storage time is longer.

A lyophilization process suitable for high efficiency hot start Taq enzyme comprising: pre-freezing, sublimation drying and analysis drying;

the pre-freezing setting is as follows: the pre-freezing temperature of the first stage reaches 0-20 ℃ within 10-40 min, and the temperature is kept for 0.5-2 h; the pre-freezing temperature of the second stage reaches-20 ℃ to-50 ℃ within 10min to 50min, and the pre-freezing temperature is kept for 1h to 5 h;

the sublimation drying is set as follows: in the first stage, the sublimation drying temperature reaches minus 45 ℃ to minus 30 ℃ within 5min to 40min, the temperature is maintained for 1 to 3 hours, and the vacuum degree is 0Pa to 30 Pa; the sublimation drying temperature of the second stage reaches-35 ℃ to-20 ℃ within 5min to 40min, the temperature is kept for 5 to 20 hours, and the vacuum degree is 0Pa to 30 Pa;

the analysis drying setting is as follows: in the first stage, the resolving and drying temperature reaches 10-20 ℃ within 10-50 min, the temperature is maintained for 1-3 h, and the vacuum degree is 0 Pa; the second stage analysis drying temperature reaches 15-30 ℃ within 10-50 min, the temperature is kept for 2-7 h, and the vacuum degree is 0 Pa.

The freeze-drying program parameters are optimized, the properties of the freeze-drying preparation are greatly improved, the stability of the freeze-drying preparation is ensured, and the obtained freeze-drying preparation has an attractive shape. The high-efficiency hot start Taq enzyme preparation is prepared into a freeze-dried preparation through freeze-drying, and the preparation method is simple, strong in specificity, high in sensitivity, good in reproducibility, easy to realize large-scale production and more convenient for customers to use and store.

Preferably, in the technical scheme, an annealing operation is also carried out after the pre-freezing is finished, and the annealing temperature is specifically controlled to reach-30 ℃ within 15min and is kept for 1.5 h. The crystal form and the size of the ice crystal particles can be changed by annealing, the crystallization is strengthened, and the uniformity of the product is improved. The annealing can improve the glass transition temperature of the amorphous phase frozen concentrated solution, has great significance to the sublimation drying stage and the desorption drying stage, and enables the property of the freeze-dried preparation to be more stable. Annealing also directly affects the quality appearance of the lyophilized product, etc.

Preferably, in the above technical solution, after the freeze-drying is finished, inert gas is injected under a vacuum state, and the box is taken out after the box is plugged.

Preferably, in the above technical solution, the inert gas is one of nitrogen, argon, helium, and carbon dioxide.

Preferably, in the technical scheme, the water content of a finished freeze-dried preparation obtained by vacuum freeze-drying of the high-efficiency Taq enzyme preparation is 0.1% -5%. The freeze-dried preparation has low water content, and can prolong the shelf life of the nucleic acid detection reagent. After the high-efficiency hot start Taq enzyme preparation is prepared into a freeze-dried preparation by freeze-drying, the limitation of cold chain transportation can be completely eliminated, and the transportation under the normal temperature condition is realized.

A preparation method of a freeze-drying preparation suitable for high-efficiency hot start Taq enzyme is characterized in that 1/3-volume freeze-drying protective agent is added in the vacuum freeze-drying process of the high-efficiency hot start Taq enzyme.

An optimized preparation method of hot start Taq enzyme is obtained by using a monoclonal antibody to seal an active center of the Taq enzyme. The hot start modification is carried out on the hot start Taq enzyme by monoclonal antibody modification, aptamer modification and single-chain binding protein modification, so that the amplification efficiency of the fluorescent PCR (particularly for a low copy number template) is obviously improved, the perfection of an amplification curve is improved, the stability is good, the repeatability is good, the specificity is strong, and the performance of the fluorescent PCR is more suitable for the market demand.

Preferably, in the above technical solution, the specific steps are as follows:

step 1, obtaining Taq enzyme;

and 2, mixing the monoclonal antibody and the obtained Taq enzyme according to a certain molar ratio, and incubating at a preset temperature to form an antigen-antibody complex, thereby obtaining the hot start Taq enzyme.

An optimized preparation method of hot start Taq enzyme is characterized in that a chemical group and a chemical group of an active center of the enzyme are subjected to chemical reaction, so that the activity of the enzyme is blocked, and the hot start Taq enzyme is obtained; when the temperature rises to a preset value, the chemical bond between the chemical group and the active center of the enzyme is broken, the activity of the enzyme is released, and the aim of hot start is fulfilled.

Preferably, in the above technical solution, the chemical reagent refers to one of acetic anhydride, benzoic anhydride, dimethyl maleic anhydride, phthalic anhydride, succinic anhydride, dimethyl maleic anhydride, citraconic anhydride or other reagents with equivalent effects.

An optimized preparation method of hot start Taq enzyme is characterized in that a single-chain binding protein gene sequence is connected to the 5' end of a Taq enzyme sequence, and fusion expression is carried out by utilizing escherichia coli to prepare a single-chain binding protein-Taq enzyme recombinant protein. The recombined Taq enzyme can obviously improve the binding capacity between the Taq enzyme and an amplification template, can detect and clone a target gene from a sample with low copy number in a lower cycle number, and has high sensitivity and strong specificity in multiplex PCR detection.

The preparation method of the high-efficiency hot start Taq enzyme liquid reagent is characterized in that the enzyme storage buffer solution is added into the hot start Taq enzyme liquid reagent. The liquid reagent of the hot start Taq enzyme has more stable performance and longer storage time. The high-efficiency hot start Taq enzyme has the advantages of good stability, good repeatability, strong specificity and simple preparation method, and can meet the market demand.

Compared with the prior art, the invention has the following beneficial effects:

the high-efficiency hot-start Taq polymerase prepared by the method can effectively avoid the occurrence of nonspecific sequences, improves the reaction specificity, reliability, uniformity and sensitivity of DNA polymerase, improves the amplification efficiency, can detect and clone a target gene from a sample with low copy number at a lower cycle number, can reduce human errors to a great extent, and is used for amplification and detection of conventional PCR reaction, complex templates and the like. The finally obtained high-efficiency hot start Taq enzyme has the advantages of good stability, good repeatability, strong specificity and simple preparation method, and can meet the market demand.

Description of the drawings:

FIG. 1 is a graph showing PCR amplification curves of Taq enzyme stored in optimized screening enzyme storage buffer and Taq enzyme not stored in enzyme storage buffer at room temperature for 24 hours in the examples of the present application;

FIG. 2 is a graph showing PCR amplification curves of Taq enzyme stored in optimized screening enzyme storage buffer and Taq enzyme not stored in enzyme storage buffer at-20 ℃ for 1 year in the examples of the present application;

FIG. 3 is a PCR amplification graph of the monoclonal antibody modified hot start Taq enzyme compared with commercial hot start Taq enzyme under the same test conditions in the example of the present application;

FIG. 4 is a PCR amplification curve chart showing that the monoclonal antibody modified hot start Taq enzyme is stored in a refrigerator at-20 ℃ for one year compared with the commercial hot start Taq enzyme in the embodiment of the present application;

FIG. 5 is a PCR amplification graph of 20 times of freezing and thawing of a monoclonal antibody modified hot start Taq enzyme and a commercial hot start Taq enzyme, respectively, in an embodiment of the present application;

FIG. 6 is a PCR amplification graph of chemically modified hot start Taq enzyme compared to commercial hot start Taq enzyme under the same test conditions in the example of the present application;

FIG. 7.1 amplification curves for FAM channels in multiplex PCR experiments;

FIG. 7.2 amplification curves for VIC channels in multiplex PCR experiments;

FIG. 7.3 amplification curves for ROX channels in multiplex PCR experiments;

FIG. 7.4 amplification curves for CY5 channel in multiplex PCR experiments;

FIG. 8 is a PCR amplification curve diagram of a hot start Taq enzyme lyophilized preparation without freeze-drying treatment placed at room temperature for 2 months and-20 ℃ in the embodiment of the present application under the same test conditions;

FIG. 9 is a PCR amplification graph of a commercial hot start Taq enzyme stored at 45 ℃ for 2 months and-20 ℃ under the same test conditions in a freeze-dried preparation of a hot start Taq enzyme vial in the example of the present application.

The specific implementation mode is as follows:

the following detailed description of specific embodiments of the invention is provided, but it should be understood that the scope of the invention is not limited to the specific embodiments.

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.

Example 1

In this example, a method for the expression and purification of Taq enzyme is provided. The Taq enzyme strains used in this example were owned by the same company.

The method comprises the following specific steps:

(1) preparing LB culture medium, sterilizing and reserving for later use.

(2) 40. mu.L of Taq enzyme strain was inoculated into 40mL of LB medium and activated by culturing overnight at 37 ℃ at 180 r/min. And inoculating 40mL of activated Taq enzyme strain into 4L of LB culture medium for amplification culture on the 2 nd day, adding IPTG (isopropyl-beta-D-thiogalactoside) with the final concentration of 0.5mmol/L when the OD value of the bacterial liquid is 0.6-0.8, carrying out shake culture at 37 ℃ and 180r/min for 8 hours, and inducing the expression of Taq enzyme. Centrifuging to remove supernatant, and collecting the thallus for later use.

(3) Performing purification pretreatment on Taq enzyme: the pellet was resuspended in 100mL of solution A (50 mmol/L Tris-HCl (pH = 8.0), 1mmol/L EDTA, 50mmol/L Dextrose, 0.1 mmol/L DTT) on ice, the cells were disrupted using an ultrasonic cell disrupter, and then centrifuged at 75 ℃ in a water bath for 60min, 8000rpm, 4 ℃ for 20min to remove the pellet and retain the supernatant for use.

(4) Purification of Taq enzyme: the supernatant was filtered through a 0.22 μm filter and the column equilibrated with solution A. The sample is pumped onto a well-balanced chromatography column. The sample was eluted with B fluid (50 mmol/L Tris-HCl (pH = 8.8), 200mmol/L imidazole, 50mmol/L NaCl), and the Taq enzyme sample was collected and placed in a dialysis bag. After dialyzing the resulting solution with solution C (50 mmol/L Tris-HCl (pH = 8.8), 100mmol/L KCl, 0.5mmol/L EDTA (pH = 8.0), 1mmol/L DTT) in a refrigerator at 4 ℃ for about 24 hours, the Taq enzyme sample was filtered through a 0.22 μm filter. Taking out a Taq enzyme sample from the dialysis bag, and desalting the sample by an ion column to obtain the purified Taq enzyme. Storing at-80 deg.C for use.

Example 2

In this example, an enzyme storage buffer for long-term storage of Taq enzyme is provided.

(1) Obtaining the Taq enzyme.

(2) Different groups of enzyme storage buffers were prepared and optimized enzyme storage buffers were selected as shown in table 1 below.

TABLE 1 preparation of different enzyme storage buffers

Group of Number (C)	Enzyme storage buffer composition
		1	20mM Tris-HCl (Ph 8.0), 0.1mM EDTA, 0.5mM DTT, 50mM KCl, 2mM sodium azide, 30mg/ml hydroxypropyl-beta-cyclodextrin Essence, 3M betaine, 0.5% BSA, 50% glycerol, diethyl pyrophosphate treated water.
2	20mM Tris-HCl (Ph 8.0), 0.1mM EDTA, 0.5mM DTT, 50mM KCl, 2% ammonium sulfate, 2mM sodium azide, 3M betaine, 0.5% BSA 60mM PVP, 50% glycerol, diethyl pyrophosphate treated water.
		3	20mM Tris-HCl (Ph 8.0), 0.1mM EDTA, 1mM DTT, 100mM KCl, 2% ammonium sulfate, 2mM sodium azide, 30mg/ml hydroxypropyl- beta-cyclodextrin, 3M betaine, 0.5% BSA, 50% glycerol, diethyl pyrophosphate treated water.
4	20mM Tris-HCl (Ph 8.0), 0.1mM EDTA, 1mM DTT, 100mM KCl, 2% ammonium sulfate, 2mM sodium azide, 3M betaine, 0.5% BSA 1.5 percent of potassium citrate, 50 percent of glycerin and diethyl pyrophosphateTreated water.
		5	20mM Tris-HCl (Ph 8.0), 0.1mM EDTA, 1mM DTT, 100mM KCl, 2% ammonium sulfate, 2mM sodium azide, 30mg/ml hydroxypropyl- beta-cyclodextrin, 3M betaine, 0.5% BSA, 60mM PVP, 1.5% potassium citrate, 50% glycerol, diethyl pyrophosphate treated water.
6	40mM Tris-HCl (Ph 8.0), 0.1mM EDTA, 0.5mM DTT, 50mM KCl, 2mM sodium azide, 30mg/ml hydroxypropyl-beta-cyclodextrin Essence, 3M betaine, 0.5% BSA, 50% glycerol, diethyl pyrophosphate treated water.
		7	40mM Tris-HCl (Ph 8.0), 0.1mM EDTA, 0.5mM DTT, 50mM KCl, 1.5% ammonium sulfate, 2mM sodium azide, 3M betaine, 0.5% BSA, 60mM PVP, 50% glycerol, diethyl pyrophosphate treated water.
8	40mM Tris-HCl (Ph 8.0), 0.1mM EDTA, 1mM DTT, 100mM KCl, 1.5% ammonium sulfate, 2mM sodium azide, 30mg/ml hydroxypropyl- beta-cyclodextrin, 3M betaine, 0.5% BSA, 50% glycerol, diethyl pyrophosphate treated water.
		9	40mM Tris-HCl (Ph 8.0), 0.1mM EDTA, 1mM DTT, 100mM KCl, 1.5% ammonium sulfate, 2mM sodium azide, 3M betaine, 0.5% BSA, 1% potassium citrate, 50% glycerol, diethyl pyrophosphate treated water.
10	40mM Tris-HCl (Ph 8.0), 0.1mM EDTA, 1mM DTT, 100mM KCl, 1.5% ammonium sulfate, 2mM sodium azide, 30mg/mlHydroxypropyl- beta-cyclodextrin, 3M betaine, 0.5% BSA, 60mM PVP, 1% potassium citrate, 50% glycerol, diethyl pyrophosphate treated water.

(3) Taq enzyme was diluted to a final concentration of 2.5U/. mu.L using an enzyme storage buffer, filtered through a sterile 0.22 μm filter, and dispensed into sterile clean centrifuge tubes for storage at-20 ℃.

(4) The screening results for the enzyme storage buffer components were optimized.

The real-time fluorescent quantitative PCR experiment is used for testing under the same experiment condition, and the screened optimized enzyme storage buffer solution component is a group number 4, which comprises the following components: 20mM Tris-HCl (Ph 8.0), 0.1mM EDTA, 1mM DTT, 100mM KCl, 2% ammonium sulfate, 2mM sodium azide, 3M betaine, 0.5% BSA, 1.5% potassium citrate, 50% glycerol.

(5) Long-term storage performance verification of enzyme storage buffer solution

The Taq enzyme stored by the optimized and screened enzyme storage buffer solution and the Taq enzyme not stored by the enzyme storage buffer solution are placed for 2 months at the temperature of 4 ℃, and a real-time fluorescence quantitative PCR experiment is used for testing under the same experiment condition, wherein the PCR amplification result shows that the Taq enzyme not stored by the enzyme storage buffer solution has a fluorescence value which is obviously reduced compared with the PCR amplification curve of the Taq enzyme diluted by the enzyme storage buffer solution when the Taq enzyme not stored by the enzyme storage buffer solution is placed for 2 months at the temperature of 4 ℃, and the result is shown in figure 1.

The Taq enzyme stored by the optimized and screened enzyme storage buffer solution and the Taq enzyme not stored by the enzyme storage buffer solution are placed for 1 year at the temperature of 20 ℃ below zero, and are tested by a real-time fluorescent quantitative PCR experiment under the same experimental condition, and the PCR amplification result shows that the Taq enzyme not stored by the enzyme storage buffer solution is placed for 1 year at the temperature of 20 ℃ below zero, and the fluorescence value of a PCR amplification curve of the Taq enzyme not stored by the enzyme storage buffer solution is obviously reduced compared with that of the Taq enzyme diluted by the enzyme storage buffer solution, and the result is shown in figure 2.

Example 3

In this embodiment, an optimized preparation method of hot-start Taq enzyme is provided, which is obtained by blocking the active center of Taq enzyme with monoclonal antibody. The monoclonal antibody used in this example was purchased from Tongxinjin pharmaceuticals Co., Ltd, Xiamen, and prepared as Taq enzyme in example 1.

The method comprises the following specific steps:

(1) obtaining the Taq enzyme.

(2) Mixing the purchased monoclonal antibody and the obtained Taq enzyme according to the molar ratio of 1:1, 1.2:1, 1.5:1, 1.7:1 and 2:1, and then respectively incubating for 1h at 37 ℃ or 50 ℃ to form an antigen-antibody complex, thereby obtaining the hot start Taq enzyme.

(3) The modified hot start Taq enzyme and the unmodified Taq enzyme are subjected to enzyme activity test by a real-time fluorescent quantitative PCR method: and testing the modified hot start Taq enzyme and the unmodified Taq enzyme under the same experimental condition, and specifically, testing the modified hot start Taq enzyme and the unmodified Taq enzyme under the condition of a template with the same concentration.

The real-time fluorescent quantitative PCR test result shows that: when the monoclonal antibody and the Taq enzyme are mixed according to the molar ratio of 1.5:1, the mixture is incubated for 1h at 50 ℃, the enzyme activity of the obtained hot start Taq enzyme is better, and the CT value and the fluorescence value of the hot start Taq enzyme are obviously improved compared with those of the hot start Taq enzyme and unmodified Taq enzyme obtained by other conditions. Therefore, the hot start Taq enzyme is prepared by mixing the monoclonal antibody and the Taq enzyme according to the molar ratio of 1.5:1 and incubating for 1h at 50 ℃.

(4) The performance test of the hot start Taq enzyme and the commercial hot start Taq enzyme (purchased from Nanjing Nozao Biotechnology Co., Ltd. (product No. P401)) of the present example includes the steps of testing the hot start Taq enzyme and the commercial hot start Taq enzyme under the same experimental condition, and specifically includes the step of testing the hot start Taq enzyme and the commercial hot start Taq enzyme under the condition of the same concentration of template.

The real-time fluorescent quantitative PCR test result shows that: the CT value and the fluorescence value of the hot start Taq enzyme are obviously improved compared with those of the commercial hot start Taq enzyme, the amplification curve is more perfect, and the result is shown in figure 3.

(5) The hot start Taq enzyme of the example was subjected to a cryopreservation stability test.

Putting the hot start Taq enzyme and the commercial hot start Taq enzyme into a refrigerator at the temperature of-20 ℃ and storing for one year; taking out the sample after one year, and carrying out enzyme activity test by a real-time fluorescent quantitative PCR method under the same experimental condition, wherein the results show that: the PCR amplification curve of the hot-start Taq enzyme frozen for one year in this example has a higher fluorescence value and a lower CT value than the PCR amplification curve of the commercial hot-start Taq enzyme frozen for one year, and the results are shown in FIG. 4.

(6) And (3) performing repeated freeze-thaw stability resistance test on the hot start Taq enzyme.

The hot start Taq enzyme and the commercial hot start Taq enzyme of the embodiment are respectively frozen and thawed 20 times under the same test condition, and the enzyme activity test is carried out by a real-time fluorescent quantitative PCR method under the same test condition, and the results show that: the fluorescence value of the PCR amplification curve of the hot-start Taq enzyme after being frozen and thawed for 20 times is higher than that of the PCR amplification curve of the commercial hot-start Taq enzyme after being frozen and thawed for 20 times, the PCR amplification curve is more perfect, and the result is shown in figure 5.

Example 4

In this embodiment, an optimized preparation method of hot-start Taq enzyme is provided, which utilizes some chemical groups to perform chemical reaction with important chemical groups of the active center of the enzyme, thereby blocking the activity of the enzyme and obtaining the hot-start Taq enzyme. When the temperature rises, the chemical bond between the chemical group and the active center of the enzyme is broken, the activity of the enzyme is released, and the purpose of hot start is achieved.

The method comprises the following specific steps:

(1) taq enzyme was obtained and prepared as in example 1.

(2) Hot start Taq enzyme was obtained according to the following Table 2 for different chemical modifications.

TABLE 2 different chemical modification modes

Chemical name	Ratio of Taq enzyme to chemical modifier	Reaction temperature	Reaction time
				Acetic anhydride (1M)	1:50, 1:100, 125, 1:500, 1:750, or 1:1000	4 deg.C, 37 deg.C, 45 deg.C or 55 deg.C	1h, 4h, 8h, 12h, 18 h or 24h
Benzoic anhydride Dimethylmaleic anhydride (1M)	1:50, 1:100, 125, 1:500, 1:750, or 1:1000	4 deg.C, 37 deg.C, 45 deg.C or 55 deg.C	1h, 4h, 8h, 12h, 18 h or 24h
				Phthalic anhydride (1M)	1:50, 1:100, 1250, 1:500, 1:750 or 1:1000	4 deg.C, 37 deg.C, 45 deg.C or 55 deg.C	1h, 4h, 8h, 12h, 18 h or 24h
Succinic anhydride (1M)	1:50, 1:100, 125, 1:500, 1:750, or 1:1000	4 deg.C, 37 deg.C, 45 deg.C or 55 deg.C	1h, 4h, 8h, 12h, 18 h or 24h
				Dimethyl maleic anhydride (1M)	1:50, 1:100, 125, 1:500, 1:750, or 1:1000	4℃37 ℃ and 45 ℃ or 55 DEG C	1h, 4h, 8h, 12h, 18 h or 24h
Citraconic anhydride (1M)	1:50, 1:100, 125, 1:500, 1:750, or 1:1000	4 deg.C, 37 deg.C, 45 deg.C or 55 deg.C	1h, 4h, 8h, 12h, 18 h or 24h

(3) The modified hot start Taq enzyme and the unmodified Taq enzyme are subjected to enzyme activity test by a real-time fluorescent quantitative PCR method: and testing the modified hot start Taq enzyme and the unmodified Taq enzyme in the table under the same experimental condition, and specifically, testing the modified hot start Taq enzyme and the unmodified Taq enzyme under the same high-concentration template and low-concentration template.

The real-time fluorescent quantitative PCR test result shows that: the dimethyl maleic anhydride (1M) and Taq enzyme are uniformly mixed according to the proportion of 1:100, and the PCR amplification effect is better when the mixture reacts for 24 hours at the temperature of 4 ℃.

(4) The performance test of the hot start Taq enzyme and the commercial hot start Taq enzyme (purchased from Nanjing Novozam Biotechnology Co., Ltd. (product No. P401)) in the present example includes the test of the hot start Taq enzyme and the commercial hot start Taq enzyme under the same experimental condition, and specifically includes the test of the hot start Taq enzyme and the commercial hot start Taq enzyme under the same concentration of template.

The real-time fluorescent quantitative PCR test result shows that: the CT value and the fluorescence value of the hot start Taq enzyme of the embodiment are obviously improved compared with those of the commercial hot start Taq enzyme, the amplification curve is more perfect, and the result is shown in figure 6.

Example 5

In this embodiment, an optimized preparation method of hot-start Taq enzyme is provided, in which a single-chain binding protein gene sequence is connected to the 5' end of the Taq enzyme sequence, and fusion expression is performed by using escherichia coli to prepare a single-chain binding protein-Taq enzyme recombinant protein. The recombined Taq enzyme can obviously improve the binding capacity between the Taq enzyme and an amplification template, can detect and clone a target gene from a sample with low copy number in a lower cycle number, and has high sensitivity and strong specificity in multiplex PCR detection.

The method comprises the following specific steps:

(1) the gene sequence of the single-chain binding protein is inquired from an NCBI website, the gene sequence of 1-110 amino acids of the single-chain binding protein is selected as a target gene, and then the target gene sequence is handed over to the company of biological engineering (Shanghai) GmbH for gene synthesis.

(2) The design connects the synthesized target gene to the 5' end of Taq enzyme gene sequence, constructs recombinant plasmid, then transfers the recombinant plasmid into escherichia coli competent cell for fusion expression, and obtains positive single colony of correct expression through colony PCR experiment.

(3) The obtained positive single colony with correct expression is inoculated in a liquid LB culture medium for amplification culture, and purified by the method in the above example 1 to obtain the single-chain binding protein-Taq enzyme recombinant protein, and the single-chain binding protein-Taq enzyme recombinant protein is stored at-80 ℃ for later use.

(4) Single-chain binding protein-Taq enzyme performance stability assay

The performance test of the single-chain binding protein Taq enzyme and the commercial hot start Taq enzyme (purchased from Nanjing Novowed Biotechnology Co., Ltd. (product number P401)) in the multiplex PCR experiment comprises the step of testing the hot start Taq enzyme and the commercial hot start Taq enzyme under the same experiment condition, and specifically comprises the step of testing the hot start Taq enzyme and the commercial hot start Taq enzyme under the condition of the same concentration of template.

The test result of the multiple PCR experiment shows that: the Taq enzyme of the embodiment has obviously improved CT value and fluorescence value compared with the commercial hot start Taq enzyme, the amplification efficiency is high, the competition among primers is small, the generation levels of amplification products of different primers are uniform, and the result is shown in figure 7.

Example 6

In this embodiment, a preparation method of a freeze-dried reagent for high-efficiency hot start of Taq enzyme is provided, and a freeze-dried preparation of a vial of Taq enzyme is prepared by a vacuum freeze-drying method.

The freeze-drying protective agent mixed liquid comprises: 10 mM-50 mM Tris-HCl (Ph 8.0), 5% to 30% trehalose, 1% to 10% mannitol, 1mg/ml to 10mg/ml bovine serum albumin, 5 mM-30 mM sodium azide, 0.01% to 0.1% Tween 20, 5% to 50% glycerol, 0.05% to 0.5% SE-15 and one or more of diethyl pyrophosphate treated water.

The method comprises the following specific steps:

(1) according to the following table 3, freeze-drying protective agent mixed liquor of different groups is prepared, and optimized components of the freeze-drying protective agent are screened out through a vacuum freeze-drying process and later-stage related detection.

TABLE 3 Freeze-drying protective agent mixture preparation for different groups

Group of Number (C)
		1	15mM Tris-HCl (Ph 8.0), 5% trehalose, 5% mannitol, 5mg/ml bovine serum albumin, 10mM sodium azide, 0.05% Tween 20, 0.1% SE-15 and diethyl pyrophosphate treated water
2	15mM Tris-HCl (Ph 8.0), 10% trehalose, 3.5% mannitol, 5mg/ml bovine serum albumin, 10mM sodium azide, 0.05% Tween 20, 0.1% SE-15 and diethyl pyrophosphate treated water.
		3	15mM Tris-HCl (Ph 8.0), 10% trehalose, 3.5% mannoseAlcohol, 5mg/ml bovine serum albumin, 10mM sodium azide, 0.05% Tween 20, 10% glycerol, 0.1% SE-15 and diethyl pyrophosphate treated water.
4	25mM Tris-HCl (Ph 8.0), 5% trehalose, 5% mannitol, 5mg/ml bovine serum albumin, 10mM sodium azide, 0.05% Tween 20, 0.1% SE-15 and diethyl pyrophosphate treated water
		5	25mM Tris-HCl (Ph 8.0), 10% trehalose, 3.5% mannitol, 5mg/ml bovine serum albumin, 10mM sodium azide, 0.05% Tween 20, 0.1% SE-15 and diethyl pyrophosphate treated water.
6	25mM Tris-HCl (Ph 8.0), 10% trehalose, 3.5% mannitol, 5mg/ml bovine serum albumin, 10mM sodium azide, 0.05% Tween 20, 10% glycerol, 0.1% SE-15 and diethyl pyrophosphate treated water.
		7	40mM Tris-HCl (Ph 8.0), 5% trehalose, 5% mannitol, 5mg/ml bovine serum albumin, 10mM sodium azide, 0.05% Tween 20, 0.1% SE-15 and diethyl pyrophosphate treated water.
8	40mM Tris-HCl (Ph 8.0), 10% trehalose, 3.5% mannitol, 5mg/ml bovine serum albumin, 10mM sodium azide, 0.05% Tween 20, 0.1% SE-15 and diethyl pyrophosphate treated water.
		9	40mM Tris-HCl (Ph 8.0), 10% trehalose, 3.5% mannitol, 5mg/ml bovine serum albumin, 10mM sodium azide, 0.05% spitThe temperature is 20 DEG, 10% glycerol, 0.1% SE-15 and diethyl pyrophosphate treated water.

(2) The hot start Taq enzyme modified by the monoclonal antibody and the prepared freeze-drying protective agent mixed solution are mixed according to the proportion of 2:1, mixing uniformly.

(3) And (3) subpackaging the uniformly mixed liquid reagent into penicillin bottles by using an accurate liquid separation system.

(4) And (5) putting the penicillin bottle into a vacuum freeze dryer for freeze drying.

(5) Freeze-drying program parameter setting:

the pre-freezing setting comprises:

in the first stage, the pre-freezing temperature reaches 0 ℃ within 20min, and the temperature is kept for 0.5 h;

the pre-freezing temperature of the second stage reaches-40 ℃ within 30min, and the temperature is kept for 3 h.

The sublimation drying device comprises:

the sublimation drying temperature of the first stage reaches-35 ℃ within 10min, the temperature is kept for 2h, and the vacuum degree is 12 Pa;

the sublimation drying temperature of the second stage reaches-30 ℃ within 20min, the temperature is maintained for 16h, and the vacuum degree is 12 Pa.

The analytical drying setup includes:

in the first stage, the resolving and drying temperature reaches 15 ℃ within 30min, the temperature is kept for 1.5h, and the vacuum degree is 0 Pa;

the second stage analysis drying temperature reaches 25 deg.C within 20min, and is maintained for 5h with vacuum degree of 0 Pa.

(6) After the freeze-drying is finished, flushing nitrogen, pressing a plug in the box, opening the box door of the freeze dryer after the temperature and the humidity of the environment meet the requirements, and taking out the hot-start Taq enzyme penicillin bottle freeze-dried preparation.

(7) Observing the appearance of the freeze-dried reagent, measuring the moisture content of the freeze-dried reagent by using a card type moisture tester, and testing the performance of the freeze-dried reagent by a PCR amplification experiment under the same experiment condition, wherein the specific results are shown in the following table 3:

TABLE 3 appearance and moisture content determination of lyophilized reagents

Group number	Appearance of the product	Determination of moisture content	PCR amplification results
				1	Lyophilized reagent collapse	2.51%	Low fluorescence value of amplification curve and delayed CT value
2	Slight collapse of lyophilized reagents	2.42%	Low fluorescence value of amplification curve
				3	Lyophilized reagent collapse	2.47%	Low fluorescence value of amplification curve and delayed CT value
4	Lyophilized reagent collapse	2.58%	Low fluorescence value of amplification curve
				5	Beautiful and full appearance	2.36%	Perfect amplification curve S and strong specificity
6	Slight collapse of lyophilized reagents	2.45%	Low fluorescence value of amplification curve
				7	Freeze-dried reagent bottom collapse	2.52%	Low fluorescence value of amplification curve
8	Layering of lyophilized reagents	2.54%	Low fluorescence value of amplification curve and delayed CT value
				9	Layering of lyophilized reagents	2.63%	Low fluorescence value of amplification curve and delayed CT value

(8) The screening results for lyophilized excipient components were optimized.

The optimized lyophilized excipient component screened by the above detection is group number 5, which comprises: 25mM Tris-HCl (Ph 8.0), 10% trehalose, 3.5% mannitol, 5mg/ml bovine serum albumin, 10mM sodium azide, 0.05% Tween 20, 0.1% SE-15, and diethyl pyrophosphate treated water.

(9) And sealing the qualified freeze-dried preparation of the hot-start Taq enzyme vial.

(10) Test of stability of freeze-dried preparation in hot-start Taq enzyme vial at room temperature

Placing the freeze-dried preparation of the hot start Taq enzyme vial at room temperature for 3 months, and carrying out a PCR amplification test on the non-freeze-dried hot start Taq enzyme under the same experimental conditions to test the room temperature stability, wherein the results show that: the fluorescence value of the PCR amplification curve of the freeze-dried preparation of the hot start Taq enzyme vial placed at room temperature for 3 months in the embodiment is slightly lower than that of the PCR amplification curve of the hot start Taq enzyme placed at-20 ℃ and not subjected to freeze-drying treatment, but the overall performance difference is not obvious, and the result is shown in figure 8.

(11) 45 ℃ accelerated stability test of hot-start Taq enzyme penicillin bottle freeze-dried preparation

The high temperature accelerated stability of the freeze-dried preparation of hot-start Taq enzyme vials was tested by performing PCR amplification on commercial hot-start Taq enzyme (purchased as purchased from tokyo kexin biotechnology limited (product number P401)) stored at 45 ℃ for 2 months and-20 ℃ under the same experimental conditions, and as a result, it was found that: the fluorescence value of the PCR amplification curve of the freeze-dried preparation of the hot start Taq enzyme vial placed at 45 ℃ for 2 months in the embodiment is not obviously different from that of the PCR amplification curve of the commercial hot start Taq enzyme, and the result is shown in figure 9.

Example 7

In this embodiment, a preparation method of a freeze-dried reagent for high-efficiency hot start Taq enzyme is provided, and an annealing operation is added in a pre-freezing stage by a vacuum freeze-drying method to prepare a freeze-dried preparation for a hot start Taq enzyme vial. The freeze-drying protective agent mixed liquor is screened and optimized in the embodiment 4, and the freeze-drying protective agent mixed liquor comprises the following specific components: the method comprises the following steps: 25mM Tris-HCl (Ph 8.0), 10% trehalose, 3.5% mannitol, 5mg/ml bovine serum albumin, 10mM sodium azide, 0.05% Tween 20, 0.1% SE-15, and diethyl pyrophosphate treated water.

The method comprises the following specific steps:

(1) and preparing optimized freeze-drying protective agent mixed liquor.

(2) The hot start Taq enzyme modified by the monoclonal antibody and the prepared freeze-drying protective agent mixed solution are mixed according to the proportion of 2:1, mixing uniformly.

(4) And (3) subpackaging the uniformly mixed liquid reagent into penicillin bottles by using an accurate liquid separation system.

(5) And (5) putting the penicillin bottle into a vacuum freeze dryer for freeze drying.

(6) Freeze-drying program parameter setting:

the pre-freezing setting is as follows:

in the first stage, the pre-freezing temperature reaches 0 ℃ within 20min, and the temperature is kept for 0.5 h;

the pre-freezing temperature (annealing operation) of the second stage reaches-30 ℃ within 15min, and the temperature is kept for 1.5 h.

In the third stage, the pre-freezing temperature reaches-45 ℃ within 30min, and the temperature is kept for 3 h.

Annealing operation can be added in the pre-freezing setting, and the annealing can change the crystal form and the size of the ice crystal particles, strengthen the crystallization and improve the uniformity of the product. The annealing can improve the glass transition temperature of the amorphous phase frozen concentrated solution, has great significance to the sublimation drying stage and the desorption drying stage, and enables the property of the freeze-dried preparation to be more stable. Annealing also directly affects the quality appearance of the lyophilized product, etc.

The sublimation drying comprises the following steps:

the sublimation drying temperature of the first stage reaches-35 ℃ within 10min, the temperature is kept for 2h, and the vacuum degree is 12 Pa;

the sublimation drying temperature of the second stage reaches-30 ℃ within 20min, the temperature is maintained for 16h, and the vacuum degree is 12 Pa.

The desorption drying comprises the following steps:

in the first stage, the resolving and drying temperature reaches 15 ℃ within 30min, the temperature is kept for 1.5h, and the vacuum degree is 0 Pa;

the second stage analysis drying temperature reaches 25 deg.C within 20min, and is maintained for 5h with vacuum degree of 0 Pa.

(7) After the freeze-drying is finished, flushing nitrogen, pressing a plug in the box, opening the box door of the freeze dryer after the temperature and the humidity of the environment meet the requirements, and taking out the hot-start Taq enzyme penicillin bottle freeze-dried preparation.

(8) And observing whether the appearance of the freeze-dried preparation is qualified.

(9) The moisture content of the lyophilized preparation was measured using a card-type moisture meter. (determination of the moisture content of the lyophilized preparation to 2.24% by means of a card-type moisture meter)

(10) And (3) sealing the freeze-dried preparation of the hot-start Taq enzyme vial.

The drawings illustrate in detail:

FIG. 1 is a graph showing PCR amplification curves of Taq enzyme stored in the optimized screening enzyme storage buffer and Taq enzyme not stored in the enzyme storage buffer at 4 ℃ for 2 months in the examples of the present application (blue curve is a PCR amplification curve of Taq enzyme stored in the optimized screening enzyme storage buffer, and red curve is a PCR amplification curve of Taq enzyme not stored in the enzyme storage buffer).

FIG. 2 is a graph showing PCR amplification curves of Taq enzyme stored in the optimized screening enzyme storage buffer and Taq enzyme not stored in the enzyme storage buffer at-20 ℃ for 1 year in the present example (blue curve is PCR amplification curve of Taq enzyme stored in the optimized screening enzyme storage buffer, and red curve is PCR amplification curve of Taq enzyme not stored in the enzyme storage buffer).

FIG. 3 is a PCR amplification graph of the monoclonal antibody modified hot start Taq enzyme to the commercial hot start Taq enzyme in the example of the present application (the blue curve is the PCR amplification curve of the monoclonal antibody modified hot start Taq enzyme in the example of the present application, and the red curve is the PCR amplification curve of the commercial hot start Taq enzyme).

FIG. 4 is a PCR amplification curve graph of the monoclonal antibody modified hot start Taq enzyme in the present embodiment, stored in a refrigerator at-20 ℃ for one year, under the same test conditions (blue curve is PCR amplification curve of the monoclonal antibody modified hot start Taq enzyme in the present embodiment, and red curve is PCR amplification curve of the commercial hot start Taq enzyme).

FIG. 5 is a PCR amplification curve of 20 times of freezing and thawing of the monoclonal antibody modified hot-start Taq enzyme and the commercial hot-start Taq enzyme respectively under the same test conditions in the example of the present application (the blue curve is the PCR amplification curve of the monoclonal antibody modified hot-start Taq enzyme in the example of the present application, and the red curve is the PCR amplification curve of the commercial hot-start Taq enzyme).

FIG. 6 is a PCR amplification graph of chemically modified hot start Taq enzyme compared with commercial hot start Taq enzyme in the example of the present application (blue curve is a PCR amplification curve of chemically modified hot start Taq enzyme in the example of the present application, and red curve is a PCR amplification curve of commercial hot start Taq enzyme).

FIG. 7 is a graph showing the amplification curve of single-stranded binding protein-Taq enzyme versus commercial hot-start Taq enzyme in a multiplex PCR experiment (blue curve is the PCR amplification curve of single-stranded binding protein-Taq enzyme and red curve is the PCR amplification curve of commercial hot-start Taq enzyme in the example).

FIG. 8 is a PCR amplification curve of a freeze-dried preparation of hot-start Taq enzyme vials placed at room temperature for 3 months in the present example compared with that of hot-start Taq enzyme not subjected to freeze-drying treatment placed at-20 deg.C (the blue curve is a PCR amplification curve of monoclonal antibody-modified hot-start Taq enzyme in the present example, and the red curve is a PCR amplification curve of hot-start Taq enzyme not subjected to freeze-drying treatment placed at-20 deg.C).

FIG. 9 is a PCR amplification curve of commercial hot start Taq enzyme stored at 45 ℃ for 2 months and-20 ℃ for the same test conditions in the lyophilized formulation of hot start Taq enzyme Vicillin according to the example of the present application (blue curve is the PCR amplification curve of the lyophilized formulation of hot start Taq enzyme stored at 45 ℃ for 2 months and red curve is the PCR amplification curve of commercial hot start Taq enzyme stored at-20 ℃).

The foregoing descriptions of specific exemplary embodiments of the present invention have been presented for purposes of illustration and description. It is not intended to limit the invention to the precise form disclosed, and obviously many modifications and variations are possible in light of the above teaching. The exemplary embodiments were chosen and described in order to explain certain principles of the invention and its practical application to enable one skilled in the art to make and use various exemplary embodiments of the invention and various alternatives and modifications as are suited to the particular use contemplated. It is intended that the scope of the invention be defined by the claims and their equivalents.