CN116064478B

CN116064478B - Thermosensitive UDG enzyme mutant and its preparation method and application

Info

Publication number: CN116064478B
Application number: CN202211389855.9A
Authority: CN
Inventors: 张晓雷; 杨俊华; 赵巧辉; 李桂林; 付光宇; 杨增利
Original assignee: Zhengzhou Immuno Biotech Co Ltd
Current assignee: Zhengzhou Immuno Biotech Co Ltd
Priority date: 2022-11-08
Filing date: 2022-11-08
Publication date: 2025-05-23
Anticipated expiration: 2042-11-08
Also published as: CN116064478A

Abstract

本发明涉及生物技术领域，尤其涉及热敏型UDG酶突变体及其制备方法与应用。本发明提供的突变体将如SEQ ID NO.1所示的氨基酸序列中的第74位甘氨酸突变为丝氨酸，突变后序列为SEQ ID NO.2。与SEQ ID NO.1所示的野生型UDG酶相比，该突变体能够发挥正常的去污染能力，热稳定性较野生型降低，95℃5min可完全失活。本发明基于大肠杆菌K‑12UDG酶结构，对酶进行突变改造，降低其结构稳定性，进行纯化方法开发，获得了活性好，纯度高的热敏型UDG酶突变体。The present invention relates to the field of biotechnology, and in particular to a thermosensitive UDG enzyme mutant and a preparation method and application thereof. The mutant provided by the present invention mutates the 74th glycine in the amino acid sequence shown in SEQ ID NO.1 to serine, and the sequence after mutation is SEQ ID NO.2. Compared with the wild-type UDG enzyme shown in SEQ ID NO.1, the mutant can exert normal decontamination ability, the thermal stability is lower than that of the wild type, and it can be completely inactivated at 95°C for 5min. Based on the structure of Escherichia coli K-12 UDG enzyme, the present invention performs mutation modification on the enzyme, reduces its structural stability, develops a purification method, and obtains a thermosensitive UDG enzyme mutant with good activity and high purity.

Description

Thermosensitive UDG enzyme mutant and preparation method and application thereof

Technical Field

The invention relates to the technical field of biology, in particular to a thermosensitive UDG enzyme mutant and a preparation method and application thereof.

Background

UDG enzyme (uracil-DNA glycosylase) can selectively hydrolyze uracil-glycosidic bonds in single-stranded or double-stranded DNA, cleave uracil and create abasic sites in DNA, and the nucleotide chain is very susceptible to hydrolytic cleavage at high temperature or high pH.

The most common and main pollutant in PCR reaction is PCR product, and the pollution-preventing qPCR kit substitutes dUTP for dTTP, so that the PCR products are DNA chains containing dU, and before PCR is started, a heat preservation step of 50 ℃ is added, and UDG enzyme can degrade uracil base in the existing U-DNA pollutant in the reaction system, and the DNA chains are broken in the subsequent denaturation step, so that amplification caused by polluted DNA is eliminated, and the specificity and accuracy of the amplification result are ensured.

The UDG enzyme wild-type UDG enzyme studied in the early stage is heat-resistant, and can be deactivated after being treated for a long time at 95 ℃, even if the UDG enzyme is treated for 5-10 min at 95 ℃, a small amount of activity still remains, so that DNA products containing dU bases generated by reverse transcription or PCR amplification are degraded, and further the subsequent PCR reaction is interfered.

There are reports of the use of thermosensitive UDG enzymes derived from psychrophilic marine bacteria that can be completely inactivated at 50 ℃. The heat-sensitive UDG enzyme is unstable to heat, has the problems of enzyme activity reduction, instability and the like in the expression and purification processes, is easy to inactivate after being transported and stored for a long time at normal temperature, and has short validity period, so that the current products on the market all show that the validity period is only one year at-20 ℃ and repeated freeze thawing needs to be avoided. Researches show that the structure of the thermosensitive enzyme is softer and more flexible, and the structural stability of the protein is poor, so that mutation transformation is urgently needed to be carried out on the enzyme, the structural stability of the enzyme is reduced, and then the purification method is developed, so that the thermosensitive UDG enzyme mutant with good activity and high purity is obtained.

Disclosure of Invention

In view of the above, the technical problem to be solved by the invention is to provide a thermosensitive UDG enzyme mutant, and a preparation method and application thereof.

The invention provides a UDG enzyme mutant, which has an amino acid sequence shown as SEQ ID NO. 2. The mutant mutates the 74 th glycine in the amino acid sequence of the wild UDG enzyme shown as SEQ ID NO.1 into serine, and the mutated sequence is SEQ ID NO. 2.

The invention also provides nucleic acids encoding the UDG enzyme mutants.

In the embodiment of the invention, the nucleic acid for encoding the UDG mutant is subjected to codon optimization, and the optimized nucleic acid sequence is shown as SEQ ID NO. 3. The nucleic acid sequences are suitable for expressing the mutants according to the invention in prokaryotes, in particular in E.coli.

The invention also provides a vector containing the nucleic acid. In some embodiments, the backbone vector of the vector is a pET-30a vector.

The invention provides hosts transformed or transfected with the vectors. In some embodiments, the host is E.coli. More specifically, the E.coli is BL21 (DE 3) strain.

The invention also provides a preparation method of the UDG enzyme mutant, which comprises the step of culturing the host to obtain a culture containing the UDG enzyme mutant.

More specifically, the preparation method of the thermosensitive UDG enzyme mutant comprises the steps of connecting nucleotide of the sequence UDG enzyme mutant shown in SEQ ID NO. 2 into a pET-30a vector to obtain an expression vector, transferring into escherichia coli for expression purification, and obtaining the thermosensitive UDG enzyme through column chromatography.

In the preparation method, the purification comprises thallus crushing, inclusion body washing, UDG enzyme renaturation, DEAE anion exchange chromatography and Heparin chromatography;

The inclusion body is washed by using an inclusion body buffer solution, wherein the buffer solution comprises 40 mM-60 mM Tris-HCl, 5 mM-10 mM DTT, 1M-3M urea and 0.5% -1% Triton X-100, and the pH is 7.8-8.2;

the UDG enzyme is denatured by using an inclusion body dissolving buffer solution, wherein the inclusion body dissolving buffer solution comprises 40 mM-60mM Tris-HCl, 5 mM-10 mM DTT and 8M urea, and the pH value is 7.8-8.2;

The UDG enzyme renaturation uses a renaturation buffer solution, wherein the renaturation buffer solution comprises 40-60 mM Tris-HCl, 100-200 mM NaCl, 1-3 mM EDTA, 1-5 mM DTT, 5-15% glycerol, 0.5-1% Tween 20 and 0.2-0.5 mg/ml protein concentration, and pH 7.8-8.2;

The DEAE anion exchange chromatography and the Heparin chromatography use column chromatography buffer solution, wherein the column chromatography buffer solution comprises 40 mM-60 mM Tris-HCl, 1 mM-3 mM EDTA, 1 mM-5 mM DTT, 5% -15% glycerol, 0.5% -1% Tween 20 and pH 7.8-8.2.

The preparation method of the thermosensitive UDG enzyme disclosed by the invention comprises the steps of taking a nucleic acid sequence shown as SEQ ID NO. 3 as a template, using an amplification primer shown as SEQ ID NO. 4 and SEQ ID NO. 5, and amplifying to obtain the nucleotide of the UDG enzyme mutant with the sequence shown as SEQ ID NO. 2.

Wherein SEQ ID NO. 4 is an upstream primer and SEQ ID NO. 5 is a downstream primer.

In the invention, the amplified product is constructed on a pET-30a vector, and the connecting sites are NdeI and EcoRI.

In the present invention, the construction of the host includes transforming the obtained expression vector into competent cells.

According to the preparation method of the thermosensitive UDG enzyme, the expression conditions comprise that LB is used for culturing until the OD value reaches 0.8-1.2 based on 37 ℃, 0.5 mM-1 mM IPTG is added, and overnight induction expression is carried out at 16 ℃ for 12-16 h.

The preparation method of the thermosensitive UDG enzyme comprises the steps of ultrasonic crushing, inclusion body washing and dissolving, dilution and renaturation of inclusion body protein, DEAE anion exchange chromatography purification and Heparin column purification. The mutant is expressed in a prokaryotic expression system in the form of inclusion bodies, and the thermosensitive UDG enzyme with normal enzyme activity function is obtained by using a method of inclusion body renaturation and further chromatography.

In the preparation method of the thermosensitive UDG enzyme, the purification process is carried out in an environment of 2-8 ℃.

The invention also provides application of the UDG enzyme mutant, the nucleic acid, the vector, the host and/or the UDG enzyme mutant prepared by the preparation method in reducing pollutants in PCR reaction.

The invention provides a preparation for reducing pollutants in a PCR reaction, which comprises at least one of the following i) to iv):

i) The UDG enzyme mutant disclosed by the invention;

ii) nucleic acid encoding a UDG enzyme mutant according to the invention;

iii) The carrier of the invention;

iv) a host according to the invention.

The invention also provides a method for reducing contaminants in a PCR reaction comprising treating the PCR amplification product with a formulation of the invention.

The thermosensitive UDG enzyme disclosed by the invention can be completely inactivated after being subjected to heat treatment at 95 ℃ for 5min, so that the PCR reaction is not interfered. And the wild-type UDG enzyme still has partial activity after being subjected to heat treatment for 5 min. The heat-sensitive UDG enzyme and the wild-type UDG enzyme were heated at 95℃for 5min, respectively, and an inactivation experiment was performed. Diluting HBV amplified product (containing dU base) to 10 ⁸ times, adding heat treated 0.5U thermosensitive UDG enzyme mutant and T manufacturer wild type UDG enzyme, and performing amplification verification. As a result, as shown in FIG. 4, the amplification curve, ct value and control of the thermosensitive UDG enzyme were identical, whereas the Ct value was 25.31 after the wild-type enzyme treatment. It shows that the heat treatment at 95 ℃ for 5min can completely inactivate the thermosensitive UDG enzyme, while the wild UDG enzyme still has partial activity, so that the thermosensitive UDG enzyme mutant is more suitable for a one-step qPCR experiment.

The mutant provided by the invention mutates the 74 th glycine in the amino acid sequence shown as SEQ ID NO.1 into serine, and the mutated sequence is SEQ ID NO. 2. Compared with the wild-type UDG enzyme shown in SEQ ID NO.1, the mutant can exert normal pollution removal capability, has lower thermal stability than that of the wild-type UDG enzyme, and can be completely inactivated at 95 ℃ for 5 min. The invention is based on the structure of the escherichia coli K-12UDG enzyme, carries out mutation transformation on the enzyme, reduces the structural stability of the enzyme, and carries out purification method development to obtain the thermosensitive UDG enzyme mutant with good activity and high purity.

Drawings

For a clearer description of embodiments of the invention or of the solutions of the prior art, the drawings that are needed in the description of the embodiments or of the prior art will be briefly described, it being obvious that the drawings in the description below are some embodiments of the invention, and that, without the inventive effort, other drawings can be obtained from them to those skilled in the art:

FIG. 1 shows the purification results of UDG enzyme heporin;

FIG. 2 shows the results of degradation of UDG enzyme, 1 is a thermosensitive UDG enzyme mutant, 2 is a T manufacturer wild-type UDG enzyme, 3 is a sterile water control, and 4 is an amplification product control;

FIG. 3 shows qPCR validating UDG decontamination capability detection;

FIG. 4 shows qPCR to verify the detection of the detergency after heat treatment with UDG enzyme;

FIG. 5 shows qPCR validation of the detection of the ability of UDG to decontaminate after 2 years of storage.

Detailed Description

The invention provides a thermosensitive UDG enzyme mutant, a preparation method and application thereof, and a person skilled in the art can properly improve the technological parameters by referring to the content of the present disclosure. It is expressly noted that all such similar substitutions and modifications will be apparent to those skilled in the art, and are deemed to be included in the present invention. While the methods and applications of this invention have been described in terms of preferred embodiments, it will be apparent to those skilled in the relevant art that the invention can be practiced and practiced with modification and alteration and combination of the methods and applications herein without departing from the spirit and scope of the invention.

The invention relates to a preparation method of thermosensitive UDG enzyme, which comprises the following steps:

constructing a nucleotide sequence shown in SEQ ID NO. 3 into a pET-30a vector to obtain an expression vector, and transferring into escherichia coli for expression purification.

In some embodiments, PCR amplification is performed using the nucleotide sequence shown in SEQ ID NO.3 as a template and the nucleotide sequences shown in SEQ ID NO. 4 and 5 as primers, and the amplified product is ligated into the pET-30a vector.

In some embodiments, the escherichia coli is BL21 (DE 3) strain, and is cultured by using an LB culture medium, and is induced when the OD value reaches 0.8-1.2, wherein the induction temperature is 16 ℃ and the induction time is 12-16 h.

In some embodiments, the method of purifying comprises:

Cell disruption, inclusion body washing, renaturation, DEAE anion exchange and Heparin purification. The inclusion body washing buffer solution comprises 40-60 mM Tris-HCl,5mM~10mM DTT,1M~3M urea, 0.5-1% Triton X-100 and pH 7.8-8.2. The inclusion body dissolving buffer solution is 40 mM-60 mM Tris-HCl,5mM~10mM DTT,8M urea, and the pH value is 7.8-8.2. The renaturation buffer solution comprises 40-60 mM Tris-HCl,100mM~200mM NaCl,1mM~3mM EDTA,1mM~5mM DTT,5-15% glycerol, 0.5-1% Tween 20, pH 7.8-8.2 and protein concentration of 0.2-0.5 mg/ml. Column chromatography buffer solution 40 mM-60 mM Tris-HCl,1mM~3mM EDTA,1mM~5mM DTT,5% -15% glycerol, 0.5% -1% Tween 20 and pH 7.8-8.2.

The purification process is carried out at low temperature, which is favorable for stabilizing protein.

SEQ ID No.1 wild type UDG enzyme amino acid sequence

SEQ ID No.2 mutant thermosensitive UDG enzyme amino acid sequence

SEQ ID No.3 mutant thermosensitive UDG enzyme nucleic acid sequence

SEQ ID No.4 upstream primer

SEQ ID No.5 downstream primer

Embodiments of the present invention will be described in detail below with reference to examples, but it will be understood by those skilled in the art that the following examples are only for illustrating the present invention and should not be construed as limiting the scope of the present invention. The specific conditions are not noted in the examples and are carried out according to conventional conditions or conditions recommended by the manufacturer. The reagents or apparatus used were conventional products commercially available without the manufacturer's attention.

Example 1

1. Cloning construction of UDG enzyme

For the sequence shown in SEQ ID No.3, the synthesis is carried out by using a total gene synthesis method, and the primer clone of SEQ ID No.4 and SEQ ID No.5 is used for constructing a pET-30a vector, and the reaction system and the conditions are as follows:

50 μl of the reaction system

The PCR reaction conditions were pre-denatured at 95℃for 5 min, denatured at 95℃for 30 sec, annealed at 58℃for 30 sec, extended at 72℃for 45 sec, 25 cycles total, final extended at 72℃for 10 min, cooled to 16℃and the gel was taken out to recover a fragment of about 700bp, digested (NdeI/EcoRI) and then ligated with vector pET30a, and incubated overnight at 37 ℃.

And (3) positive clone screening, namely selecting a monoclonal to perform colony PCR screening, extracting plasmids, sequencing, and selecting clones with correct sequencing to perform subsequent expression and purification.

2. UDG enzyme expression

Transferring the positive clone into BL21DE3 host cell, culturing in LB culture medium at 37 deg.C until OD value reaches 1.0, adding 1mM IPTG, and expressing at 16 deg.C for 16 hr.

3. UDG enzyme purification

Thallus breaking and inclusion body washing, namely re-suspending BL21 (DE 3) thallus by PBS according to the proportion of 1:20, collecting precipitate after ultrasonic breaking, expressing the mutant as inclusion body, washing the inclusion body by 50mM Tris-HCl,5mM DTT,2M urea, 0.5% Triton X-100 and pH 8.0 for 3 times, and removing part of impurities such as impurity proteins and nucleic acid;

UDG enzyme denaturation, centrifugal precipitation is carried out by using 50mM Tris,5mM DTT,8M urea, pH is 8.0, the re-suspension volume is the same as the re-suspension volume of the broken bacteria, and ice bath stirring is carried out for 3h after re-suspension. Centrifugation at 15000rpm, filtration of the supernatant with a 0.45 μm filter, protein concentration determination by A280, dilution with 50mM Tris,5mM DTT,8M urea pH 8.0 to a concentration of 1mg/ml.

UDG enzyme renaturation, namely, a renaturation buffer solution 50mM Tris-HCl,150mM NaCl,1mM EDTA,1mM DTT,10% glycerol, 0.5% Tween 20 and pH 8.0 are used for slowly diluting a modified sample, the modified sample is diluted for 4 times, the final concentration of urea is 2M, the protein concentration is 0.25mg/ml, the temperature is 4 ℃, and the mixture is stirred for 4 hours, so that the protein is gradually renatured. The renatured samples were further dialyzed into 50mM Tris-HCl,150mM NaCl,1mM EDTA,1mM DTT,10% glycerol, 0.5% Tween 20, pH 8.0 buffer, dialyzed overnight at 4 ℃.

DEAE anion exchange chromatography, namely dialyzing a renaturation sample to remove salt, wherein a binding buffer solution is 50mM Tris-HCl,1mM EDTA,1mM DTT,10% glycerol, 0.5% Tween 20 and pH 8.0, and 1M NaCl,150mM NaCl is added into an elution buffer solution on the basis of the binding buffer solution to dissociate target proteins;

and (3) Heparin chromatography, namely, dialyzing and desalting DEAE dissociated samples, wherein the binding buffer is 50mM Tris-HCl,1mM EDTA,1mM DTT,10% glycerol, 0.5% Tween 20 and pH 8.0, and adding 1M NaCl;20mM NaCl into the eluting buffer on the basis of the binding buffer to wash impurities, and dissociating target proteins by 100mM NaCl, wherein the protein purity is more than 99% as shown in figure 1.

4. Activity detection of thermosensitive UDG enzyme mutant (shown as SEQ ID NO: 2)

4.1 Agarose gel electrophoresis

The following system (10. Mu.l) was used for PCR amplification using dUTP-containing products:

1 μl of 10-reaction buffer

1. Mu.l 1. Mu.g/. Mu.l dUTP PCR reaction product

1U UDG (SEQ ID NO: 2)

The mixture was made up to 10. Mu.l with water and reacted in a 37℃water bath for 1 hour, followed by electrophoresis.

The agarose gel electrophoresis result shows that the activity of the thermosensitive UDG enzyme mutant (shown as SEQ ID NO: 2) is equivalent to that of the wild type, and the thermosensitive UDG enzyme mutant can effectively degrade amplified products.

4.2QPCR detection

40. Mu.l of the reaction system was diluted 10 ⁸ th power as a sample, 15. Mu.l of HBV amplified product (containing dU base), 2 XqPCR mix, 20. Mu.l of HBV upstream primer (100. Mu.M) 0.6. Mu.l, downstream primer (100. Mu.M) 0.6. Mu.l, probe (100. Mu.M) 0.3. Mu.l, ultrapure water 3. Mu.l, and 0.5. Mu.l of thermosensitive UDG enzyme mutant (1U/. Mu.l, shown in SEQ ID NO: 2) and T manufacturer wild type UDG enzyme (1U/. Mu.l) were added respectively for amplification verification, and the results are shown in FIG. 3 below, the wells to which thermosensitive mutant and T manufacturer wild type UDG enzyme were added were not amplified, indicating that 0.5U UDG enzyme was able to effectively degrade amplified product.

5. Thermo-sensitive UDG enzyme thermostable assay

The heat sensitive mutant (SEQ ID NO: 2) and the wild-type UDG enzyme were heated at 95℃for 5min, respectively, and an inactivation experiment was performed. Diluting HBV amplified product (containing dU base) to 10 ⁸ times, adding heat-treated 0.5U thermosensitive UDG enzyme mutant (shown as SEQ ID NO: 2) and T manufacturer wild type UDG enzyme, and performing amplification verification, wherein the system is shown as 4.2. As a result, FIG. 4 shows that the amplification curve, ct value and control of the thermosensitive UDG enzyme (SEQ ID NO: 2) are identical, whereas the Ct value is 25.31, which is greater after the wild-type enzyme treatment. It shows that the heat treatment at 95 ℃ for 5min can completely inactivate the thermosensitive UDG enzyme (shown as SEQ ID NO: 2), and the wild UDG enzyme still has partial activity, so that the thermosensitive UDG enzyme mutant (shown as SEQ ID NO: 2) is more suitable for an anti-pollution qPCR experiment system.

6. Validity period verification

The experimenter carries out preservation validity tracking verification on the thermosensitive UDG enzyme mutant from escherichia coli. A2020060701 lot of UDG enzyme (shown as SEQ ID NO: 2) was used and after 2 years of storage at 20℃the ability to remove contamination was tested by qPCR. Diluting HBV amplified product (containing dU base) to 10 ⁸ times, adding 0.5U 2020060701 batch thermosensitive UDG enzyme mutant (shown as SEQ ID NO: 2), and preserving the UDG enzyme at-20deg.C for 2 years as shown in FIG. 5.

It should be noted that the above embodiments are merely for illustrating the technical solution of the present invention and not for limiting the same, and although the present invention has been described in detail with reference to the above embodiments, it should be understood by those skilled in the art that the technical solution described in the above embodiments may be modified or some or all of the technical features may be equivalently replaced, and these modifications or substitutions do not deviate from the essence of the corresponding technical solution from the scope of the technical solution of the embodiments of the present invention.

Claims

1. A UDG enzyme mutant, characterized in that it has an amino acid sequence as shown in SEQ ID NO: 2.

2. A nucleic acid encoding the UDG enzyme mutant according to claim 1.

3. The nucleic acid according to claim 2, characterized in that its nucleic acid sequence is shown in SEQ ID NO:3.

4. A vector containing the nucleic acid according to claim 2 or 3.

5. Transform or transfect a host with the vector according to claim 4.

6. The method for preparing the UDG enzyme mutant according to claim 1, characterized in that it comprises culturing the host according to claim 5 to obtain a culture containing the UDG enzyme mutant.

7. Use of the UDG enzyme mutant according to claim 1, the nucleic acid according to claim 2 or 3, the vector according to claim 4, the host according to claim 5 and/or the UDG enzyme mutant prepared by the preparation method according to claim 6 in reducing pollutants in PCR reactions.

8. A preparation for reducing contaminants in a PCR reaction, characterized in that it comprises at least one of the following i) to iv):

i), the UDG enzyme mutant according to claim 1;

ii), the nucleic acid according to claim 2 or 3;

iii) the vector according to claim 4;

iv) The host according to claim 5.

9. A method for reducing contaminants in a PCR reaction, comprising treating a PCR amplification product with the preparation of claim 8.