Thermosensitive UDG enzyme mutant and its preparation method and application

Abstract

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

Technical Field

[0001] The invention relates to the field of biotechnology, and in particular to a thermosensitive UDG enzyme mutant and a preparation method and application thereof. Background Art

[0002] UDG enzyme (uracil-DNA glycosylase) can selectively hydrolyze the uracil-glycosidic bond in single-stranded or double-stranded DNA, remove uracil and generate abasic sites in DNA. This nucleotide chain is easily hydrolyzed and broken under high temperature or high pH conditions.

[0003] The most common and main contaminant in PCR reactions is PCR products. The anti-contamination qPCR kit replaces dTTP with dUTP, so its PCR products are all DNA chains containing dU. By adding a 50°C insulation step before the start of PCR, the UDG enzyme can degrade the uracil bases in the existing U-DNA contaminants in the reaction system, and break the DNA chain in the subsequent denaturation step, eliminating the amplification caused by the contaminating DNA, thereby ensuring the specificity and accuracy of the amplification results.

[0004] The wild-type UDG enzyme studied in the early stage is relatively heat-resistant and needs to be treated at 95°C for a long time to be inactivated. Even if it is treated at 95°C for 5 to 10 minutes, a small amount of activity will still remain, causing the degradation of the DNA products containing dU bases produced by reverse transcription or PCR amplification, thereby interfering with the subsequent PCR reaction.

[0005] At present, there are reports of using thermosensitive UDG enzymes derived from cold-loving marine bacteria, which can be completely inactivated at 50°C. Since thermosensitive UDG enzymes are unstable to heat, there are problems such as decreased enzyme activity and instability during expression and purification; and they are easily inactivated during long-term transportation and storage at room temperature, resulting in a short shelf life. Therefore, the products currently on the market all show a shelf life of only one year at -20°C, and repeated freezing and thawing need to be avoided. Studies have shown that the structure of thermosensitive enzymes is relatively soft and flexible, and the protein structure stability is poor. Therefore, it is urgent to mutate the enzyme to reduce its structural stability, and then develop a purification method to obtain a thermosensitive UDG enzyme mutant with good activity and high purity. Summary of the invention

[0006] In view of this, the technical problem to be solved by the present invention is to provide a thermosensitive UDG enzyme mutant and a preparation method and application thereof.

[0007] The present invention provides a UDG enzyme mutant having an amino acid sequence as shown in SEQ ID NO: 2. The mutant mutates the 74th glycine in the amino acid sequence of the wild-type UDG enzyme as shown in SEQ ID NO.1 to serine, and the sequence after mutation is SEQ ID NO: 2.

[0008] The present invention also provides a nucleic acid encoding the UDG enzyme mutant.

[0009] In the embodiment of the present invention, the nucleic acid encoding the UDG mutant is codon optimized, and the optimized nucleic acid sequence is shown in SEQ ID NO: 3. The nucleic acid sequence is suitable for expressing the mutant of the present invention in prokaryotes, especially in Escherichia coli.

[0010] The present invention also provides a vector containing the nucleic acid. In some embodiments, the backbone vector of the vector is a pET-30a vector.

[0011] The present invention provides a host for transforming or transfecting the vector. In some embodiments, the host is Escherichia coli. More specifically, the Escherichia coli is a BL21 (DE3) strain.

[0012] The present invention also provides a method for preparing the UDG enzyme mutant, which comprises culturing the host to obtain a culture containing the UDG enzyme mutant.

[0013] More specifically, the method for preparing the thermosensitive UDG enzyme mutant comprises: connecting the nucleotide encoding the UDG enzyme mutant of the sequence shown in SEQ ID NO: 2 into the pET-30a vector to obtain an expression vector, and transferring it into Escherichia coli for expression and purification; and obtaining the thermosensitive UDG enzyme by column chromatography.

[0014] In the preparation method of the present invention, the purification includes: bacterial cell crushing, inclusion body washing, UDG enzyme renaturation, DEAE anion exchange chromatography and Heparin chromatography;

[0015] The inclusion body washing is performed using an inclusion body buffer, wherein the buffer comprises: 40 mM to 60 mM Tris-HCl, 5 mM to 10 mM DTT, 1 M to 3 M urea and 0.5% to 1% Triton X-100, pH 7.8 to 8.2;

[0016] The UDG enzyme denaturation uses an inclusion body dissolution buffer, which includes: 40mM-60mM Tris-HCl, 5mM-10mM DTT and 8M urea, pH 7.8-8.2;

[0017] The UDG enzyme is renatured using a renaturation buffer, which includes: 40mM-60mM Tris-HCl, 100mM-200mM NaCl, 1mM-3mM EDTA, 1mM-5mM DTT, 5%-15% glycerol, 0.5%-1% Tween20 and a protein concentration of 0.2mg / ml-0.5mg / ml, pH7.8-8.2;

[0018] The DEAE anion exchange chromatography and Heparin chromatography use a column chromatography buffer, which includes: 40mM-60mM Tris-HCl, 1mM-3mM EDTA, 1mM-5mM DTT, 5%-15% glycerol and 0.5%-1% Tween 20, pH 7.8-8.2.

[0019] In the preparation method of the thermosensitive UDG enzyme of the present invention, the preparation method of the nucleotide encoding the UDG enzyme mutant of the sequence shown in SEQ ID NO: 2 comprises: using the nucleic acid sequence shown in SEQ ID NO: 3 as a template, using SEQ ID NO: 4 and SEQ ID NO: 5 amplification primers, and obtaining it by amplification.

[0020] Among them, SEQ ID NO: 4 is the upstream primer, and SEQ ID NO: 5 is the downstream primer.

[0021] In the present invention, the amplified product is constructed into the pET-30a vector, and the connection sites are NdeI and EcoRI.

[0022] In the present invention, the construction of the host comprises transforming the obtained expression vector into competent cells.

[0023] In the preparation method of the thermosensitive UDG enzyme of the present invention, the expression conditions include: culturing with LB medium at 37°C until the OD value reaches 0.8-1.2, adding 0.5mM-1mM IPTG, and inducing expression overnight at 16°C for 12h-16h.

[0024] In the preparation method of the thermosensitive UDG enzyme of the present invention, the purification method includes: ultrasonic disruption, inclusion body washing and dissolution, inclusion body protein dilution and renaturation, DEAE anion exchange chromatography purification and Heparin column purification. The mutant is expressed in the form of inclusion bodies in a prokaryotic expression system. The present invention uses the inclusion body renaturation method and further chromatography to obtain a thermosensitive UDG enzyme with normal enzyme activity.

[0025] In the method for preparing the thermosensitive UDG enzyme of the present invention, the purification process is carried out in an environment of 2°C to 8°C.

[0026] The present invention also provides the use 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 reactions.

[0027] The present invention provides a preparation for reducing contaminants in a PCR reaction, comprising at least one of the following i) to iv):

[0028] i), the UDG enzyme mutant of the present invention;

[0029] ii), a nucleic acid encoding the UDG enzyme mutant of the present invention;

[0030] iii), the vector of the present invention;

[0031] iv) the host described in the present invention.

[0032] The present invention also provides a method for reducing contaminants in a PCR reaction, which comprises treating the PCR amplification product with the preparation of the present invention.

[0033] The "thermosensitive UDG enzyme" described in the present invention can be completely inactivated after heat treatment at 95°C for 5 minutes, thus having no interference with the PCR reaction. However, the "wild-type UDG enzyme" still has some activity after heat treatment for 5 minutes. The thermosensitive UDG enzyme and the wild-type UDG enzyme were heated at 95°C for 5 minutes respectively to perform an inactivation experiment. The HBV amplification product (containing dU base) was diluted 10 8 Then, 0.5 U of heat-treated thermosensitive UDG enzyme mutant and wild-type UDG enzyme from manufacturer T were added for amplification verification. Figure 4 As shown in the figure, the amplification curve and Ct value of the thermosensitive UDG enzyme are consistent with the control, while the curve of the wild-type enzyme treatment is later and the Ct value is larger, which is 25.31. This shows that the thermosensitive UDG enzyme can be completely inactivated by heat treatment at 95℃ for 5 minutes, while the wild-type UDG enzyme still has some activity. Therefore, the thermosensitive UDG enzyme mutant is more suitable for one-step qPCR experiments.

[0034] 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 5 minutes. Based on the structure of Escherichia coli K-12 UDG enzyme, the present invention mutates the enzyme, reduces its structural stability, develops a purification method, and obtains a thermosensitive UDG enzyme mutant with good activity and high purity. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] In order to more clearly illustrate the specific implementation of the present invention or the technical solutions in the prior art, the drawings required for use in the specific implementation or the prior art description are briefly introduced below. Obviously, the drawings in the following description are some implementations of the present invention. For those of ordinary skill in the art, other drawings can be obtained based on these drawings without creative work:

[0036] Figure 1 Shows the results of purification of UDG enzyme Heparin;

[0037] Figure 2 The results of UDG enzyme degradation are shown, 1 is a thermosensitive UDG enzyme mutant, 2 is a wild-type UDG enzyme from manufacturer T, 3 is a sterile water control, and 4 is an amplification product control;

[0038] Figure 3 qPCR was used to verify the decontamination ability of UDG;

[0039] Figure 4 qPCR was used to verify the decontamination ability of UDG enzyme after heat treatment;

[0040] Figure 5 Shown is qPCR validation of the decontamination ability of UDG after 2 years of storage. DETAILED DESCRIPTION

[0041] The present invention provides a thermosensitive UDG enzyme mutant and a preparation method and application thereof. Those skilled in the art can refer to the content of this article and appropriately improve the process parameters for implementation. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art, and they are all considered to be included in the present invention. The methods and applications of the present invention have been described through preferred embodiments, and relevant personnel can obviously modify or appropriately change and combine the methods and applications of this article without departing from the content, spirit and scope of the present invention to implement and apply the technology of the present invention.

[0042] The present invention relates to a method for preparing a thermosensitive UDG enzyme, comprising:

[0043] The nucleotide sequence shown in SEQ ID NO: 3 was constructed into pET-30a vector to obtain an expression vector, which was then transformed into Escherichia coli for expression and purification.

[0044] 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.

[0045] In some embodiments, the Escherichia coli is a BL21 (DE3) strain, cultured using LB medium, induced when the OD value reaches 0.8 to 1.2, with an induction temperature of 16° C. and a time of 12 h to 16 h.

[0046] In some embodiments, the purification method comprises:

[0047] Bacteria disruption, inclusion body washing, renaturation, DEAE anion exchange and Heparin purification. The inclusion body washing buffer: 40mM-60mM Tris-HCl, 5mM-10mM DTT, 1M-3M urea, 0.5%-1% Triton X-100, pH7.8-8.2. Inclusion body dissolution buffer: 40mM-60mM Tris-HCl, 5mM-10mM DTT, 8M urea, pH 7.8-8.2. Renaturation buffer: 40mM-60mM Tris-HCl, 100mM-200mM NaCl, 1mM-3mM EDTA, 1mM-5mM DTT, 5%-15% glycerol, 0.5%-1% Tween 20, pH 7.8-8.2, protein concentration is 0.2mg / ml-0.5mg / ml. Column chromatography buffer: 40mM~60mM Tris-HCl, 1mM~3mM EDTA, 1mM~5mM DTT, 5%~15% glycerol, 0.5%~1% Tween 20, pH 7.8~8.2.

[0048] The purification process of the present invention is carried out under low temperature conditions, which is beneficial to protein stability.

[0049] SEQ ID.No.1 Wild-type UDG enzyme amino acid sequence

[0050]

[0051] SEQ ID.No.2 amino acid sequence of mutant thermosensitive UDG enzyme

[0052]

[0053] SEQ ID.No.3 Mutant thermosensitive UDG enzyme nucleic acid sequence

[0054]

[0055] SEQ ID.No.4 Upstream primer

[0056]

[0057] SEQ ID.No.5 downstream primer

[0058]

[0059] The embodiments of the present invention will be described in detail below in conjunction with the examples, but it will be appreciated by those skilled in the art that the following examples are only used to illustrate the present invention and should not be construed as limiting the scope of the present invention. If specific conditions are not specified in the examples, they are carried out according to conventional conditions or conditions recommended by the manufacturer. If the manufacturer is not specified for the reagents or instruments used, they are all conventional products that can be purchased commercially.

[0060] Example 1

[0061] 1. Cloning and construction of UDG enzyme

[0062] The sequence shown in SEQ ID No.3 was synthesized by whole gene synthesis and cloned into the pET-30a vector using primers SEQ ID No.4 and SEQ ID No.5. The reaction system and conditions were as follows:

[0063] Reaction system 50 μl

[0064]

[0065]

[0066] PCR reaction conditions: pre-denaturation, 95°C, 5 minutes; denaturation, 95°C, 30 seconds; annealing, 58°C, 30 seconds; extension, 72°C, 45 seconds; 25 cycles in total; final extension, 72°C, 10 minutes; cool to 16°C, take out, gel-recover fragments of about 700 bp, digest with NdeI / EcoRI, and connect to vector pET30a, and culture at 37°C overnight.

[0067] Positive clone screening: single clones were selected for colony PCR screening, plasmids were extracted and sent for sequencing, and clones with correct sequencing were selected for subsequent expression and purification.

[0068] 2. UDG enzyme expression

[0069] The positive clones were transferred into BL21DE3 host cells; the cells were cultured in LB medium at 37°C. When the OD value reached 1.0, 1 mM IPTG was added and the cells were induced to express overnight at 16°C for 16 h, and the cells were collected.

[0070] 3. UDG enzyme purification

[0071] Bacterial cell disruption and inclusion body washing: BL21 (DE3) bacterial cells were resuspended in PBS at a ratio of 1:20, and the precipitate was collected after ultrasonic disruption. The mutant was expressed in inclusion bodies. The inclusion bodies were washed with 50mM Tris-HCl, 5mM DTT, 2M urea, 0.5% Triton X-100, pH 8.0 for 3 times to remove some impurities such as miscellaneous proteins and nucleic acids;

[0072] UDG enzyme denaturation: resuspend the centrifugal precipitate with 50mM Tris, 5mM DTT, 8M urea, pH 8.0, the resuspension volume is the same as the resuspension volume of the bacterium, and stir in an ice bath for 3 hours after resuspension. Centrifuge at 15000rpm, filter the supernatant with a 0.45μm filter membrane, determine the protein concentration by A280, and dilute with 50mM Tris, 5mM DTT, 8M urea pH 8.0 to a concentration of 1mg / ml.

[0073] UDG enzyme refolding: Use refolding buffer 50mM Tris-HCl, 150mM NaCl, 1mM EDTA, 1mM DTT, 10% glycerol, 0.5% Tween 20, pH 8.0 to slowly dilute the denatured sample 4 times, so that the final concentration of urea is 2M and the protein concentration is 0.25mg / ml. Stir for 4h at 4℃ to gradually refold the protein. Further dialyze the refolded sample into 50mM Tris-HCl, 150mM NaCl, 1mM EDTA, 1mM DTT, 10% glycerol, 0.5% Tween 20, pH 8.0 buffer and dialyze overnight at 4℃.

[0074] DEAE anion exchange chromatography: The renatured sample was dialyzed for desalting, the binding buffer was 50mM Tris-HCl, 1mM EDTA, 1mM DTT, 10% glycerol, 0.5% Tween 20, pH 8.0, and the elution buffer was 1M NaCl added to the binding buffer, and 150mM NaCl was added to dissociate the target protein;

[0075] Heparin chromatography: DEAE dissociation sample dialyzed to desalt, binding buffer is 50mM Tris-HCl, 1mM EDTA, 1mM DTT, 10% glycerol, 0.5% Tween 20, pH 8.0, elution buffer is based on binding buffer with 1M NaCl added; 20mM NaCl wash, 100mM NaCl dissociate target protein, such as Figure 1 As shown, the protein purity reached more than 99%.

[0076] 4. Activity detection of thermosensitive UDG enzyme mutant (shown in SEQ ID NO: 2)

[0077] 4.1 Agarose gel electrophoresis

[0078] Use the dUTP-containing PCR amplification product to prepare the following system (10 μl):

[0079] 1μl 10* reaction buffer

[0080] 1μl 1μg / μl dUTP PCR reaction product

[0081] 1U UDG (shown in SEQ ID NO: 2)

[0082] Add water to 10 μl and react in a 37°C water bath for 1 h, followed by electrophoresis.

[0083] The results of agarose gel electrophoresis are as follows: the activity of the thermosensitive UDG enzyme mutant (shown in SEQ ID NO: 2) is equivalent to that of the wild type and can effectively degrade the amplified product.

[0084] 4.2qPCR detection

[0085] Reaction system 40 μl: HBV amplification product (containing dU base) diluted 10 8 The second power was used as a sample, 15 μl; 2×qPCRmix, 20 μl; HBV upstream primer (100 μM) 0.6 μl; downstream primer (100 μM) 0.6 μl; probe (100 μM) 0.3 μl; ultrapure water 3 μl; and 0.5 μl of thermosensitive UDG enzyme mutant (1 U / μl, shown in SEQ ID NO: 2) and T manufacturer wild-type UDG enzyme (1 U / μl) were added respectively for amplification verification, and the results are as follows Figure 3 As shown, there was no amplification in the wells added with the thermosensitive mutant and wild-type UDG enzymes from manufacturer T, indicating that 0.5U UDG enzyme can effectively degrade the amplified product.

[0086] 5. Thermal stability test of thermosensitive UDG enzyme

[0087] The thermosensitive mutant (shown in SEQ ID NO: 2) and wild-type UDG enzymes were heated at 95°C for 5 min to perform inactivation experiments. The HBV amplified product (containing dU bases) was diluted 10 8 Second, 0.5 U of the heat-sensitive UDG enzyme mutant (shown in SEQ ID NO: 2) after heat treatment and the wild-type UDG enzyme of manufacturer T were added for amplification verification, and the system was the same as 4.2. Figure 4 As shown, the amplification curve and Ct value of the thermosensitive UDG enzyme (shown in SEQ ID NO: 2) are consistent with the control, while the curve treated with the wild-type enzyme is later and the Ct value is larger, which is 25.31. This indicates that the thermosensitive UDG enzyme (shown in SEQ ID NO: 2) can be completely inactivated by heat treatment at 95°C for 5 minutes, while the wild-type UDG enzyme still has some activity. Therefore, the thermosensitive UDG enzyme mutant (shown in SEQ ID NO: 2) is more suitable for the anti-pollution qPCR experimental system.

[0088] 6. Validity Verification

[0089] The experimenter verified the shelf life of the thermosensitive UDG enzyme mutant derived from Escherichia coli in the present invention. The UDG enzyme of batch 2020060701 (shown in SEQ ID NO: 2) was used and stored at -20°C for 2 years, and then the decontamination ability was tested by qPCR. The HBV amplified product (containing dU base) was diluted 10 8 Second, 0.5 U of the thermosensitive UDG enzyme mutant (shown in SEQ ID NO: 2) from batch 2020060701 was added. Figure 5 The results showed that the UDG enzyme could still effectively degrade the amplified product when stored at -20°C for 2 years.

[0090] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or replace some or all of the technical features therein by equivalents. However, these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. UDG enzyme mutants, It is 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, It is 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. A method for preparing the UDG enzyme mutant according to claim 1, It is characterized in that The method comprises culturing the host according to claim 5 to obtain a culture containing a 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. Preparations for reducing contaminants in PCR reactions, It is characterized in that Including 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 PCR reactions, It is characterized in that The method comprises treating a PCR amplification product with the preparation of claim 8.

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