Endonuclease and application thereof

Abstract

The invention provides an endonuclease mutant, which comprises (a) a protein which is subjected to amino acid mutation and has an endonuclease activity function and contains at least one of the following sites compared with an amino acid sequence as shown in SEQ ID NO.1: T43, G67, Q69, W89, Q99, G101, G132, S135, N136, F137, S140, N143, S145, F149, Q152, A154, Q169, N188 or G222; or (b) an amino acid sequence which is obtained by substitution and / or deletion and / or addition of one or more conserved amino acid residues of the endonuclease mutant and the amino acid sequence shown in (a) and has the same function. The endonuclease mutant obtained by the invention has the capability of tolerating higher salt concentration, and has higher activity in low-temperature and room-temperature environments.

Description

[0001] The present invention is a divisional application of the Chinese application with the application date of July 24, 2023, application number 202310905132.8, and the name of the invention being “A Nucleic Acid Endonuclease and Its Application”. Technical Field

[0002] The present invention relates to the technical field of nucleases, and in particular to an endonuclease and an application thereof. Background Art

[0003] In recent years, with the development of biotechnology, more and more biological products such as antibodies, recombinant proteins, recombinant protein vaccines, viral vector vaccines, cell therapy and gene therapy drugs have entered the therapeutic field, and the quality control of biological products has become increasingly strict. Host nucleic acid residues have a variety of biosafety risks. Excessive host nucleic acid residues may cause infection, tumorigenesis, immune side effects, gene transposition and recombination, etc. Therefore, host nucleic acid residues have become the top priority of biological product quality control. The Chinese Pharmacopoeia clearly stipulates that the DNA residue in yeast and Escherichia coli expressed biological products shall not exceed 10ng / dose. In 2020, the Chinese Pharmacopoeia will update the DNA residue standard of human rabies vaccine (Vero cells) to ≤3ng / dose. In addition to the regulations on residues, the relevant guidance documents issued by FDA and CDE recommend that the residual cell host DNA fragments should not exceed the length of a functional gene (about 200bp). Therefore, the production process of biological products must contain a step to remove nucleic acids and conduct inspections and verifications to ensure that the nucleic acid residues in biological products meet the requirements of relevant regulations. Commonly used methods for removing nucleic acids, such as precipitation, will cause aggregation and encapsulation, which will affect the removal efficiency and is difficult to meet the standards when the requirements for nucleic acid residues are high. In order to control the amount of nucleic acid residues in biological products, various types of nucleases have been widely used in the purification process of biological products in recent years and have achieved good results.

[0004] Nuclease is an enzyme that can cut the phosphodiester bond of a polynucleotide chain. Nuclease is a hydrolase that acts on the PO position of the phosphodiester bond. Nucleases from different sources have different specificities and modes of action. Some nucleases can only act on RNA, called ribonucleases (RNase), some nucleases can only act on DNA, called deoxyribonucleases (DNase), and some nucleases have lower specificity and can act on both RNA and DNA, so they are called non-specific nucleases. According to the different positions of the nuclease, nucleases can be divided into exonucleases and endonucleases.

[0005] At present, the mainstream commercial nucleases on the market are mainly Benzonase (Serratia marcescens nuclease), Omnicleave (Epicentre) or DNase I. Benzonase and Omnicleave are universal nucleases that can digest DNA and RNA, while DNase I can only digest DNA. Under normal circumstances, nucleases have high nuclease activity under conditions below 100mM NaCl, and when the salt concentration is higher than 100mM, the enzyme activity is significantly reduced. Nucleases have a wide range of uses. For example, the main applications of Benzonase universal nuclease are as follows: 1) Removal of nucleic acids in viral vaccines, viral vectors for cell and gene therapy, and oncolytic viruses; 2) Removal of nucleic acids in biological products such as proteins; 3) Reducing the viscosity caused by nucleic acids; 4) Can be added to cell culture medium to prevent cell aggregation; 5) Reduce the viscosity of Escherichia coli cell lysate to improve filterability; 6) Pretreatment of particles (viruses, inclusion bodies, etc.); 7) Sample preparation for ELISA, column chromatography, two-dimensional electrophoresis and western blot analysis, which can improve resolution and recovery after nuclease treatment. Nucleases can be used to efficiently remove nucleic acid residues in biological products, but the activity of mainstream nucleases is highly inhibited or even inactivated at high salt concentrations, which cannot meet the needs. Therefore, the development of a salt-tolerant and efficient nuclease is of great significance for the removal of host nucleic acid residues in biological samples. Nucleases with high salt tolerance can be more widely used in genetic engineering, biomacromolecule production, especially the production of nucleic acid drugs. Commercially available nucleases are generally expressed in yeast or Escherichia coli supernatant, with complex production processes and high costs.

[0006] The tolerance of proteins to salt concentration is affected by many factors. Among them, changes in the physical and chemical properties of the protein surface will significantly affect the tolerance of proteins to salt concentration. The trends of protein surface changes include lower isoelectric point, lower hydrophobicity, higher heat capacity, lower compressibility and higher flexibility parameters.

[0007] At present, sequence analysis of halophilic bacterial proteins has found a statistical increase in negatively charged amino acids in halophilic proteins. The enrichment of Asp and Glu leads to a decrease in the isoelectric point, while lysine is relatively reduced. The accumulation of acidic amino acids on the surface is the most significant feature of enzymes adapted to high salinity, and this increase in acidity is also believed to prevent protein self-aggregation. In addition, the increase in acidic amino acids also promotes protein solubility, and the repulsive force between negative charges provides flexibility for protein function under high salt concentrations. Studies have found that the salt tolerance of carbonic anhydrase II and choline kinase has been significantly improved after acidic amino acids were replaced on the surface.

[0008] However, in addition to the increase of acidic amino acids on the protein surface, the increase of basic amino acids has also been reported to enhance the salt tolerance of proteins. In seawater with a salinity of 3.5%, which contains about 470 mM Na + and 540 mM Cl - . Therefore, most marine prokaryotes can be considered as slightly halophilic organisms, and their periplasmic / extracellular proteins are exposed to the external environment with high salt concentration. Vibrio is a Gram-negative bacterium mainly distributed in rivers, lakes, estuaries and marine environments. It grows well in an environment with a salinity of 2-4% and cannot grow in a salt-free environment. Studies have found that the nuclease I (VsEndA) from the marine bacterium Vibrio salmoniphila is more adaptable to higher salt concentrations than the homologous nuclease I (VcEndA) from the saltwater bacterium Vibrio cholerae. VsEndA has the highest nuclease activity at a salt concentration of 425mM NaCl, and still has about 60% activity at a salt concentration of 600mM. In contrast, VcEndA only has the highest activity at 200mM salt, and the activity of VcEndA is only 10% under 500mM salt conditions. The protein structures of VsEndA and VcEndA are highly similar, but the number of positively charged amino acids on the surface of VsEndA has increased significantly, such as lysine and arginine. The researchers speculate that the increase in positive charge on the surface of VsEndA can be related to the Cl - The interaction stabilizes the structure of the protein itself. In addition, the more positive charges on the surface of VsEndA are also conducive to the interaction with negatively charged DNA.

[0009] Although VsEndA exhibits good salt tolerance, the enzyme activity drops sharply when the salt concentration in the solution exceeds 500mM. At a salt concentration of 700mM, the enzyme activity is only about 30%, which still cannot meet the high salt tolerance requirements in the production of biological products. Therefore, a nuclease with better salt tolerance is urgently needed. Summary of the invention

[0010] In view of the deficiencies in the prior art, the present invention provides a nuclease endonuclease and its application, which solves the problem that the salt tolerance in the prior art is not high and cannot meet the high salt tolerance requirement in the production of biological products.

[0011] In one aspect, the present invention provides a nuclease mutant, comprising: (a) compared with the amino acid sequence shown in SEQ ID NO.1, the nuclease mutant contains at least one of the following sites: T43, G67, Q69, W89, Q99, G101, G132, S135, N136, F137, S140, N143, S145, F149, Q152, A154, Q169, N188 or G222, which has undergone amino acid mutation and has a nuclease activity function; or

[0012] (b) The endonuclease mutant has the same function as the amino acid sequence shown in (a) through substitution and / or deletion and / or addition of one or more conservative amino acid residues.

[0013] Further, the nuclease mutant comprises at least one site substitution selected from the group consisting of T43E, G67E or G67K, Q69E, W89D, Q99K, G101K, S135K, N136R, F137E, S140E, N143D or N143K, S145D, F149D, Q152K, Q169D, N188E, G222E or G222K.

[0014] Furthermore, the endonuclease mutant comprises any one of the following amino acid mutations:

[0015] G67E+Q69E+S140E+N143D+S145D+F149D+Q169D+N188E+G222E;

[0016] Q69E+S140E+N143D+G222E;

[0017] G67K+N143K+Q152K+G222K;

[0018] T43E;

[0019] F137E;

[0020] W89D+N136R;

[0021] Q99K+G101K+S135K.

[0022] In a second aspect, the present invention provides a DNA molecule encoding the above-mentioned endonuclease mutant.

[0023] Furthermore, the amino acid sequence corresponding to the nucleotide sequence of the DNA molecule includes those shown in SEQ ID NO.2-7.

[0024] The third aspect of the present invention provides a recombinant vector, an expression cassette or a host cell containing the above DNA molecule.

[0025] The fourth aspect of the present invention provides a kit comprising the above-mentioned nuclease mutant.

[0026] In a fifth aspect, the present invention provides a method for cutting nucleic acid, using the above-mentioned nuclease mutant, DNA molecule, recombinant vector, expression cassette or host cell.

[0027] In a sixth aspect, the present invention provides a method for preparing the above-mentioned nuclease mutant, culturing the above-mentioned host cell and obtaining the nuclease mutant from the culture; preferably, the host cell includes Escherichia coli.

[0028] In a seventh aspect, the present invention provides the use of the above-mentioned nuclease mutant, the above-mentioned DNA molecule, the above-mentioned recombinant vector, the expression cassette or host cell, and the above-mentioned kit in removing residual nucleic acids in biological products.

[0029] The term "biological products" refers to biological materials such as microorganisms, cells, and various animal and human tissues and fluids obtained by ordinary or genetic engineering, cell engineering, protein engineering, fermentation engineering and other biotechnologies, including bacteria, vaccines, toxins, toxoids, immune sera, blood products, immunoglobulins, antigens, allergens, cytokines, hormones, enzymes, fermentation products, monoclonal antibodies, recombinant DNA products, in vitro immunodiagnostic products, etc.

[0030] The term "kit" refers to a box consisting of chemical reagents that can be used to detect chemical components, drug residues, virus species, etc.

[0031] Since the first 21 amino acids of the wild-type VsEndA protein are signal peptides, the protein synthesized in the body will automatically remove the signal peptide, so during recombinant expression, expression starts from the 22nd position. In the present invention, SEQ ID NO.1 is the VsEndA protein with the signal peptide removed, so the first amino acid is numbered from 22.

[0032] The technical principle of the present invention is: based on the computer-aided design method, the present invention performs modification without affecting the overall structural stability of VsEndA, and replaces multiple polar amino acids at specific sites on its surface to reduce the hydrophobicity and electrical neutrality of the VsEndA surface. After computer virtual screening and experimental final verification, reasonable amino acid replacement changes the surface charge, hydrophobicity and stability of the protein, thereby enabling VsEndA to tolerate higher salt concentrations and have higher activity in low temperature and room temperature environments. In addition, the nucleases of the present invention are all expressed in Escherichia coli in the form of inclusion bodies, and the final products are obtained by directed denaturation technology.

[0033] Compared with the prior art, the present invention has the following beneficial effects:

[0034] (1) The present invention obtains a new nuclease with high salt tolerance and low temperature activity by performing point mutation on VsEndA. The screened nuclease reaches the highest enzyme activity in a 700mM NaCl environment and can maintain a high nuclease activity in a 400-900mM NaCl environment, which basically covers the high-salt environment in the production process of biological products. The modified molecule still maintains a high biological activity in low temperature and room temperature environments, and reaches the maximum enzyme activity at 25°C, which is just suitable for the environmental temperature of biological product production.

[0035] (2) The new nucleases of the present invention are all expressed by Escherichia coli into inclusion bodies and purified by protein denaturation and renaturation methods to obtain highly active nucleases. The production process is simple, the production cost is low, and there is a cost advantage.

[0036] (3) The nuclease of the present invention is a new all-purpose nuclease that acts not only on DNA but also on RNA. Its substrates are extensive and universal, and it has huge economic benefits. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] Figure 1 The amino acid sequence and three-dimensional structure comparison diagram of three homologous nucleases in Example 1 of the present invention;

[0038] Figure 2 This is a surface potential analysis diagram of VsEndA in Example 1 of the present invention;

[0039] Figure 3 This is the electrophoresis diagram of the E. coli expression of VsEndA and its mutants in Example 2 of the present invention, wherein T represents the whole bacteria, S represents the supernatant, and P represents the precipitate;

[0040] Figure 4 This is the electrophoresis diagram of pure VsEndA and its mutants in Example 2 of the present invention;

[0041] Figure 5 This is a diagram showing the results of enzyme activity detection of different nucleases at different salt ion concentrations in Example 3 of the present invention;

[0042] Figure 6 This is a diagram showing the enzyme activity detection results of 1527 at different temperatures in Example 4 of the present invention;

[0043] Figure 7 This is a diagram showing the enzyme cleavage effect of 1527 on DNA and RNA in Example 5 of the present invention. DETAILED DESCRIPTION

[0044] The technical scheme of the present invention will be further described in detail below in conjunction with specific embodiments. It should be understood that the following embodiments are only exemplary descriptions and explanations of the present invention and should not be construed as limiting the scope of protection of the present invention. All technologies implemented based on the above content of the present invention are included in the scope that the present invention is intended to protect.

[0045] Unless otherwise specified, the raw materials and reagents used in the following examples are commercially available or can be prepared by known methods.

[0046] Example 1 Protein structure modification and mutant screening based on computer-aided design

[0047] 1. Screening of halophilic microorganisms

[0048] (1) Comparison of amino acid sequences and three-dimensional structures of different halophilic microorganisms

[0049] Three nucleases of different halophilic Vibrio species, VnnEndA (vibrio vulnificus), VcEndA (Vibriocholerae) and VsEndA (Vibrio salmonicida), were selected as research objects. The amino acid sequence and protein three-dimensional structure of the three nucleases I were compared. Figure 1 shown.

[0050] Depend on Figure 1 It can be seen that the three nucleases have a high degree of sequence homology (up to 75% homology) when Clustal Omega is used to align the homologous sequences of the three nucleases, and the key amino acids related to metal ion interaction and activity are all consistent. The three-dimensional structures of the three also have a high degree of similarity, and the whole is a spherical shape with a gap, and the gap area is the enzyme activity center. The overall structure of the three nucleases is relatively rigid, containing a large number of α helices and two pairs of small β sheets, including four pairs of disulfide bonds. The enzyme activity center is located in a narrow area composed of α helices and β sheets, and the metal ions are also located in it. Taking VsEndA as an example, the conserved His84 and Arg103 are essential for enzyme activity. In particular, the five pairs of salt bonds Asp40–Arg216, Glu81–Arg76, Glu83–Arg134, Glu117–Arg103 and Glu210–Arg176 are highly conserved in the nucleases of the three halotolerant Vibrio, which are of great significance for maintaining the stability of the overall structure of the nuclease.

[0051] (2) Surface amino acid comparison of three nucleases

[0052] The surface amino acids of the three nucleases were analyzed in PyMOL. It was found that a large number of basic amino acids were substituted on the surface of VsEndA compared with the other two nucleases, which made the surface of VsEndA carry a large amount of positive charge. On the one hand, it helps to improve the solubility of VsEndA and inhibit its own aggregation. On the other hand, it helps VsEndA to bind to DNA and to Cl in high salt solution. - The interactions maintain the stability of their structures.

[0053] Since the salt tolerance of VsEndA is significantly better than that of the other two nucleases, VsEndA was selected as the research object and further modified.

[0054] 2. Transformation of VsEndA

[0055] Point mutations were performed on VsEndA to reduce the hydrophobicity and charge neutrality of the protein surface.

[0056] (1) Surface potential analysis of VsEndA

[0057] The surface potential analysis of VsEndA is as follows Figure 2 As shown. Figure 2 It can be seen that the active center of VsEndA is rich in lysine and arginine, which has a strong positive charge, which is conducive to the negatively charged DNA approaching the active center. There are many negatively charged acidic amino acids in the back area of ​​the active center, which is conducive to the Na+ in the high salt environment. + The distribution of surface charge of VsEndA has obvious two-sidedness, that is, the active center is positively charged and the inactive center is negatively charged, which has a wide space for transformation.

[0058] (2) Selection of amino acid mutation sites in VsEndA

[0059] In order not to affect the enzymatic activity and structural stability of VsEndA, the flexible region away from the active center was selected for modification when VsEndA was modified. Combined with the relatively low conserved regions in the homologous sequence alignment, T43, G67, Q69, W89, Q99, G101, G132, S135, N136, F137, S140, N143, S145, F149, Q152, A154, Q169, N188, G222 and other sites were finally selected for mutant screening.

[0060] (3) Screening of VsEndA mutants

[0061] When selecting mutants, reasonable polar amino acid substitutions were made at different mutation sites to design several different mutants. First, a computer was used to simulate the change in free energy after mutation, and mutants with reduced or slightly increased free energy after mutation were selected to reduce the impact of amino acid substitution on the overall stability of the structure. The homology modeling method was further used to simulate the three-dimensional structures of different mutants, and the predicted structural conformation was optimized in a certain force field. Finally, the molecular docking method was used to molecularly dock each mutant with DNA from the VnnEndA crystal structure (PDB ID: 1OUP), and mutants with reasonable spatial conformation and high affinity for DNA binding were selected. The screened VsEndA mutants are shown in Table 1.

[0062] Table 1 Amino acid mutation information of different mutants

[0063]

[0064]

[0065] Example 2 Plasmid construction, protein expression and purification of VsEndA and its mutants

[0066] 1. Plasmid construction

[0067] In this patent, 7 different mutants were obtained by subjecting the wild-type VsEndA to various mutation combinations as shown in Table 1, namely 1525 (Q69E, S140E, N143D, G222E), 1526 (G67E, Q69E, S140E, N143D, S145D, F149D, Q169D, N188E, G222E), 1527 (G67K, N143K, Q152K, G222K), 1692 (T43E), 1699 (F137E), 1700 (W89D, N136R) and 1701 (Q99K, G101K, S135K). The specific amino acid sequence information of each molecule is shown in Table 2. The first 21 amino acids of VsEndA from the marine bacterium Vibrio salmoniphila are a signal peptide, so the wild-type VsEndA synthesized in this example starts from the 22nd alanine.

[0068] Beijing Qingke Biotechnology Co., Ltd. was commissioned to synthesize the gene sequences of VsEndA (hereinafter marked as WT) and its mutants (1525, 1526, 1527, 1692, 1699, 1700, 1701). According to the operation method in "Molecular Cloning", PCR was used to obtain the target fragment. Then the target fragment was recombined with the universal vector pET28a, transformed, sequenced and maintained to obtain a plasmid capable of expressing the corresponding protein. According to the operation method in "Qiagen Mini-prep Kit", the plasmid of the nuclease was prepared.

[0069] Table 2 Endonuclease amino acid sequence

[0070]

[0071]

[0072] 2. Protein Expression

[0073] The plasmid constructed above was transformed into the E. coli expression strain BL21 (DE3), and a single clone colony was picked from the plate and transferred to a 500 mL TB medium containing Kana resistance at a volume ratio of 1:50. The initial OD600 was about 0.1, 37°C, 220rpm, and cultured until OD600 was 2.0. IPTG was added at a final concentration of 0.5mM, and cultured at 37°C, 220rpm for 4h before harvesting the bacteria. Protein expression was detected by SDS-PAGE, as shown in Figure 3 shown.

[0074] Depend on Figure 3 It can be seen that all proteins were successfully expressed in the form of inclusion bodies with high expression levels.

[0075] 3. Bacteria cell crushing and inclusion body recovery

[0076] The cells were crushed by high pressure, and the inclusion bodies were recovered after washing to prepare for the next step of protein renaturation. The buffer used for cell crushing was 20mM Tris, pH 8.0, 0.5M NaCl.

[0077] 4. Protein purification

[0078] (1) Protein renaturation

[0079] The inclusion bodies in step 3 were dissolved at room temperature using 10 times (v / w) volume of denaturation solution (8M Urea, 50mM Tris, pH 9.0), and DTT was added after 30 minutes, and reduction was continued for 30 minutes. The denaturation solution was centrifuged at 12000g, and the supernatant was dropped into 50 times volume of renaturation solution (20mM Tris, 0.5M arginine, 2.5mM Cysteine, pH 8.0), stirred at room temperature overnight, and the renaturation effect was detected by mass spectrometry the next day.

[0080] (2) Protein purification

[0081] The pH of the renatured protein in step (1) was adjusted to 7.5 with hydrochloric acid, and the conductivity was diluted to below 10 ms / cm with deionized water. After centrifugation, the supernatant was directly passed through a SPHP column (GE) for capture.

[0082] The operation is as follows: before purification, the SPHP column is equilibrated with 5 column volumes of equilibration solution (20 mM Tris, pH 7.5); the renaturation solution is passed through the column, and then the column is washed with 5 column volumes of equilibration solution to remove non-specific binding proteins; linear gradient elution is performed with 15 column volumes of elution buffer (20 mM Tris, pH 7.5, 1 M NaCl), and the eluate containing the target protein is collected to obtain an enzyme solution.

[0083] The final product is Figure 4 As shown, after purification by SPHP, the bands of the eight target proteins were clear and of high purity, with only 1526 having slightly poor yield and purity.

[0084] Example 3 Detection of mutant enzyme activity at different salt concentrations

[0085] Using commercially available Benzonase and WT as controls, the nuclease activity of each mutant at different salt ion concentrations was detected. The specific detection method is as follows:

[0086] First, calf thymus DNA was dissolved in 5mM MgCl2, 0.1mg / ml BSA, 50mM Tris, pH 8.0, with a final concentration of 0.1mg / ml. Take 2.5ml of DNA sample, add a certain amount of NaCl and 0.125ml of the enzyme solution obtained in Example 2 according to the set salt concentration, add 0.125ml of 1×PBS to the control group, mix well and place in a 37°C water bath, and take samples at 15min, 30min, 45min and 60min respectively. After each step of the reaction, take 0.5ml of the product after the reaction, add 0.5ml of 4% perchloric acid solution, mix well, and incubate on ice for 30min. After centrifuging the final reactant at 14000rpm, measure the OD value of the supernatant at 260nm (ultraviolet spectrophotometer) and calculate the enzyme activity.

[0087] The calculation formula is:

[0088] △A: measure the absorbance of the solution at time t. t: measure the reaction time of the solution

[0089] The results are as follows Figure 5 As shown in the figure, Benzonase has high nuclease activity under the condition of less than 100mM NaCl. When the salt concentration is higher than 100mM, the enzyme activity decreases significantly. When the salt concentration reaches above 300mM, Benzonase is basically inactive. In contrast, WT showed the strongest nuclease activity at a salt concentration of 400mM, and the enzyme activity gradually decreased with the increase of salt concentration in the reaction system. 1525 and 1527 showed better salt tolerance. 1525 had high nuclease activity in the salt concentration range of 400-800mM, and the optimal salt concentration was 600mM, while 1527 had high activity in the salt concentration range of 500-800mM, and the optimal salt concentration was 700mM. After the modification, the tolerance of 1525 and 1527 to NaCl was significantly improved, meeting the salt tolerance requirements for the production of biological products.

[0090] Example 4 Enzyme activity at different temperatures

[0091] In the actual production process of biological products, biological products are generally kept at room temperature. The activity of nucleases at room temperature significantly affects the nucleic acid residues in the final product. Conventional nucleases usually have high enzyme activity at 37-42°C, and the enzyme activity is greatly reduced at room temperature. Based on the needs of actual production, this example detects the activity of nuclease 1527 at a lower temperature. The NaCl concentration of the experiment is selected as 700mM, and the experimental method is basically the same as Example 3. The experimental results are shown in Figure 3. Figure 6 shown.

[0092] The results show that at low temperatures, 1527 is relatively stable and maintains a certain nuclease activity. As the temperature rises, 1527 exhibits the highest enzyme activity at 25°C (close to room temperature). As the reaction temperature is further increased, the activity of 1527 gradually decreases. This shows that at room temperature, 1527 has the highest activity, which is very consistent with the needs of actual production and is conducive to large-scale application in industrial production.

[0093] Example 5 Nuclease activity of 1527 against DNA and RNA

[0094] In a reaction system of 5 mM MgCl2, 0.7 M NaCl, 20 mM Tris, pH 8.0, 5 μg DNA and 5 μg RNA were digested with 1527. After reacting at room temperature for 30 minutes, the digestion results were detected by 1% agarose gel electrophoresis. The results are shown in Figure 7 shown.

[0095] The results show that compared with the control group, DNA and RNA can be completely removed by enzyme cleavage in 1527. 1527 nuclease is a universal enzyme with a wider range of applications.

[0096] Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present invention rather than to limit it. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solution of the present invention can be modified or replaced by equivalents without departing from the purpose and scope of the technical solution of the present invention, which should be included in the scope of the claims of the present invention.

Claims

1. A nuclease mutant, characterized in that: The amino acid sequence of the nuclease mutant is shown in SEQ ID NO.

4.

2. A DNA molecule, characterized in that The DNA molecule encodes the endonuclease mutant according to claim 1.

3. A recombinant vector, expression cassette or host cell containing the DNA molecule of claim 2. A kit comprising the endonuclease mutant according to claim 1.

5. A method for cutting nucleic acid, characterized in that: The nucleic acid is cut using the endonuclease mutant of claim 1, the DNA molecule of claim 2, the recombinant vector, the expression cassette or the host cell of claim 3.

6. A method for preparing the endonuclease mutant according to claim 1, characterized in that: Cultivate the host cell according to claim 3, and obtain the endonuclease mutant from the culture.

7. The method according to claim 6, characterized in that The host cell includes Escherichia coli.

8. Use of the endonuclease mutant according to claim 1, the DNA molecule according to claim 2, the recombinant vector, expression cassette or host cell according to claim 3, and the kit according to claim 4 in removing residual nucleic acids from biological products.

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