All-round nuclease mutant as well as preparation method and application thereof

Abstract

The invention provides an all-round nuclease mutant as well as a preparation method and application thereof, and relates to the technical field of biology. Compared with a wild type, the totipotent nuclease mutant provided by the invention has higher enzyme activity and temperature resistance and better stability, can be applied to industrial applications such as vaccine purification, biological pharmacy residual DNA removal, cell lysis solution treatment and the like, and has obvious technical advantages and popularization value. The preparation method of the totipotent nuclease mutant provided by the invention is simple and convenient, the expression quantity of the totipotent nuclease mutant is effectively improved by further optimizing host cells, an expression vector, an inclusion body inducing expression method and a renaturation mode, and the problem that the activity of the totipotent nuclease is obviously reduced after renaturation is relieved.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and in particular to a totipotent nuclease mutant, its preparation method, and its application. Background Technology

[0002] Traditional totipotent nucleases (Benzonases or similar nucleases) are mostly derived from natural microbial secretion systems or expressed in eukaryotic systems. In industrial production, they generally suffer from low expression levels, difficulty in refolding, incomplete recovery of enzyme activity, and high costs. While existing E. coli recombinant expression systems offer advantages in high-density fermentation and cost, totipotent nucleases often exist as inclusion bodies within E. coli, leading to a significant decrease in activity after refolding, thus limiting their application in nucleic acid degradation or residual DNA removal.

[0003] In view of this, the present invention is hereby proposed. Summary of the Invention

[0004] The primary objective of this invention is to provide a totipotent nuclease mutant with high activity, thereby addressing the problem of low activity in existing totipotent nucleases.

[0005] A second objective of this invention is to provide a nucleic acid molecule.

[0006] A third objective of this invention is to provide a carrier.

[0007] A fourth objective of this invention is to provide a cell.

[0008] The fifth objective of this invention is to provide a method for preparing a totipotent nuclease mutant, in order to solve the problem that the activity of totipotent nucleases decreases significantly after renaturation in existing preparation methods.

[0009] The sixth objective of this invention is to provide the application of the above-mentioned pluripotent nuclease mutant in nucleic acid degradation.

[0010] To achieve the above objectives, the following technical solution is adopted: In a first aspect, the present invention provides a totipotent nuclease mutant, the amino acid sequence of which is shown in SEQ ID NO.1: MRFNNKMLALAALLFAAQASADTLESIDNCAVGCPTGGSSNVSIVRHAYTLNNNSTTKFANWVAYHITKDTPASGKTRNWKTDPALNPADTLAPADYTGANAALKVDRGHQAPLASLAGVSDWESLNYLSNITPQ KSDLQQGAWARLEDQERKLIDRADISSVYTVTGPLYERDMGKLPGTQKAHTIPSAYWKVIFINNSPAVNHYAAFLFDQNTPKGADFCQFRVTVDEIEKRTGLIIWAGLPDDVQASLKSKPGVLPELMGCKN (SEQ ID NO.1).

[0011] In a second aspect, the present invention provides a nucleic acid molecule comprising a nucleic acid sequence encoding the pluripotent nuclease mutant.

[0012] As a further technical solution, the nucleic acid sequence encoding the totipotent nuclease mutant is shown in SEQ ID NO.2: (SEQ ID NO.2).

[0013] Thirdly, the present invention provides a carrier containing the aforementioned nucleic acid molecule.

[0014] As a further technical solution, the carrier includes pET-28a.

[0015] Fourthly, the present invention provides a cell that carries the nucleic acid molecule, or contains the vector, or expresses the pluripotent nuclease mutant.

[0016] As a further technical solution, the cells include Rosetta (DE3).

[0017] Fifthly, the present invention provides a method for preparing a totipotent nuclease mutant, comprising the following steps: The cells were cultured to induce the expression of inclusion bodies of the totipotent nuclease mutant, and the inclusion bodies were obtained. The inclusion bodies were then refolded and separated to prepare the totipotent nuclease mutant.

[0018] As a further technical solution, the cells are induced under culture conditions containing 0.1-1 mM IPTG at a temperature of 37°C.

[0019] As a further technical solution, the reversibility includes the following steps: The inclusion bodies were fully dissolved at pH 11 to obtain a solution. The solution and refolding solution were then mixed and incubated at a volume ratio of 3:1. The refolding solution was then added to bring the volume ratio of the solution and refolding solution to 2:1 and incubated. Finally, the refolding solution was added to bring the volume ratio of the solution and refolding solution to 1:1 and incubated.

[0020] Sixthly, the present invention provides the application of the above-mentioned pluripotent nuclease mutant in nucleic acid degradation.

[0021] Compared with the prior art, the present invention has the following beneficial effects: The totipotent nuclease mutant provided by this invention has higher enzyme activity and temperature resistance than the wild type, and better stability. It can be used in industrial applications such as vaccine purification, residual DNA removal in biopharmaceuticals, and cell lysate treatment, and has significant technical advantages and promotional value.

[0022] The method for preparing totipotent nuclease mutants provided by this invention is simple and convenient. Through further optimization of host cells, expression vectors, induced inclusion body expression methods, and refolding methods, the expression level of totipotent nuclease mutants is effectively improved, alleviating the problem of significant decrease in activity of totipotent nucleases after refolding. Attached Figure Description

[0023] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the specific embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0024] Figure 1 SDS-PAGE images of recombinant wild-type and mutant totipotent nuclease proteins; Figure 2 Agarose gel electrophoresis images of nucleic acid degradation by recombinant wild-type and mutant totipotent nucleases. Detailed Implementation

[0025] The embodiments and examples of the present invention will be described in detail below. However, those skilled in the art will understand that the following embodiments and examples are for illustrative purposes only and should not be considered as limiting the scope of the present invention. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are within the scope of protection of the present invention. Unless otherwise specified, conventional conditions or conditions recommended by the manufacturer shall apply. Reagents or instruments whose manufacturers are not specified are all commercially available conventional products.

[0026] In this article, the term "amino acid" refers to naturally occurring amino acids and synthetic amino acids, as well as amino acid analogs and amino acid mimics that function in a similar manner to naturally occurring amino acids. Naturally occurring amino acids include those encoded by the genetic code and their modified forms, such as hydroxyproline, γ-carboxyglutamic acid, and O-phosphoserine. Common naturally occurring amino acids include: alanine (Ala; A), arginine (Arg; R), asparagine (Asn; N), aspartic acid (Asp; D), cysteine ​​(Cys; C); glutamic acid (Glu; E), glutamine (Gln; Q), glycine (Gly; G); histidine (His; H), isoleucine (Ile; I), leucine (Leu; L), lysine (Lys; K), methionine (Met; M), phenylalanine (Phe; F), proline (Pro; P), serine (Ser; S), threonine (Thr; T), tryptophan (Trp; W), tyrosine (Tyr; Y), and valine (Val; V).

[0027] The term "vector" refers to a nucleic acid delivery vehicle into which nucleotides can be inserted. When a vector enables the expression of a protein encoded by the inserted polynucleotide, it is called an expression vector. Vectors can be introduced into host cells through transformation, transduction, or transfection, allowing the genetic material they carry to be expressed in the host cells.

[0028] In a first aspect, the present invention provides a totipotent nuclease mutant, the amino acid sequence of which is shown in SEQ ID NO.1.

[0029] This invention improves key amino acid residues through site-directed mutagenesis (a site-directed mutagenesis was performed at Asn140 in the metal ion binding / substrate recognition region, replacing Asn140 with Gln), making the enzyme more stable at the metal ion binding site or active site structure, and ultimately obtaining a totipotent nuclease mutant with higher catalytic activity and stronger temperature resistance than the wild type, which can be used for DNA / RNA degradation.

[0030] The pluripotent nuclease mutant provided by this invention has broad application potential in vaccine production, antibody preparation, and nucleic acid removal, and can meet the needs of higher-standard biological agent production. Compared with existing technologies, it has significant advantages in industrial application.

[0031] In a second aspect, the present invention provides a nucleic acid molecule comprising a nucleic acid sequence encoding the pluripotent nuclease mutant.

[0032] This nucleic acid molecule can be expressed and translated to obtain the pluripotent nuclease mutant of the present invention.

[0033] In some alternative embodiments, the nucleic acid sequence encoding the pluripotent nuclease mutant is shown in SEQ ID NO. 2.

[0034] The sequence was optimized by the inventors, who found that the gene sequence has higher translation efficiency and expression level in Rosetta (DE3) host cells.

[0035] Thirdly, the present invention provides a carrier containing the aforementioned nucleic acid molecule.

[0036] By introducing the vector into recipient cells, the recipient cells are able to express the pluripotent nuclease mutant of the present invention.

[0037] In some preferred embodiments, the carrier is pET-28a.

[0038] Fourthly, the present invention provides a cell that carries the nucleic acid molecule, or contains the vector, or expresses the pluripotent nuclease mutant.

[0039] In some alternative embodiments, the cells include, but are not limited to, Rosetta (DE3), preferably Rosetta (DE3).

[0040] Fifthly, the present invention provides a method for preparing a totipotent nuclease mutant, comprising the following steps: The cells were cultured to induce the expression of inclusion bodies of the totipotent nuclease mutant, and the inclusion bodies were obtained. The inclusion bodies were then refolded and separated to prepare the totipotent nuclease mutant.

[0041] This preparation method is simple, convenient, and low in cost. It can be used to prepare pluripotent nuclease mutants and is easy to scale up industrially.

[0042] In some alternative embodiments, the cell is Rosetta (DE3) carrying pET-28a containing a nucleic acid molecule with the nucleic acid sequence SEQ ID NO.2.

[0043] This invention utilizes the combination of Rosetta(DE3) and pET-28a to compensate for rare codon (tRNA) defects, thereby significantly improving the translation efficiency and overall expression level of heterologous nuclease genes in *E. coli*. Compared to the yeast or conventional BL21 system used in some literature, this approach offers advantages in prokaryotic expression levels and process compatibility (facilitating scale-up and purification).

[0044] In some alternative embodiments, cells are induced in a culture containing 0.1-1 mM IPTG at a temperature of 37°C.

[0045] The inventors have discovered that the induction conditions provided by this invention can effectively increase the expression level of inclusion bodies.

[0046] In some alternative implementations, the complexification includes the following steps: The inclusion bodies were fully dissolved at pH 11 to obtain a solution. The solution and refolding solution were then mixed and incubated at a volume ratio of 3:1. The refolding solution was then added to bring the volume ratio of the solution and refolding solution to 2:1 and incubated. Finally, the refolding solution was added to bring the volume ratio of the solution and refolding solution to 1:1 and incubated.

[0047] This method differs from existing methods that use conventional dilution / urea or guanidine hydrochloride refolding (or ordinary dilution refolding). This invention systematically proposes a refolding process that dissolves inclusion bodies under alkaline conditions and uses a stepwise reduction of denaturant, which can reduce misfolding / mispairing of disulfide bonds, thereby significantly improving refolding yield and activity recovery (implementation data can support >70–80% activity recovery).

[0048] In some alternative implementations, the separation method includes Ni² + -NTA affinity purification.

[0049] The process of this invention eliminates the dependence on expensive or complex folding aids (such as large amounts of GSH / GSSG or dedicated chaperone proteins), avoids the low-yield problem of additional secretory expression (common in yeast / secretionary systems), and uses conventional Ni² + -NTA affinity purification can yield the target product, reducing the technical barriers and costs for pilot-scale and industrial-scale production.

[0050] Sixthly, the present invention provides the application of the above-mentioned pluripotent nuclease mutant in nucleic acid degradation.

[0051] The totipotent nuclease mutant provided by this invention has good heat / salt stability and catalytic efficiency, and can be used for the removal of nucleic acid residues in the production of vaccines, antibodies and recombinant proteins.

[0052] The present invention will be further illustrated below with specific embodiments. However, it should be understood that these embodiments are merely for the purpose of more detailed illustration and should not be construed as limiting the present invention in any way.

[0053] Example 1: Construction of a wild-type totipotent nuclease recombinant expression system 1) Enzyme digestion and ligation The pMD-18-WT-NP plasmid previously developed by our team was used as a template (the nucleic acid sequence of the wild-type totipotent nuclease (WT-NP) fragment is shown in SEQ ID NO.5).

[0054] SEQ ID NO.5: ATGCGCTTTAACAACAAAATGCTGGCGCTGGCGGCGCTGCTGTTTGCGGCGCAGGCGAGCGCGGATACCCTGGAAAGCATTGATAACTGCGCGGTGGGCTGCCCGACCGGCGGCAGCAGCAACGTGAGCATTGTGCGCCATGCGTATACCCTGAACAACAACAGCACCACCAAATTTGCGAACTGGGTGGCGTATCATATTACCAAAGATACCCCGGCGAGCGGCAAAACCCGCAACTGGAAAACCGATCCGGCGCTGAACCCGGCGGATACCCTGGCGCCGGCGGATTATACCGGCGCGAACGCGGCGCTGAAAGTGGATCGCGGCCATCAGGCGCCGCTGGCGAGCCTGGCGGGCGTGAGCGATTGGGAAAGCCTGAACTATCTGAGCAACATTACCCCGCAGAAAAGCGATCTGAACCAGGGCGCGTGGGCGCGCCTGGAAGATCAGGAACGCAAACTGATTGATCGCGCGGATATTAGCAGCGTGTATACCGTGACCGGCCCGCTGTATGAACGCGATATGGGCAAACTGCCGGGCACCCAGAAAGCGCATACCATTCCGAGCGCGTATTGGAAAGTGATTTTTATTAACAACAGCCCGGCGGTGAACCATTATGCGGCGTTTCTGTTTGATCAGAACACCCCGAAAGGCGCGGATTTTTGCCAGTTTCGCGTGACCGTGGATGAAATTGAAAAACGCACCGGCCTGATTATTTGGGCGGGCCTGCCGGATGATGTGCAGGCGAGCCTGAAAAGCAAACCGGGCGTGCTGCCGGAACTGATGGGCTGCAAAAAC。

[0055] The pMD-18-WT-NP plasmid and the pET-28a empty vector were digested separately with NdeI / XhoI restriction endonucleases according to the following system:

[0056] The enzyme digestion products were subjected to agarose gel electrophoresis and gel extraction was performed using the ATGPure Gel DNA Extraction Mini Kit (D103) from Nanjing Jujiang Biotechnology Co., Ltd. to obtain the WT-NP gene fragment and the linearized pET-28a vector.

[0057] The purified WT-NP gene fragment and the linearized pET-28a vector were prepared according to the following system, and the ligation reaction was carried out overnight at 16°C:

[0058] 2) Conversion and Screening The ligation product obtained above was converted into 100 μl of Rosetta(DE3) E. coli competent cells and incubated on ice for 30 min. Then, the cells were heat-shocked at 42 °C for 45 sec, and incubated on ice for another 5 min. 890 μl of antibiotic-free LB medium was added, and the cells were incubated at 37 °C and 200 rpm for 1 h. The cells were collected by centrifugation at 4000 rpm for 1 min, and 100 μl of the supernatant was resuspended and plated onto Kanamycin-resistant plates. The plates were incubated overnight at 37 °C, and positive clones were screened.

[0059] Colony PCR was performed using T7 primers for verification. Positive clones were selected, transferred, and sequenced to confirm that the inserted fragment was correct and without mutations. This yielded the Rosetta(DE3)-pET-28a-WT-NP recombinant wild-type totipotent nuclease expression engineered bacteria and the pET-28a-WT-NP recombinant wild-type totipotent nuclease expression vector.

[0060] 3) Fermentation expression The correctly sequenced recombinant engineered strain was inoculated into Kanamycin-resistant LB medium tubes for culture. Then, 1% of the inoculum was transferred to 50 ml of LB medium to obtain secondary seed culture. 5 ml of the secondary seed culture was then transferred to 500 ml of LB medium and cultured at 37°C and 220 rpm for 2-4 hours until OD600 = 0.2-0.8. At this point, 0.1-1 mM IPTG inducer was added to induce the expression of wild-type totipotent nuclease.

[0061] After induced fermentation, the bacterial culture was collected by centrifugation, and the bacterial sludge was weighed. The bacterial cells were resuspended in suspension buffer (20-50mM Tris-HCl, 100-800mM KCl, 0.1-0.5mM DTT, 0.1mM-2mM EDTA, pH=7.8) at a dilution ratio of 1:5 to 1:10. After the bacterial cells were thoroughly mixed, they were homogenized by high pressure homogenizer at 600-750 bar for three cycles. The supernatant and precipitate were collected by centrifugation at 13000g.

[0062] Example 2: Mutant Construction 1) Site-directed mutagenesis primer design Using the pET-28a-WT-NP recombinant wild-type totipotent nuclease expression vector plasmid as a template, forward primers (SEQ ID NO.3) and reverse primers (SEQ ID NO.4) were designed to replace Asn140 (original codons such as AAC or AAT) with Gln (CAG or CAA).

[0063] SEQ ID NO:3: GAAAAGCGATCTGCAGCAGGGCGCGTGGGCGCGCCTGGA; SEQ ID NO:4: CACGCGCCCTGCTGCAGATCGCTTTTCTGCGGGGTAATG.

[0064] 2) PCR site-directed mutagenesis The pET-28a-WT-NP recombinant wild-type totipotent nuclease expression vector plasmid was used as a template. High-fidelity PCR amplification was performed using Proofast® Max Master Mix (P202-2) from Nanjing Jujiang Biotechnology Co., Ltd., and the PCR reaction was prepared according to the following system:

[0065] Reaction procedure: pre-denaturation 95℃ 1min, denaturation 95℃ 10s, annealing 63℃ 15s, extension 72℃ 2min, cycle 30, complete extension 72℃ 5min.

[0066] 3) DpnI digestion of methylated template plasmids The PCR products were digested with DpnI to degrade the methylated template plasmid. The reaction was prepared according to the following DpnI digestion reaction system:

[0067] The mixture was thoroughly mixed and incubated at 37°C for 3 h for digestion. After the reaction, the reaction tube was placed in an 85°C water bath for 20 min to inactivate the DpnI enzyme. The digested reaction product was then subjected to agarose gel electrophoresis, and the gel was extracted using the ATGPureGel DNA Extraction Mini Kit (D102) from Nanjing Jujiang Biotechnology Co., Ltd., to obtain the linearized vector of the pET-28a-MT-NP recombinant mutant totipotent nuclease.

[0068] 4) Recombination of connections The pET-28a-MT-NP recombinant mutant totipotent nuclease linearized vector DNA fragment obtained above was subjected to self-circularization recombination using the CloneUFO One Step Cloning Kit (C101) from Nanjing Jujiang Biotechnology Co., Ltd., and the reaction was prepared according to the following recombination reaction system:

[0069] Mix well and place at 37°C for 30 min for recombination reaction.

[0070] 5) Conversion and Screening 10 μl of the recombinant product obtained above was transformed into 100 μl of Rosetta(DE3) Escherichia coli competent cells and incubated on ice for 30 min. Then, the cells were heat-shocked at 42 °C for 45 sec, followed by incubation on ice for 5 min. 890 μl of antibiotic-free LB medium was added, and the cells were incubated at 37 °C and 200 rpm for 1 h on a shaker. The cells were collected by centrifugation at 4000 rpm for 1 min, and 100 μl of the supernatant was resuspended and plated onto Kanamycin-resistant plates. The plates were incubated overnight at 37 °C, and positive clones were screened.

[0071] Colony PCR was performed using T7 primers for verification. Positive clones were selected, transferred, and sequenced to confirm that the inserted fragment was correct and without mutation. This yielded the Rosetta(DE3)-pET-28a-MT-NP recombinant mutant totipotent nuclease expression engineered bacteria and the pET-28a-MT-NP recombinant mutant totipotent nuclease expression vector (the nucleic acid sequence of the mutant is shown in SEQ ID NO.2).

[0072] 6) Fermentation expression The correctly sequenced recombinant engineered strain was inoculated into Kanamycin-resistant LB medium tubes for culture. Then, 1% of the inoculum was transferred to 50 ml of LB medium to obtain secondary seed culture. 5 ml of the secondary seed culture was then transferred to 500 ml of LB medium and cultured at 37°C and 220 rpm for 2-4 hours until OD600 = 0.2-0.8. At this point, 0.1-1 mM IPTG inducer was added to induce the expression of wild-type totipotent nuclease.

[0073] After induced fermentation, the bacterial culture was collected by centrifugation, and the bacterial sludge was weighed. The bacterial cells were resuspended in suspension buffer (20-50mM Tris-HCl, 100-800mM KCl, 0.1-0.5mM DTT, 0.1mM-2mM EDTA, pH=7.8) at a dilution ratio of 1:5 to 1:10. After the bacterial cells were thoroughly mixed, they were homogenized by high pressure homogenizer at 600-750 bar for three cycles. The supernatant and precipitate were collected by centrifugation at 13000g.

[0074] Example 3: Alkali-induced refolding and purification 1) Inclusion body dissolution The inclusion bodies obtained from Examples 1 and 2 were thoroughly and uniformly resuspended in dissolution buffer (50 mM Tris-HCl, 8 M urea, 2 mM DTT, pH 11.0). The mixtures were then magnetically stirred and gently incubated in a chromatography cabinet at 4°C for 1.5 h until completely dissolved.

[0075] 2) Gradient recombination The solutions containing the inclusion bodies obtained in Examples 1 and 2 were gradually diluted to refolding buffer (50 mM Tris-HCl, 0.5 M NaCl, 1 mM MgCl2, 5% glycerol, pH 8.5).

[0076] The specific dilution steps are as follows: 1. Add refolding solution to the solutions containing inclusion bodies obtained in Examples 1 and 2 at a ratio of 3:1 and incubate for 1 hour.

[0077] 2. Add refolding solution to the solution from step 1 at a ratio of 2:1 and incubate for 1 hour.

[0078] 3. Add the refolding solution to the solution from step 2 at a ratio of 1:1 and incubate for 1 hour.

[0079] 4. Use the refolding solution as a replacement buffer to desalt the solution from step 3 until there is no urea in the buffer solution, thus completing the refolding process.

[0080] 3) Purification The refolded solution was centrifuged at high speed (20,000 g) for 30 min at 4°C, and the insoluble precipitate was discarded. The supernatant was collected. The recombinant wild-type and mutant totipotent nuclease proteins, namely WT-NP and MT-NP, were obtained by Ni column affinity chromatography.

[0081] The above proteins were validated by SDS-PAGE gel running, and the results are as follows: Figure 1 As shown.

[0082] Example 4: Determination of nucleic acid degradation efficiency The nucleic acid degradation efficiency of the recombinant wild-type and mutant totipotent nuclease solutions obtained above, namely WT-NP and MT-NP, was determined. A 5× Reaction Buffer was prepared: 100 mM Tris-HCl, 5 mM MgCl2, 50 mM KCl, 5 mM DTT, pH 8.0. The reaction was carried out according to the following nucleic acid degradation reaction system:

[0083] The above reaction system was incubated at 37°C for 30 min. After the reaction was complete, 10 mM EDTA was added to terminate the reaction, and the system was placed in an ice bath to reduce enzyme activity. A portion of the degradation reaction solution was taken for nucleic acid concentration determination. The nucleic acid degradation efficiency of WT-NP and MT-NP under room temperature conditions was compared by measuring the absorbance (OD260) of dsDNA, and their respective degradation rates were calculated. The degradation rate of WT-NP was 46%, and the degradation rate of MT-NP was 97%.

[0084] A portion of the degradation reaction solution was subjected to agarose gel electrophoresis. The degree of DNA band degradation was observed under UV light, and the degradation efficiency was compared with that of wild-type and mutant. The results are as follows: Figure 2 (The DNA group consists of only the addition of a nucleic acid template; the WT-NP+DNA group consists of the addition of a wild-type totipotent nuclease and a nucleic acid template; and the MT-NP+DNA group consists of a mutant totipotent nuclease and a nucleic acid template.) The results show that the mutants have better degradation efficiency.

[0085] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features therein. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

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

1.

2. A nucleic acid molecule, characterized in that, The nucleic acid molecule includes a nucleic acid sequence encoding the pluripotent nuclease mutant of claim 1.

3. The nucleic acid molecule according to claim 2, characterized in that, The nucleic acid sequence encoding the pluripotent nuclease mutant is shown in SEQ ID NO.

2.

4. A carrier, characterized in that, The carrier contains the nucleic acid molecule as described in claim 2 or 3.

5. The carrier according to claim 4, characterized in that, The carrier includes pET-28a.

6. A cell, characterized in that, The cell carries the nucleic acid molecule of claim 2 or 3, or contains the vector of claim 4 or 5, or expresses the pluripotent nuclease mutant of claim 1.

7. The cell according to claim 6, characterized in that, The cells include Rosetta (DE3).

8. A method for preparing a totipotent nuclease mutant, characterized in that, Includes the following steps: The cells described in claim 6 or 7 are cultured to induce the expression of inclusion bodies of the totipotent nuclease mutant, and the inclusion bodies are obtained. The inclusion bodies are then refolded and separated to prepare the totipotent nuclease mutant.

9. The preparation method according to claim 8, characterized in that, Cells were induced in a culture containing 0.1-1 mM IPTG at a temperature of 37°C. The reversal includes the following steps: The inclusion bodies were fully dissolved at pH 11 to obtain a solution. The solution and refolding solution were then mixed and incubated at a volume ratio of 3:

1. The refolding solution was then added to bring the volume ratio of the solution and refolding solution to 2:1 and incubated. Finally, the refolding solution was added to bring the volume ratio of the solution and refolding solution to 1:1 and incubated.

10. The application of the totipotent nuclease mutant of claim 1 in nucleic acid degradation.

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