A recombinant nuclease and its application

Abstract

The present invention provides a recombinant nuclease and its applications. The amino acid sequence thereof is shown as any one of SEQ ID NO:1, SEQ ID NO:9, and SEQ ID NO:10; or a sequence having at least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99%, or at least 99.5%, or at least 99.9% sequence identity with the amino acid sequence shown as any one of SEQ ID NO:1, SEQ ID NO:9, and SEQ ID NO:10. The recombinant nuclease of the present invention still retains good non-specific nucleic acid cleavage activity under conditions of a relatively wide temperature range (4-40 °C), a relatively wide pH range (5.5-10), and a relatively wide NaCl ion concentration (250 mM-1000 mM), and can significantly reduce the nucleic acid content in the above solution and reduce the increase in solution viscosity caused by a large amount of nucleic acid.

Description

Technical Field

[0001] The present invention relates to the technical field of nucleases, and in particular to a recombinant nuclease and its application. Background Art

[0002] Currently, the non-specific nuclease widely used in the market is from Merck & Co., Inc. It is an enzyme genetically engineered from Serratia Marcescens. It can degrade all forms of DNA and RNA, including single-stranded, double-stranded, linear, circular, native, and denatured nucleic acids, digesting them into 5'-monophosphate oligonucleotides with a length of 3-8 bases, and having no base recognition specificity. Its optimal activity temperature is 37°C. It is widely used as an enzyme preparation in various scientific research and in the vaccine, protein, and polysaccharide pharmaceutical industries to remove nucleic acid residues in samples or products, improving the purity of samples and the biological efficacy of products. The enzyme can be inactivated by heating at 70°C for 10 minutes or 65°C for 20 minutes, and metal ion chelators can inhibit its activity. is produced in another Bacillus, and the CyanaseTM nuclease of RiboSolutions, Inc. in the United States is derived from another microorganism. They are all similar in performance. The main disadvantages include the inability to be effectively inactivated by high temperature, very low enzyme activity under low temperature conditions, and extremely limited tolerance to the increased salt ion concentration in the reaction system. To make up for the above disadvantages of Benzonase, three non-specific nucleases derived from psychrophilic and halophilic microorganisms have been developed in the market, hoping to maintain good enzyme activity at temperatures below 37°C or even under refrigerated conditions (4°C - 8°C), in a wide range of salt concentrations and a wide range of pH values, and to have irreversible enzyme inactivation at a certain temperature to increase its safety. These three products are CryonaseTM of Takara Bio Inc. in Japan, HL-SAN of AecticZymes AS in Norway, and Ppr nuclease of Brita Sp. z o.o. in Poland. Their problems lie in the poor tolerance to high salt, low temperature, and low pH under certain conditions, or the requirement for a relatively high Mg 2+ concentration for maintaining enzyme activity, resulting in being too sensitive to metal chelators.

[0003] Host DNA residual contamination is a very important and urgent problem in the industrial production of recombinant protein or cell and gene therapy (CGT) drugs and enzymes used for diagnosis, treatment and scientific research. For example, in nucleic acid-based diagnosis, if good sensitivity and specificity are to be maintained and there are no false positive results, the content of exogenous DNA in the system is extremely high. The production process of therapeutic proteins, vaccines and other products for medical use has extremely strict requirements for host DNA residues. Depending on the amount of product used, it is generally controlled in the range of 10pg-10ng / dose. In addition, these drugs are usually very sensitive to temperature. In order to maximize the biological activity of the sample, the sample processing process is usually carried out at 25℃ or even at 4℃. Viral vectors used in gene and cell therapy also need to effectively remove plasmid DNA and host cell DNA residues during the viral packaging process. And these values ​​are strictly regulated and supervised by regulatory agencies such as the World Health Organization (WHO), the US Food and Drug Administration (FDA), the European Medicines Agency (EMEA) and the China National Medical Products Administration (NMPA).

[0004] The ideal tool for removing nucleic acid contamination is a non-specific nuclease, which should maintain high activity at low temperatures (4°C-25°C), a wide pH range (5.5-10.0), and high salt and / or additive conditions in common purification processes (downstream purification treatment). And the nuclease should be inactivated under temperature conditions that are safe for the purified biological product.

[0005] International patent WO2006095769 describes a polypeptide with endonuclease activity from psychrophilic strain of Shewanella sp., which is a psychrophilic microorganism that exhibits high enzyme activity at low temperatures. It can be used to remove nucleic acids in protein solutions and reduce the viscosity of protein extracts. However, it needs to be incubated at 70°C for 30 minutes to be inactivated (doi:10.3389 / fbioe.2015.00148), which is very unfavorable for removing nuclease activity from biological samples after nuclease treatment, because many biological samples are inactivated after incubation at 70°C for 30 minutes.

[0006] International patent WO2013 / 121228 proposes a non-specific endonuclease and its active fragment from Aliivibriosalmonicida, and names it HL-SAN. Its enzyme activity is poor under low temperature conditions (25°C and below) except for pH=8.5.

[0007] International Patent WO2021 / 049960 describes a non-specific endonuclease from Photobacterium profundum and names it Ppr. Its optimal enzyme activity requires 50 - 150 mM MgCl 2 to be present, especially under refrigeration conditions at 6 °C, 500 mM NaCl and 100 mM MgCl 2 need to be present to maintain optimal enzyme activity. Such a high concentration of Mg 2+ proves that the enzyme is very sensitive to metal chelators, and the increased MgCl 2 brings certain uncertainties to downstream purification and the biological activity of the target protein.

[0008] Because of the various deficiencies still existing in the above-mentioned nuclease, it is necessary to develop an endonuclease with high activity, thermal instability, high efficiency and low cost under a wide temperature range, wide pH range and wide ion concentration tolerance. Summary of the Invention

[0009] Aiming at the deficiencies existing in the prior art, the present invention provides a recombinant nuclease and its application, which is a non-specific thermally unstable and highly efficient and low-cost endonuclease with high activity under a wide temperature range, wide pH range and wide ion concentration.

[0010] The present invention relates to a method for directed screening of nucleases, including obtaining a non-specific endonuclease variant from Vibrio salmonicida with directed optimization through bioinformatics and convolutional neural networks. This directed screening method uses the primary structure molecular descriptors of existing nuclease sequences as the training set to construct a convolutional neural network model; constructs a random sequence library with at least 50% identity to the non-specific endonuclease from Vibrio salmonicida and inputs the above convolutional neural network model to predict the active sequences.

[0011] In one embodiment of the present invention, the directed screening method includes: 1) screening sequences with an identity greater than 60% to the non-specific endonuclease derived from Vibrio salmonicida, performing cross-comparison and performing structural superposition clustering analysis on amino acid residues; 2) according to the results of sequence identity and amino acid residue structure matching, dividing the sequences into a framework region and a variable region, and then splitting the sequences in the variable region into five categories of short sequences including single amino acids, double amino acids, triple amino acids, quadruple amino acids, and penta amino acids; 3) by means of random sequence growth and combination, assembling the short sequences in the variable region on the basis of the framework region to establish a potential sequence library containing random sequences, and having a certain identity (50% < identity < 85%) with the sequence of the non-specific endonuclease (WP_044583181.1) derived from Vibrio salmonicida; 4) calculating the primary structure molecular descriptors of the existing nuclease sequences ((https: / / www.rcsb.org / 3d-sequence / 2PU3?assemblyId=1)) and performing normalization as the training set to pre-train a convolutional neural network including an input layer, a hidden layer, and an output layer, the hidden layer including 4 convolutional layers and 2 pooling layers, and the output layer being a densely connected layer; 5) calculating the primary structure molecular descriptors of the sequences in the potential sequence library obtained in step 3) and performing normalization, using the model trained in step 4) for prediction, and selecting the top 5 sequences most likely to have nuclease activity according to the predicted values, as shown in Table 2, SEQ ID NO:1, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10. And performing solubility prediction, as well as expression level and enzyme activity detection on these 5 sequences.

[0012] In the first aspect of the present invention, there is provided a recombinant nuclease, the amino acid sequence of which is shown as any one of SEQ ID NO:1, SEQ ID NO:9, and SEQ ID NO:10; or a sequence having at least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99%, or at least 99.5%, or at least 99.9% identity with the amino acid sequence shown as any one of SEQ ID NO:1, SEQ ID NO:9, and SEQ ID NO:10.

[0013] In one embodiment of the present invention, the recombinant nuclease is a non-specific endonuclease. The recombinant nuclease has no strict requirement for the sequence of nucleic acids and can non-specifically degrade almost all forms of nucleic acids. In one embodiment of the present invention, the substrates of the recombinant nuclease include any one or several of single-stranded, double-stranded, linear and circular DNA and RNA, RNA-DNA hybrids, and genomic DNA. The substrate can also be a nucleic acid containing base derivatives such as deoxyinosine, deoxyuridine, or hydroxymethyldeoxyuridine.

[0014] In one embodiment of the present invention, the recombinant nuclease has high nucleic acid degradation activity within a wide range of conditions and can completely digest nucleic acids into oligonucleotides with a length of 2 - 5 bases below the hybridization limit. In one embodiment of the present invention, the recombinant nuclease has nucleic acid degradation activity within the temperature range of (4 - 50 °C) or the pH range of (5.0 - 10.0). For example, in one embodiment of the present invention, the recombinant nuclease still has good activity at low temperature (4 °C). For example, in one embodiment of the present invention, the recombinant nuclease has excellent activity at pH (5.5 - 10.0). In one embodiment of the present invention, the recombinant nuclease has good enzymatic hydrolysis activity at low pH (5.5).

[0015] In one embodiment of the present invention, the recombinant nuclease can tolerate a certain concentration of salt ions (0 - 1.5 M). The recombinant nuclease has nucleic acid degradation activity within the salt ion concentration range of 0 - 1.5 M. In one embodiment of the present invention, the recombinant nuclease has excellent enzymatic hydrolysis activity when the salt ions are in the range of (0 - 1.0 M). The recombinant nuclease still has excellent enzymatic hydrolysis activity at high salt (1.0 M).

[0016] In one embodiment of the present invention, the recombinant nuclease has nucleic acid degradation activity within the Mg 2+ concentration range of 0 - 80 mM. The recombinant nuclease has excellent nucleic acid degradation activity within the Mg 2+ concentration range of 5 - 60 mM. The recombinant nuclease does not have to be activated by Mg 2+ (0 mM) and still can have excellent enzymatic hydrolysis activity.

[0017] In one embodiment of the present invention, the recombinant nuclease can tolerate a certain concentration of inhibitors, and the inhibitors include but are not limited to NaCl (0.25 - 1.5 M), urea (0 - 8 M), guanidine hydrochloride (0 - 6 M), ammonium sulfate (0 - 0.2 M), imidazole (0 - 0.5 M).

[0018] In one embodiment of the present invention, the recombinant nuclease is applicable to a variety of culture media or buffers. The culture media or buffers include RPMI1640, MEM, DMEM, CD - CHO, PBS, TBS, Tris - HCl, NaCl, MgCl 2 . The recombinant nuclease of the present invention has enzymatic activity in a variety of culture media or buffers. For example, it has enzymatic activity in culture media such as RPMI1640 medium, MEM, DMEM, CD - CHO, etc. Also, for example, it has enzymatic activity in PBS buffer, TBS buffer, Tris - HCl buffer.

[0019] In one embodiment of the present invention, compared with the high-salt-tolerant HL-SAN nuclease, the recombinant nuclease can tolerate lower concentrations of salt ions, has significant activity at 250 mM and 500 mM, and can tolerate a wider pH range (5.5 - 10) and temperature range (4 - 50 °C).

[0020] In one embodiment of the present invention, the recombinant nuclease further comprises a tag, a signal peptide, a leader peptide, and / or a nuclear localization sequence. Preferably, the tag comprises a His- or HQ-tag. In one embodiment of the present invention, the amino acid sequence of the signal peptide is Mkyllptaaagllllaaqpama (SEQ ID NO.2).

[0021] In the second aspect of the present invention, there is provided a polynucleotide encoding the above-mentioned recombinant nuclease.

[0022] In one embodiment of the present invention, the sequence of the polynucleotide is as shown in SEQ ID NO:4, or a sequence having at least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99%, or at least 99.5%, or at least 99.9% sequence identity with the sequence shown in SEQ ID NO:4.

[0023] In the third aspect of the present invention, there is provided a biological material containing the above-mentioned polynucleotide or expressing the above-mentioned recombinant nuclease.

[0024] In one embodiment of the present invention, the biological material includes a nucleic acid construct, an expression vector, and a host cell.

[0025] In one embodiment of the present invention, the nucleic acid construct further comprises one or more control sequences operably linked to the polynucleotide, and the polynucleotide is expressed in a host cell under the action of the one or more control sequences.

[0026] In one embodiment of the present invention, the control sequence includes but is not limited to a promoter and a terminator.

[0027] In one embodiment of the present invention, the promoter includes but is not limited to lac, tac, trp, trc, CMV, EF1α, H1, T7, SV40, pMC1, PGK. Preferably, the promoter is the T7 promoter.

[0028] In one embodiment of the present invention, the terminator includes but is not limited to T0, T7, rrnB, SV40, hGH, BGH, rbGlob. Preferably, the terminator is the T7 terminator.

[0029] In one embodiment of the present invention, the expression vector comprises the polynucleotide or nucleic acid construct. In one embodiment of the present invention, the expression vector can be obtained by ligating the polynucleotide and the control sequence together. In one embodiment of the present invention, the expression vector may include one or more restriction sites to allow insertion or substitution of the polynucleotide at such sites. In one embodiment of the present invention, the polynucleotide or the nucleic acid construct containing the polynucleotide is inserted into the expression vector to express the nuclease.

[0030] In one embodiment of the present invention, the expression vector can be any vector that has been subjected to DNA recombination procedures and can cause the expression of the nucleotide, for example, a plasmid or a virus. In one embodiment of the present invention, the plasmid or virus includes but is not limited to plasmid pBR322, pUC19, pACYC177, pET32a, pACYC184, pUB110, pE194, pTA1060, pAMβ1, AAV, adenovirus vector, lentiviral vector, etc.

[0031] In one embodiment of the present invention, the expression vector preferably contains one or more selectable markers that allow for convenient selection of transformed cells, transfected cells, transduced cells, etc. The selectable markers include antibiotic resistance genes, heavy metal resistance genes, nutritional marker genes, biochemical marker genes, etc. For example, ampicillin resistance gene (Ampr), kanamycin resistance gene (Kanr), tetracycline resistance gene (Tetr), streptomycin resistance gene (Strr), chloramphenicol resistance gene (Cmlr), G418 resistance gene (G418r), copper resistance gene (Cur), zinc resistance gene (Znr), cadmium resistance gene (Cdr), tryptophan synthase gene (TRP1), uracil synthase gene (URA3), leucine synthase gene (LEU2), histidine synthase gene (HIS4), β-galactosidase gene (lacZ), glucuronidase gene (GUS), chloramphenicol acetyltransferase gene (CAT), glutamine synthetase gene (glutamine synthetase, GS), dihydrofolate reductase gene (dihydrofolatereductase, DHFR), etc.

[0032] In one embodiment of the present invention, the host cells include but are not limited to bacteria, fungi, viruses, mammalian cells or plant cells.

[0033] In the fourth aspect of the present invention, there is provided a composition containing the above-mentioned recombinant nuclease, polynucleotide and / or biological material.

[0034] In one embodiment of the present invention, the composition may further contain any one or more of the following other enzymes, including DNA polymerase, protease, phosphatase, T4 PNK, RNase H, and deoxynucleotide kinase.

[0035] In one embodiment of the present invention, the composition may further include one or more of a buffer, magnesium ions, guanine nucleotides, uracil nucleotides, thymine nucleotides, ATP, sodium ions, and surfactants. The buffer includes Tris-HCl buffer, PBS (phosphate buffer), TE buffer, etc.

[0036] In the fifth aspect of the present invention, there is provided a kit including the above nuclease, polynucleotide, biomaterial, and composition.

[0037] In the sixth aspect of the present invention, there is provided an application of the above nuclease, polynucleotide, biomaterial, composition, and kit in the preparation of any one of the following reagent uses: 1) drug purification, preferably the drug includes a viral vector and a recombinant protein; 2) improving the sensitivity of gene analysis, preferably the gene analysis includes PCR, qPCR, 3SR, SDA, NGS, LAR / LCR, and LAMP; 3) reducing the viscosity of the protein lysate, preferably the protein lysate includes protein lysates of cells, tissues, and microorganisms; 4) lysing or degrading nucleic acids, where the nucleic acids include DNA or RNA.

[0038] In the seventh aspect of the present invention, there is provided a method for producing the above recombinant nuclease, which includes culturing the above host cells and collecting the recombinant nuclease from the culture.

[0039] In the eighth aspect of the present invention, there is provided a method for lysing or degrading nucleic acids, including adding the above recombinant nuclease, polynucleotide, composition, biomaterial, or kit to the nucleic acids.

[0040] In one embodiment of the present invention, the nucleic acids include single-stranded, double-stranded, linear, and circular DNA and RNA, RNA-DNA hybrids, and genomic DNA.

[0041] In the ninth aspect of the present invention, there is provided a method for removing nucleic acids from a sample, including adding the above nuclease, polynucleotide, composition, biomaterial, or kit to the sample.

[0042] In one embodiment of the present invention, the sample is a biological macromolecule sample. The sample includes nucleic acids, proteins, viral vectors, carbohydrates, polymers, or lipids. In one embodiment of the present invention, the sample includes double-stranded nucleic acids, target DNA:RNA duplexes, or recombinantly produced proteins, especially enzymes, vaccines, vaccination antigens, antibodies, and other therapeutic proteins. In one embodiment of the present invention, the sample is a reactant for nucleic acid amplification reactions or reverse transcription reactions. In one embodiment of the present invention, the nucleic acid amplification reaction is selected from PCR, qPCR, 3SR, SDA, NGS, LAR / LCR, and LAMP. In one embodiment of the present invention, the sample includes samples for protein analysis, and the protein analysis includes ELISA, column chromatography, two-dimensional electrophoresis, and blotting analysis. In one embodiment of the present invention, the sample includes lentiviral vectors (LV) in CAR-T cell therapy, adenoviral vectors (AdV) in gene therapy, and adeno-associated viral vectors (AAV).

[0043] In the tenth aspect of the present invention, a method for reducing the viscosity of a protein lysate is provided, the method including adding the nuclease, polynucleotide, composition, biomaterial, or kit to the protein lysate. The protein lysate includes protein lysates of cells, tissues, and / or microorganisms.

[0044] Compared with the prior art, the present invention has the following beneficial effects:

[0045] The thermally unstable recombinant nuclease of the present invention still retains good non-specific nucleic acid cleavage activity under conditions where salts and / or additives are present in the buffer used during the purification of macromolecular drugs (such as vaccines, antibodies, virus particles, etc.) or other processes such as PCR, cell and stem cell therapy, within a wide temperature range (4 - 40 °C), a wide pH range (5.5 - 10), and a wide NaCl ion concentration range (250 mM - 1000 mM). It can significantly reduce the nucleic acid content in the above solutions and reduce the increase in solution viscosity caused by a large amount of nucleic acids.

[0046] This nuclease can be used in the purification processes of viral vectors for gene and cell therapy (including lentiviral vectors (LV) in CAR-T cell therapy, adenoviral vectors (AdV) in gene therapy, and adeno-associated viral vectors (AAV)); it can also be used in the purification of recombinant proteins, especially in the purification processes of various enzymes, vaccines, vaccination antigens, antibodies, and other therapeutic proteins; in addition, it can be used to purify PCR, qPCR, 3SR, SDA, NGS, LAR / LCR, and LAMP reaction reagents and mixtures to reduce interfering nucleic acids (including DNA and RNA) and improve the sensitivity and specificity of related gene analysis. Or it can be used to reduce the viscosity of protein lysate samples such as cells, tissues, and microorganisms (it can be used for chemical cell lysis to eliminate the need for an ultrasonic instrument or high-pressure homogenizer, or in combination with chemical cell lysis to greatly reduce the workload of the ultrasonic instrument or high-pressure homogenizer). Description of the Drawings

[0047] Figure 1 Sequences screened with more than 60% homology to the non-specific endonuclease derived from Vibrio salmonicida

[0048] Figure 2 Plasmid map of the recombinant nuclease S1P2 constructed using the pET32a expression vector

[0049] Figure 3 Activity diagram of the recombinant nuclease S1P2 under different pH conditions

[0050] Figure 4 For different Mg 2+ Activity diagram of the recombinant nuclease S1P2 under value conditions

[0051] Figure 5 Activity diagram of the recombinant nuclease S1P2 under different temperature conditions

[0052] Figure 6 Comparison diagram of S1P2 and HL-SAN nucleases at 37°C

[0053] Figure 7 Comparison diagram of S1P2 and HL-SAN nucleases at 25°C

[0054] Figure 8 Comparison diagram of S1P2 and HL-SAN nucleases at 4°C

[0055] Figure 9 Result diagram of the viscosity influence of the S1P2 nuclease on Escherichia coli after disruption

[0056] Figure 10 Result diagram of the degradation of Escherichia coli genomic DNA by the S1P2 nuclease Detailed Description of the Invention

[0057] The technical solution 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 for illustrative explanation of the present invention and should not be construed as limiting the protection scope of the present invention. All technologies implemented based on the above content of the present invention are covered within the scope of protection intended by the present invention.

[0058] Unless otherwise specified, the raw materials and reagents used in the following embodiments are all commercially available products or can be prepared by known methods.

[0059] The term "expression" includes any step involved in polypeptide production, including but not limited to: transcription, post-transcriptional modification, translation, post-translational modification, and secretion.

[0060] The term "expression vector" means a linear or circular DNA molecule that contains a polynucleotide encoding a polypeptide and is operably linked to a control sequence that provides for its expression.

[0061] A "His-tag" refers to a polyhistidine tag typically containing at least 6 histidine residues, which can be added to the N- or C-terminus. His tags are known in the art for, for example, protein purification, but can also be used to improve solubility at low pH values. Similarly, as known in the art, an "HQ-tag" (i.e., a histidine-glutamine tag) can also be used for purification.

[0062] The term "host cell" means any cell type that is easily transformed, transfected, transduced, etc. with a nucleic acid construct or expression vector containing the polynucleotide of the present invention. The term "host cell" encompasses any progeny of a parental cell that is not completely identical to the parental cell due to mutations that occur during replication.

[0063] The term "microorganism" generally means a small organism visible through a microscope. Microorganisms typically exist in the form of single cells or cell colonies. Some microorganisms may be multicellular. Microorganisms include prokaryotes (e.g., bacteria and archaea) and eukaryotes (e.g., some fungi, algae, protozoa). In this article, viruses can be considered microorganisms.

[0064] The term "nucleic acid construct" means a single-stranded or double-stranded nucleic acid molecule that is isolated from a naturally occurring gene, or is modified in a way that does not exist in nature to contain a nucleic acid segment, or is synthetic, and the nucleic acid molecule contains one or more control sequences.

[0065] The term "operably linked" means a configuration in which a control sequence is placed at an appropriate position relative to the coding sequence of a polynucleotide such that the control sequence directs the expression of the coding sequence.

[0066] The degree of relatedness between two amino acid sequences or two nucleotide sequences is described by the parameter "sequence identity". For the purposes of the present invention, the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol. [Journal of Molecular Biology] 48:443-453) as implemented in the Needle program of the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite [EMBOSS: European Molecular Biology Open Software Suite], Rice et al., 2000, Trends Genet. [Trends in Genetics] 16:276-277) (preferably version 5.0.0 or later) is used to determine the sequence identity between two amino acid sequences. The parameters used are a gap opening penalty of 10, a gap extension penalty of 0.5, and the EBLOSUM62 (BLOSUM62 version of EMBOSS) substitution matrix. The output of Needle labeled "longest identity" (obtained using the nobrief option) is used as the percentage identity and is calculated as follows: (number of identical residues x 100) / (alignment length - total number of gaps in the alignment).

[0067] Example 1 Directed screening and optimization method of nuclease

[0068] For the optimization goal of improving the tolerance of non-specific nucleases to temperature, pH, and salt ion strength, the present invention uses bioinformatics and convolutional neural networks to obtain a directed-optimized non-specific endonuclease variant from Vibrio salmonicida. Sequences with a homology greater than 60% to the non-specific endonuclease (WP_044583181.1) from Vibrio salmonicida in the NCBI database are selected using the protein-protein Blast function (https: / / blast.ncbi.nlm.nih.gov / Blast.cgi?PROGRAM=blastp&PAGE_TYPE=BlastSearch&LINK_LOC=blasthome), and their sequence alignments are shown in Figure 1 and Table 1.

[0069] Table 1 Endonuclease sequences with homology greater than 60%

[0070]

[0071]

[0072] The specific technical solution of this method includes the following steps:

[0073] Step 1: Perform cross-alignment on the initial sequences and conduct structural superposition clustering analysis on amino acid residues.

[0074] Step 2: According to the results of sequence identity and amino acid residue structure matching, divide the sequences into a framework region and a variable region. The framework region is used as the conserved sequence ( Figure 1 the sequence covered by yellow in the sequence alignment), and the variable region is used as the fragmented sequence. Then, split the sequences in the variable region into five categories of short sequences including single amino acid, double amino acid, triple amino acid, quadruple amino acid, and quintuple amino acid.

[0075] The basic amino acids in the framework region sequence include the following amino acids or amino acid combinations. The numbers in parentheses are the amino acid positions of the polypeptide:

[0076] P(3) / F(6) / AK(9,10) / IY(16,17) / D(19) / SFYCGC(23 - 28) / I(30) / W(32) / KK(35,36) / G(38) / P(40) / L(42) / CGY(45 - 47) / RKQ(50 - 52) / RA(55,56) / RIEWEH(58 - 63) / VPA(65 - 67) / FG(70 - 71) / W(77) / W(80) / M(95) / D(98) / HNL(100 - 102) / P(104) / GE(107,108) / NGDR(110 - 113) / N(115) / F(118) / W(121) / G(126) / YGQC(129 - 132) / F(138) / PP(145,146) / G(151) / IAR(153 - 155) / Y(157) / YM(159,160) / Y(164) / L(168) / Q(172) / LM(175,176) / AW(178,179) / WEC(188 - 190) / RD(192,193) / R(195) / Q(200) / N(204) / C(211).

[0077] The basic amino acids in the variable region sequence include:

[0078] AP / SS / AVK / HPT / QV / WQ / HQ / QC / QD / GRKNC / DK / FK / EA / NGN / KVD / RRAD / SR / QE / RQ / QYP / IA / NH.

[0079] Step 3: Combine and assemble short sequences in the fragment region on the basis of the backbone region by means of random sequence growth and combination to establish a potential sequence library containing random sequences. The sequences in this library are 213 - 216 amino acids in length and have a homology (50% < homology < 85%) that meets certain conditions with the sequence of a non-specific endonuclease (WP_044583181.1) derived from Vibrio salmonicida.

[0080] Step 4: Obtain nuclease sequences (https: / / www.rcsb.org / 3d-sequence / 2PU3?assemblyId=1) with Mg 2+ binding ability and nucleic acid catalytic cleavage ability through literature research and the RCSB-PDB database, calculate the primary structure molecular descriptors, and then use them as a training set after normalization to pre-train a convolutional neural network containing an input layer, a hidden layer, and an output layer. The hidden layer contains 4 convolutional layers and 2 pooling layers, and the output layer is a densely connected layer.

[0081] Step 5: Calculate the primary structure molecular descriptors of the sequences in the potential sequence library obtained in Step 3 and perform normalization processing. Use the model trained in Step 4 for prediction, and select the top 5 sequences (see Table 2) that are most likely to have nuclease activity according to the prediction values. Use an online protein solubility prediction tool (https: / / www.novopro.cn / tools / prot-sol.html; doi: 10.1093 / bioinformatics / btx345) to predict the solubility of the candidate nucleases. Use Alphafold2 to predict the similarity of the structures of the selected sequences to the non-specific endonuclease (WP_044583181.1) derived from Vibrio salmonicida, and select four optimal sequences for expression and detect their enzyme activities.

[0082] Table 2 Five selected sequences

[0083]

[0084]

[0085] Example 2 Construction of the S1P2 recombinant nuclease expression plasmid

[0086] Construct the S1P2 recombinant nuclease expression plasmid using the pET32a expression vector, as Figure 2As shown in the figure. Among them, the amino acid sequence of S1P2 (SEQ ID NO: 1) is shown in Table 2, and the amino acid sequence of the PelB signal peptide (SEQ ID NO: 2) is Mkyllptaaagllllaaqpama, with a length of 22 AA; the sequence of the His tag (SEQ ID NO: 3) with a TEV protease cleavage site is Asmehhhhhhgsenlyfqs, with a length of 19 AA.

[0087] The nucleotide sequence encoding pelB-His-TEVsite-S1P2 is shown in SEQ ID NO.4, with a length of 783 bp. The nucleotide sequence of pET32a-pelB-His-TEVsite-S1P2 is shown in SEQ ID NO.5, with a length of 6162 bp.

[0088] Example 3 Obtaining of Escherichia coli strains and expression of S1P2 recombinant nuclease

[0089] Obtaining of Escherichia coli recombinant strains: In order to obtain the pET32a-pelB-His-TEVsite-S1P2 recombinant strain of Escherichia coli BL21(DE3). The pET32a-pelB-His-TEVsite-S1P2 plasmid synthesized by GenScript was used for the transformation of BL21(DE3). After picking monoclonal colonies and identification, the pET32a-pelB-His-TEVsite-S1P2 recombinant strain of BL21(DE3) in LB medium was obtained for the expression of S1P2 nuclease.

[0090] Expression of S1P2 recombinant nuclease: The overnight-activated bacterial solution (1:50) was inoculated into a conical flask containing LB medium. When OD600 = 0.4 - 0.5 at 30 °C, IPTG was added with a final concentration of 0.2 mM, and induction was carried out at 18 °C for 20 - 22 h. The bacterial solution was centrifuged at 12000 r / min for 10 min at 4 °C. The bacterial precipitate was washed twice with buffer (25 mM Tris-HCl PH7.5, 0.5 M NaCl, 5 mM MgCl2, 10 mM imidazole), weighed for wet weight, resuspended with 5 - 10 ml / g buffer, PMSF was added to prevent protein degradation, and ultrasonic disruption was carried out at 350 w, with the ultrasound on for 2 s and off for 4 s. Ultrasonic treatment was carried out for 30 min, and fresh ice was changed every 10 minutes. Centrifugation was carried out at 12000 r / min for 10 min to remove insoluble proteins, and the protein supernatant was filtered through a 0.45 um filter membrane.

[0091] Purification of S1P2 recombinant nuclease: Purification was carried out using an affinity chromatography nickel column. The sample was immobilized on the nickel column, and the S1P2 nuclease was eluted with 25 mM Tris-HCl pH 7.5, 0.5 M NaCl, 5 mM MgCl2, and 300 mM imidazole. Washing to remove impurities could be performed with 60 mM imidazole. The S1P2 nuclease sample eluted from the nickel column was further purified by SEC molecular sieve (select G200), then ultrafiltered and concentrated to finally obtain S1P2 nuclease with a purity of over 98%. The obtained S1P2 was measured for concentration using a nanodrop with the molar extinction coefficient and molecular weight. Finally, the enzyme was stored in 50% (v / v) (25 mM Tris-HCl pH 7.5, 0.5 M NaCl, 5 mM MgCl2), 50% (v / v) Glycerol, 0.01% (v / v) Tween 20, and frozen at -20°C.

[0092] Example 4 Influence of S1P2 Recombinant Nuclease Activity under Different Conditions

[0093] 1. Detection method for the enzymatic activity characteristics of S1P2 recombinant nuclease: The nuclease was added to 1 ml of calf thymus DNA (50 μg / mL) (dissolved in 25 mM Tris-HCl pH 8.5, 0.5 M NaCl, 5 mM MgCl 2 ), incubated at 37°C for 30 min, and the reaction was terminated by rapidly adding 4% perchloric acid (to a final concentration of 2% perchloric acid to terminate the reaction), placed on ice for 60 min, centrifuged at 14000 r / min for 10 min, and then the OD260nm value was measured (the undegraded DNA precipitate was separated by centrifugation, and the content of free nucleotides and oligonucleotide fragments less than 10 bp in the supernatant was confirmed by measuring OD260nm). The blank without the enzyme solution was used as a control. The amount of enzyme when the difference in absorbance at 260 nm of the nucleotide amount generated under the above fixed conditions was 1.0 was defined as one enzyme activity unit.

[0094] 2. Determination of the optimal buffer pH condition for S1P2 recombinant nuclease

[0095] To determine the optimal pH of the nuclease, the reaction was carried out as described in step 1, and 1 U of the enzyme (pH 5.5 - 10) was added to the reaction mixtures at 0 M, 0.25 M, 0.5 M, 0.75 M, and 1 M. The results are as Figure 3 shown. It can be seen from the results that S1P2 nuclease exhibits the highest activity in a buffer with pH 8.5 at a 250 mM NaCl concentration.

[0096] 3. Determination of the optimal Mg 2+ for S1P2 recombinant nuclease

[0097] To determine the optimal Mg2+ for the nuclease, the reaction was carried out as described in step 1, and 1 U of the enzyme (25 mM Tris-HCl PH8.5, 0.5 M NaCl, 37 °C) was added to the reaction mixtures at 0 mM, 5 mM, 10 mM, 15 mM, 20 mM, 50 mM, 60 mM, 80 mM, 100 mM, 150 mM, and 200 mM. The results are as Figure 4 shown. It can be seen from the results that the S1P2 nuclease exhibits the highest activity in a buffer with a Mg 2+ concentration of 10 mM, the optimal range is 5 - 60 mM Mg 2+ concentration, and there is still a certain enzyme activity (34%) at 0 mM Mg 2+ concentration.

[0098] 4. Confirmation of the potential effects of various additives on the enzyme activity of S1P2 recombinant nuclease

[0099] To determine the tolerance range of common ionic components in the reaction buffer during the purification process, the reaction was carried out as described in step 1 in different inhibitor solutions (NaCl, urea, guanidine hydrochloride, ammonium sulfate, imidazole), as shown in Table 3. It can be seen from the results that S1P2 has a certain activity at the following concentrations.

[0100] Table 3 Concentrations of different inhibitor solutions

[0101]

[0102] 5. Thermal inactivation temperature of S1P2 recombinant nuclease

[0103] To determine the activity of the nuclease at different temperatures, the reaction was carried out as described in step 1, and 1 U of the enzyme was added to the reaction mixtures at different temperatures (4 - 50 °C). The results are as Figure 5 shown. It can be seen from the results that the S1P2 nuclease exhibits the highest activity in a buffer of approximately 35 °C, 25 mM Tris-HCl PH8.5, 0.5 M NaCl, 5 mM MgCl 2 .

[0104] 6. Comparison of S1P2 and HL-SAN nucleases

[0105] The enzyme activities of S1P2 and HL-SAN were determined under the following conditions. Different temperatures (4 °C, 25 °C, 37 °C), different NaCl concentrations (0 - 1 M), different pH values (5, 5 - 10), in 25 mM Tris-HCl, 5 mM MgCl 2 . Compared with the high-salt-tolerant HL-SAN, S1P2 has a certain activity at low salt, significant activity at 250 mM and 500 mM, and a wider pH tolerance range, as shown respectively in Figure 6(37 °C), Figure 7 (25 °C) and Figure 8 (4 °C) are shown. Among them, Figure 6 a, Figure 6 b, Figure 6 c, Figure 6 d, Figure 6 e, Figure 6 f, Figure 6 g, Figure 6 h have pH values of 5.5, 6, 7, 7.5, 8, 8.5, 9, 10 respectively; Figure 7 a, Figure 7 b, Figure 7 c, Figure 7 d, Figure 7 e, Figure 7 f, Figure 7 g, Figure 7 h have pH values of 5.5, 6, 7, 7.5, 8, 8.5, 9, 10 respectively; Figure 8 a, Figure 8 b, Figure 8 c, Figure 8 d, Figure 8 e, Figure 8 f, Figure 8 g, Figure 8 h have pH values of 5.5, 6, 7, 7.5, 8, 8.5, 9, 10.

[0106] 7. Enzyme activity of S1P2 nuclease in different culture media and common buffer solutions PBS and TBS

[0107] At 37 °C, the reaction was carried out in the manner described in step 1, and the activity in different culture media and buffer solutions was determined. The results are shown in Table 4.

[0108] Table 4 Enzyme activity in different culture media and buffer solutions

[0109]

[0110]

[0111] 8. S1P2 nuclease can significantly reduce the viscosity after the disruption of Escherichia coli

[0112] The recombinant Escherichia coli constructed with pET28a (0.2 g) was suspended in cell lysis buffer (25 mM Tris-HCl 250 mM NaCl 5 mM MgCl 2 25 mg / ml lysozyme, with a concentration of 0.4 g / L), and then the cell lysate was incubated with S1P2 at 4 °C for 1 h and centrifuged at 12,000 r / min for 2 min. The control was without enzyme. As Figure 9The results of photographing after centrifugation are shown. A is the lysate containing 30 US1P2 nuclease, and B is the lysate without nuclease.

[0113] 9. S1P2 nuclease can degrade the genomic DNA of Escherichia coli

[0114] Genomic DNA was extracted from recombinant Escherichia coli constructed with pET28a. 1 U of S1P2 nuclease was added to 1000 ng of gDNA (50 ng / ul) (dissolved in 25 mM Tris / HCl PH8.5, 5 mM MgCl 2 ) and different NaCl concentrations, and incubated at 37 °C for 30 min. The control was incubated at 37 °C for 30 min without adding the enzyme. A 1% agarose gel was quickly run and separated at a voltage of 120 V for 35 min. The photographing results are as Figure 10 shown. From the results, it can be seen that S1P2 nuclease can degrade the genomic DNA of Escherichia coli at different NaCl concentrations.

[0115] 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 preferred embodiments, those of ordinary skill in the art should understand that the technical solutions of the present invention can be modified or equivalently replaced without departing from the purpose and scope of the technical solutions of the present invention, and they should all be covered within the scope of the claims of the present invention.

[0116] SEQ ID NO.1: The amino acid sequence of S1P2 is shown in Table 2: S1P2

[0117] SEQ ID NO.2: The amino acid sequence of PelB signal peptide (length: 22 AA)

[0118]

[0119] SEQ ID NO.3: His tag with TEV protease cleavage site (length: 19 AA)

[0120]

[0121] SEQ ID NO.4

[0122]

[0123] SEQ ID NO.5: pET32a-pelB-His-TEVsite-S1P2 (length: 6162 bp)

[0124]

[0125]

[0126]

Claims

1. A recombinant nuclease, characterized in that: Its amino acid sequence is the sequence shown in SEQ ID NO:

1.

2. A recombinant nuclease according to claim 1, characterized in that: The recombinant nuclease is a non-specific endonuclease.

3. A recombinant nuclease according to claim 1, characterized in that: The substrate of the recombinant nuclease includes any one or more of single-stranded, double-stranded, linear and circular DNA and / or RNA, RNA-DNA hybrid, and genomic DNA.

4. A recombinant nuclease according to claim 1, characterized in that: The recombinant nuclease has nucleic acid degradation activity within the temperature range of 4 to 50° C. or the pH value range of 5.0 to 10.

0.

5. The recombinant nuclease according to claim 1, characterized in that: The recombinant nuclease has nucleic acid degradation activity within the range of salt ion concentration of 0 to 1.5M.

6. A recombinant nuclease according to claim 1, characterized in that: The recombinant nuclease is in Mg 2+ It has nucleic acid degradation activity in the concentration range of 0 to 80M.

7. A recombinant nuclease according to claim 1, characterized in that: The recombinant nuclease has nucleic acid degradation activity under the action of a certain concentration of inhibitor.

8. A recombinant nuclease according to claim 7, characterized in that: The inhibitors include NaCl, urea, guanidine hydrochloride, ammonium sulfate, and imidazole.

9. A recombinant nuclease according to claim 8, characterized in that: The concentration of the inhibitor is 0-8M urea, 0-6M guanidine hydrochloride, 0-0.2M ammonium sulfate, and 0-0.5M imidazole.

10. The recombinant nuclease according to claim 1, characterized in that: The recombinant nuclease has nucleic acid degradation activity in a culture medium or a buffer.

11. A recombinant nuclease according to claim 10, characterized in that: The culture medium or buffer includes RPMI1640, MEM, DMEM, CD-CHO, PBS, TBS, Tris-HCl, NaCl, and MgCl2.

12. A recombinant nuclease according to claim 1, characterized in that: The recombinant nuclease further comprises a tag, a signal peptide, a leader peptide and / or a nuclear localization sequence.

13. A recombinant nuclease according to claim 12, characterized in that: Such tags include His- or HQ-tags.

14. A recombinant nuclease according to claim 12, characterized in that: The amino acid sequence of the signal peptide is shown in SEQ ID NO.

2.

15. A polynucleotide encoding the recombinant nuclease according to any one of claims 1 to 14.

16. A biological material containing the polynucleotide according to claim 15 or expressing the recombinant nuclease according to any one of claims 1 to 14.

17. The biomaterial according to claim 16, characterized in that: The biological materials include nucleic acid constructs, expression vectors, and host cells.

18. The biomaterial according to claim 17, characterized in that: The nucleic acid construct further comprises one or more control sequences operably linked to the polynucleotide, and the polynucleotide is expressed in a host cell under the action of the one or more control sequences.

19. The biomaterial according to claim 18, characterized in that: The control sequence includes but is not limited to a promoter and a terminator.

20. The biomaterial according to claim 17, characterized in that: The expression vector comprises the polynucleotide or nucleic acid construct.

21. The biomaterial according to claim 17, characterized in that: The expression vector includes a plasmid or a virus.

22. The biomaterial according to claim 17, characterized in that: The expression vector includes one or more selectable markers.

23. The biomaterial according to claim 22, characterized in that: The selective markers include antibiotic resistance genes, heavy metal resistance genes, nutritional marker genes, and biochemical marker genes.

24. The biomaterial according to claim 17, characterized in that: The host cell includes bacteria, fungi, viruses, mammalian cells or plant cells.

25. A composition, characterized in that Containing the recombinant nuclease according to any one of claims 1 to 14, the polynucleotide according to claim 15 and / or the biological material according to any one of claims 16 to 24.

26. The composition of claim 25, wherein: The composition further contains any one or more of the following other enzymes: DNA polymerase, protease, phosphatase, T4 PNK, RNase H, deoxynucleotide kinase.

27. The composition of claim 25, wherein: The composition further comprises one or more of a buffer, magnesium ions, guanine nucleotides, uracil nucleotides, thymine nucleotides, ATP, sodium ions, and a surfactant.

28. The composition of claim 27, wherein: The buffer includes Tris-HCl buffer, PBS and TE buffer.

29. A kit comprising the recombinant nuclease of claim 1, the polynucleotide of claim 15, the biological material of any one of claims 16 to 24, and the composition of any one of claims 25 to 28.

30. Use of the recombinant nuclease according to any one of claims 1 to 14, the polynucleotide according to claim 15, the biological material according to any one of claims 16 to 24, the composition according to any one of claims 25 to 28, and the kit according to claim 29 in the preparation of any of the following reagents: 1) drug purification; 2) improving the sensitivity of gene analysis; 3) reducing the viscosity of protein lysate; 4) cleaving or degrading nucleic acids, wherein the nucleic acids include DNA or RNA.

31. The use according to claim 30, characterized in that: The drugs include viral vectors and recombinant proteins.

32. The use according to claim 30, characterized in that: The genetic analysis includes PCR, qPCR, 3SR, SDA, NGS, LAR / LCR and LAMP.

33. The use according to claim 30, characterized in that: The protein lysate includes protein lysate of cells, tissues and microorganisms.

34. A method for producing a recombinant nuclease, characterized in that: The method comprises culturing the biological material according to claim 17, and collecting the recombinant nuclease from the culture.

35. A method for cleaving or degrading nucleic acid, characterized in that: The method comprises adding the recombinant nuclease according to any one of claims 1 to 14, the polynucleotide according to claim 15, the biological material according to any one of claims 16 to 24, the composition according to any one of claims 25 to 28, and the kit according to claim 29 to the nucleic acid.

36. A method for cleaving or degrading nucleic acid according to claim 35, characterized in that: The nucleic acids include single-stranded, double-stranded, linear and circular DNA and RNA, RNA-DNA hybrids, and genomic DNA.

37. A method for removing nucleic acid from a sample, characterized in that: The method comprises adding the recombinant nuclease according to any one of claims 1 to 14, the polynucleotide according to claim 15, the biological material according to any one of claims 16 to 24, the composition according to any one of claims 25 to 28, and the kit according to claim 29 to a sample.

38. A method for removing nucleic acids from a sample as claimed in claim 37, characterized in that: The sample comprises protein.

39. A method for removing nucleic acids from a sample as claimed in claim 38, characterized in that: The protein includes a double-stranded nucleic acid, a DNA:RNA duplex of interest, or a recombinantly produced protein.

40. A method for removing nucleic acid from a sample as claimed in claim 39, characterized in that: Such proteins include enzymes, vaccines, vaccination antigens, antibodies, and other therapeutic proteins.

41. A method for removing nucleic acid from a sample as claimed in claim 37, characterized in that: The sample includes a viral vector.

42. A method for removing nucleic acids from a sample as claimed in claim 41, characterized in that: The viral vectors include lentiviral vectors in CAR-T cell therapy, adenoviral vectors and adeno-associated viral vectors in gene therapy.

43. A method for removing nucleic acids from a sample as claimed in claim 37, characterized in that: The sample includes reactants of a nucleic acid amplification reaction or a reverse transcription reaction.

44. A method for removing nucleic acids from a sample as claimed in claim 43, characterized in that: The nucleic acid amplification reaction is selected from PCR, qPCR, 3SR, SDA, NGS, LAR / LCR and LAMP.

45. A method for removing nucleic acids from a sample as claimed in claim 37, characterized in that: The samples include samples for protein analysis.

46. ​​A method for removing nucleic acids from a sample as claimed in claim 45, characterized in that: The protein analysis includes ELISA, column chromatography, two-dimensional electrophoresis and western blot analysis.

47. A method for reducing the viscosity of a protein lysate, characterized in that: The method comprises adding the recombinant nuclease according to any one of claims 1 to 14, the polynucleotide according to claim 15, the biological material according to any one of claims 16 to 24, the composition according to any one of claims 25 to 28, and the kit according to claim 29 to the protein lysate.

48. A method for reducing the viscosity of a protein lysate according to claim 47, characterized in that: The protein lysate includes protein lysate of cells, tissues and / or microorganisms.

Images