SSB protein mutant and preparation method thereof

Abstract

The invention discloses an SSB protein mutant and a preparation method thereof. The invention provides a single-stranded DNA binding protein (SSB) mutant, which is characterized in that a high-purity and high-temperature-resistant SSB protein mutant is obtained through a protein engineering modification platform by taking SSB protein from escherichia coli as a template through the following multi-site mutated amino acid sequence combination K136L / K224F / E230W, so that the SSB protein mutant can be used for preparing the SSB protein mutant under the environmental conditions of normal-temperature or higher-temperature transportation and storage and the like. Therefore, a series of application scenes and use limitations of the SSB protein in nucleic acid amplification and sequencing can be expanded.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to an SSB protein mutant and its preparation method. Background Technology

[0002] Single-stranded DNA-binding proteins (SSBs) are a class of proteins that specifically bind to single-stranded DNA (ssDNA) and RNA. Their main function is to participate in DNA replication and recombination in vivo. They function by forming nucleoprotein complexes with ssDNA in the system, while exhibiting low preference for single-stranded DNA (dsDNA). DNA mainly exists in a double helix structure in cells. During DNA replication, DNA helicase moves along the replication fork, producing a segment of ssDNA. ssDNA is unstable and quickly re-pairs to form double-stranded DNA or is degraded by nucleases. SSBs bind to the ssDNA produced by DNA helicase, effectively maintaining the presence of ssDNA and ensuring DNA replication.

[0003] SSB proteins derived from *E. coli* have a wide range of functional applications in molecular biology, playing crucial roles in DNA replication, repair, recombination, and telomere maintenance. They are widely used in various PCR-based DNA sequencing, multiplex PCR, RNA reverse transcription, DNA sequencing experiments, RT-PCR, and isothermal amplification. SSB can specifically bind to ssDNA to form homotetramers, thereby protecting ssDNA from nuclease degradation. Therefore, SSB proteins must undergo high-temperature denaturation during PCR amplification, requiring thermal stability to maintain activity under high-temperature conditions. Under high-temperature conditions, SSB should maintain stable structure and function, improving PCR yield and specificity, increasing reverse transcription yield and extension in RT-PCR, and enhancing DNA sequencing of regions with strong secondary structure. Therefore, developing SSB protein mutants with higher thermal stability and better preservation of biological activity is crucial for gene detection. Summary of the Invention

[0004] To overcome the shortcomings of existing technologies, the purpose of this invention is to provide an SSB protein mutant with higher thermal stability, its preparation method, and its application.

[0005] To achieve the above objectives, the present invention adopts the following technical solution: The first aspect of the present invention provides an SSB protein mutant, which is obtained by mutation based on wild-type SSB protein; wherein the amino acid sequence of the wild-type SSB protein is shown in SEQ ID NO:1.

[0006] Furthermore, the nucleotide sequence of the wild-type SSB protein is shown in SEQ ID NO:2.

[0007] Furthermore, the amino acid sequence of the wild-type SSB protein is shown in SEQ ID NO:3.

[0008] Furthermore, the nucleotide sequence of the wild-type SSB protein is shown in SEQ ID NO:4.

[0009] Furthermore, the mutation site of the mutant is selected from any one or a combination of more than one of K136L, K224F, E230W, and N232K.

[0010] Furthermore, the mutation site of the mutant is selected from a combination of K136L / K224F / E230W.

[0011] In this invention, "AxxxB" refers to the mutation of amino acid A at position xxx to amino acid B. For example, "E30I" indicates that glutamic acid (E) at position 30 is mutated to isoleucine (I), and so on. For mutants with dual or multiple mutation sites, each mutation site is separated by " / ". For example, K136L / K224F / E230W indicates that, relative to the amino acid sequence of the wild-type SSB protein, lysine (K) at position 136 is replaced by leucine (L), lysine (K) at position 224 is replaced by phenylalanine (F), and glutamic acid (E) at position 230 is replaced by tryptophan (W). All three mutations are present in the specific SSB protein mutant.

[0012] In some embodiments, the SSB protein mutants described in this invention also include SSB protein mutants corresponding to amino acid sequences that have at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with the SSB protein mutants described in this invention, and their functions include being equivalent to, slightly reduced in, slightly improved in, or significantly improved in the SSB protein mutants described in this invention. All of the above-mentioned SSB protein mutants are also included within the protection scope of this invention.

[0013] In some embodiments, the term "identity" as used in this invention refers to the sequence identity between two nucleic acid molecules or polypeptides, the same as "homology." Identity can be determined by comparing positions in the sequences that can be aligned for comparison purposes. When a position in the compared sequences is occupied by the same base, the molecules are identical at that position. The degree of identity or similarity between nucleic acid or amino acid sequences is a function of the number of identical or matching nucleotides at common positions between the nucleic acid sequences. Various alignment algorithms and / or procedures can be used to calculate the identity between two sequences, including FASTA or BLAST, which are available as part of the GCG sequence analysis package (University of Wisconsin, Madison, Wis.) and can be used, for example, by default.

[0014] In some implementations, the “identity” is calculated using a global alignment (i.e., comparing two sequences over their full length). Methods for comparing the identity of two or more sequences are well known to those skilled in the art. For example, when performing a global alignment, the “Needle” procedure can be used, which employs the Needleman-Wunsch global alignment algorithm (Needleman and Wunsch, 1970 J. Mol. Biol. 48: 443-453) to find the optimal alignment (including the gap) of two sequences while considering their full length. This Needle procedure is available, for example, on the World Wide Web website ebi.ac.uk. The identity percentage according to the invention is preferably calculated using the EMBOSS: needle (global) procedure with a “gap open” parameter equal to 10.0, a “gap extension” parameter equal to 0.5, and a Blosum62 matrix.

[0015] This invention demonstrates, through recombinase transcription-mediated amplification (RTMA), that the SSB protein mutant of this invention possesses higher thermal stability, can stably exert its activity within the range of 20-45℃, and can maintain high reactivity even after long-term storage at 45℃.

[0016] A second aspect of the present invention provides a polynucleotide molecule that encodes the SSB protein mutant described in the first aspect of the present invention.

[0017] As used in this invention, the term "polynucleotide molecule" is intended to include polymeric forms of nucleotides of any length containing deoxyribonucleotides, ribonucleotides, and / or their analogues, including DNA, RNA, and DNA / RNA hybrids, and also including DNA or RNA analogues, such as those containing a modified backbone (e.g., peptide nucleic acid (PNA) or phosphate thioester) or modified bases. Therefore, nucleic acids of this invention include DNA, cDNA, mRNA, recombinant nucleic acids, etc.

[0018] When applied to polynucleotide molecules, the term "encoding" refers to a polynucleotide that, if in its natural state or when manipulated by methods known to those skilled in the art, can be transcribed and / or translated to produce an mRNA containing a polypeptide and / or fragments thereof, is called "encoding" the polypeptide. The antisense strand is the complement of this nucleic acid, and the coding sequence can be deduced from it.

[0019] In some embodiments, the polynucleotide molecule is isolated or purified. The sequence of the nucleic acid molecule can be obtained using conventional techniques. Once the relevant sequence is obtained, it can be obtained in large quantities using recombinant methods. This typically involves cloning it into a vector, transforming it into cells, and then isolating the relevant sequence from the proliferated host cells using conventional methods. Alternatively, the relevant sequence can be synthesized artificially, especially when the fragment length is short. Generally, longer fragments can be obtained by first synthesizing multiple small fragments and then ligating them.

[0020] A third aspect of the present invention provides a carrier comprising the polynucleotide molecule described in the third aspect of the present invention.

[0021] In this invention, a vector refers to an artificial construct capable of delivering and preferably expressing one or more target genes or sequences in a host cell. The vector used in this invention is not limited and can be an expression vector, viral vector, etc. Known vectors or self-constructed vectors can be used. Known vectors include plasmid vectors, lentiviral vectors, adenovirus vectors, AAV viral vectors, etc.

[0022] In some embodiments, the expression vector may contain expression regulatory sequences, such as transcription and translation start and stop codons, which are specific to the type of host cell (e.g., bacteria, fungi, plants, or animals) into which the vector is to be introduced, depending on the circumstances and whether the vector is DNA-based or RNA-based. Recombinant expression vectors may contain restriction sites to facilitate cloning.

[0023] In some embodiments, the vector may also contain one or more marker genes that allow selection of host cells for transformation or transfection. Marker genes include biocidal resistance (e.g., resistance to antibiotics, heavy metals, etc.); prototrophic complementation in auxotrophic hosts, etc. Suitable marker genes for the expression vector of the present invention include, for example, neomycin / G418 resistance genes, hygromycin resistance genes, histidine resistance genes, tetracycline resistance genes, ampicillin resistance genes, kanamycin resistance genes, and puromycin resistance genes.

[0024] In some implementations, the expression vector can be constructed using methods well known to those skilled in the art. These methods include, but are not limited to, recombinant DNA technology, DNA synthesis technology, etc. DNA encoding the SSB protein mutant can be effectively ligated to a multiple cloning site in the vector to guide mRNA synthesis and thus protein expression, or for homologous recombination.

[0025] A fourth aspect of the present invention provides a host cell comprising the polynucleotide molecule described in the second aspect of the present invention or the vector described in the third aspect of the present invention.

[0026] Furthermore, the host cell includes prokaryotic cells or eukaryotic cells.

[0027] In some embodiments, the prokaryotic cells include bacteria, actinomycetes, cyanobacteria, mycoplasma, chlamydia, and rickettsiae. The bacteria include *Escherichia coli*, *Bacillus subtilis*, *Salmonella typhimurium*, *Pseudomonas*, *Streptomyces*, and *Staphylococcus*. The eukaryotic cells include mammalian cells, insect cells, plant cells, and yeast cells. Any cell known to those skilled in the art as a host cell may be used.

[0028] In some embodiments, the host cells are prepared by introducing the nucleic acid molecules or expression vectors of the present invention as described above into the host cells. The methods of introduction include, but are not limited to, physical, chemical, and biological methods. The physical methods include, but are not limited to, microinjection, electroporation, calcium phosphate precipitation, lipid transfection, and particle bombardment. The chemical methods include, but are not limited to, colloidal dispersion systems and lipid-based systems. The colloidal dispersion systems include, but are not limited to, macromolecular complexes, nanocapsules, microspheres, and beads. The lipid-based systems include, but are not limited to, oil-in-water emulsions, micelles, mixed micelles, and liposomes. The biological methods include, but are not limited to, DNA vectors, lentiviral vectors, poxvirus vectors, herpes simplex virus vectors, adenovirus vectors, and adeno-associated virus vectors.

[0029] A fifth aspect of the present invention provides a complex comprising the SSB protein mutant described in the first aspect of the present invention and a single-stranded target nucleic acid molecule that binds to the SSB protein mutant.

[0030] Furthermore, the single-stranded target nucleic acid molecule is single-stranded DNA or RNA.

[0031] The sixth aspect of the present invention provides any of the following products: 1) A derivative comprising the SSB protein mutant described in the first aspect of the present invention and a peptide tag that can be attached to the SSB protein mutant.

[0032] 2) A kit comprising the SSB protein mutant described in the first aspect of the present invention, and one or more of the following: DNA polymerase, dNTP or a mixture of dNTP and ddNTP, nuclease, buffer, recombinase, reducing agent, recombinase helper protein, crowding agent, ATP or ATP analog, and salt ions.

[0033] 3) A reaction system comprising the SSB protein mutant described in the first aspect of the present invention and one or more of the following: primer pair, recombinase, polymerase, nuclease, dNTP, crowding agent, recombinase helper protein, ATP or ATP analog, and salt ions.

[0034] Furthermore, the peptide tags include detection tags, purification tags, localization tags, and functional research tags.

[0035] In some implementations, the detection tags include fluorescent protein tags (such as GFP, RFP, YFP / CFP), bioluminescent tags (such as luciferase), and chemiluminescent tags (such as APEX); the purification tags include His tags, GST tags, FLAG tags, and Strep tags; the localization tags include nuclear localization signals (NLS), membrane localization tags (such as palmitoylated sequences or cardamomylated sequences), and secretion signal peptides (such as IgG signal peptides); and the functional study tags include HA tags, Myc tags, SBP tags, and SUMO tags.

[0036] Furthermore, the application scenarios for the reagent kit or reaction system include nucleic acid amplification, sequencing, and gene editing.

[0037] Furthermore, the kit also includes instructions.

[0038] In some embodiments, the DNA polymerase includes, but is not limited to, Taq, Bst, Vent, Phi29, Pfu, Tru, Tth, Tl1, Tac, Tne, Tma, Tih, Tf1, Pwo, Kod, Sac, Sso, Poc, Pab, Mth, Pho, ES4 DNA polymerase, and the Klenow fragment.

[0039] In some implementations, the dNTPs are nucleoside sources for PCR-based DNA amplification; dATP, dGTP, dCTP, and dTTP are all necessary. Additionally, dNTPs can be chemically modified for hot-start methods, such as CleanAmp™ dNTPs manufactured by TriLink BioTechnologies, Inc.

[0040] In some embodiments, the recombinase is selected from T4 UvsX, RecA / Rad51, RadA, or a combination thereof.

[0041] In some embodiments, the recombinase auxiliary protein is selected from T4 UvsY, Escherichia coli recO, Escherichia coli recR, or a combination thereof.

[0042] In some embodiments, the crowding agent is selected from polyethylene glycol, polyethylene oxide, polystyrene, Ficoll, dextran, PVP, albumin, or combinations thereof.

[0043] In some embodiments, the salt ion is selected from Tris, magnesium ions, potassium ions, or combinations thereof.

[0044] In some implementations, the primer pair refers to a 7-50 nucleic acid sequence capable of forming a base pair complementary to the template strand and serving as a starting point for template strand replication. Primers are typically synthesized, but naturally occurring nucleic acids can also be used. The primer sequence does not necessarily need to be identical to the template sequence, as long as it is sufficiently complementary to hybridize with the template.

[0045] In some implementations, the technologies used for "nucleic acid amplification" include polymerase chain reaction (PCR), ligase chain reaction (LCR), strand displacement amplification (SDA), transcription-mediated amplification (TMA), loop-mediated isothermal amplification (LAMP), branched DNA signal amplification (bDNA), rolling circle amplification (RCA), and recombinase transcription-mediated amplification (RTMA). The technologies used for "sequencing" include first-generation sequencing (Sanger sequencing), second-generation sequencing (NGS, high-throughput sequencing), and third-generation sequencing. The technologies used for "gene editing" include CRISPR / Cas technology, transcription activator-like effector nuclease technology (TALEN), and zinc finger nuclease technology (ZFN).

[0046] The seventh aspect of the present invention provides any of the following methods: 1) A method for preparing the host cell according to the fourth aspect of the present invention, the method comprising the following steps: introducing the polynucleotide molecule according to the second aspect of the present invention or the vector according to the third aspect of the present invention into the host cell.

[0047] 2) A method for preparing the SSB protein mutant according to the first aspect of the present invention, the method comprising the following steps: culturing the host cell according to the fourth aspect of the present invention under suitable conditions, inducing host cell expression, separating and purifying the expression product, thereby obtaining the SSB protein mutant.

[0048] 3) A method for stabilizing a single-stranded nucleic acid molecule, the method comprising: contacting the single-stranded nucleic acid molecule with the SSB protein mutant described in the first aspect of the present invention.

[0049] 4) A method for improving the efficiency of nucleic acid amplification reaction, the method comprising: adding the SSB protein mutant described in the first aspect of the present invention to the reaction system.

[0050] 5) A method for enhancing gene editing efficiency, the method comprising: adding the SSB protein mutant of the first aspect of the present invention to the gene editing system before or simultaneously with introducing the gene editing system into a cell.

[0051] 6) A method for reducing template secondary structure interference in DNA sequencing, the method comprising: binding a DNA template with an SSB protein mutant as described in the first aspect of the present invention during a sequencing reaction.

[0052] In some embodiments, expression vectors containing the coding nucleotide sequence of the SSB protein mutant of the present invention and suitable transcription / translation control signals can be constructed using methods well known to those skilled in the art, preferably commercially available vectors such as pET28a. These methods include in vitro recombinant DNA technology, DNA synthesis technology, in vivo recombination technology, etc.

[0053] The eighth aspect of the present invention provides any of the following applications: 1) The application of the SSB protein mutant described in the first aspect of the present invention in nucleic acid amplification reactions.

[0054] 2) Application of the SSB protein mutant described in the first aspect of the present invention in sequencing reactions.

[0055] 3) The application of the SSB protein mutant described in the first aspect of the present invention in gene editing technology.

[0056] 4) The use of the SSB protein mutant described in the first aspect of the present invention in the preparation of kits for nucleic acid amplification, sequencing or gene editing.

[0057] 5) The application of the SSB protein mutant described in the first aspect of the present invention in the preparation of reaction systems for nucleic acid amplification, sequencing or gene editing.

[0058] Advantages and beneficial effects of the present invention: This invention provides a mutant of single-stranded DNA-binding protein (SSB). Using a protein engineering platform and an SSB protein derived from E. coli as a template, a high-purity, heat-resistant SSB protein mutant is obtained through the following multi-site mutated amino acid sequence combination K136L / K224F / E230W. This mutant can still function under environmental conditions such as transportation and storage at room temperature or higher, which will help expand a series of application scenarios and usage limitations of SSB protein in nucleic acid amplification and sequencing. Detailed Implementation

[0059] The present invention will be further illustrated below with reference to specific embodiments. These specific embodiments are for illustrative purposes only and should not be construed as limiting the invention. Those skilled in the art will understand that various changes, modifications, substitutions, and variations can be made to these embodiments without departing from the principles and spirit of the invention. The scope of the invention is defined by the claims and their equivalents. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this invention pertains.

[0060] The reagents, raw materials, and experimental consumables used in this invention are readily available to those skilled in the art and, unless otherwise specified, can be obtained commercially. Experimental methods not specifying particular conditions in this invention are typically performed under conventional conditions or according to the manufacturer's recommendations. In particular, the following examples are for illustrative purposes only and should not be construed as limiting the scope of the invention in any way. It should be noted that the experimental conditions and results described in the following examples are for illustrative purposes only and should not, and will not, limit the invention as described in the claims.

[0061] Example 1: Preparation of a mutant strain of the single-stranded DNA-binding protein SSB The amino acid sequence of wild-type SSB protein is as follows: MFKRKSTAELAAQMAKLNGNKGFSSEDKGEWKLKLDNAGNGQAVIRFLPSKNDEQAPFAILVNHGFKKNGKWYIETCSSTHGDYDSCPVCQYISKNDLYNTDNKEYSLVKRKTSYWANILVVKDPAAPENEGKVFKYRFGKKIWDKINAMIA VDVEMGETPVDVTCPWEGANFVLKVKQVSGFSNYDESKFLNQSAIPNIDDESFQKELFEQMVDLSEMTSKDKFKSFEELNTKFGQVMGTAVMGGAAATAAKKADKVADDLDAFNVDDFNTKTEDDFMSSSSGSSSSADDTDLDDLLNDL (SEQ ID NO:1) The amino acid sequence of plasmid pET28a-SSB-6×His is as follows: MFKRKSTAELAAQMAKLNGNKGFSSEDKGEWKLKLDNAGNGQAVIRFLPSKNDEQAPFAILVNHGFKKNGKWYIETCSSTHGDYDSCPVCQYISKNDLYNTDNKEYSLVKRKTSYWANILVVKDPAAPENEGKVFKYRFGKKIWDKINAMIAVDVE MGETPVDVTCPWEGANFVLKVKQVSGFSNYDESKFLNQSAIPNIDDESFQKELFEQMVDLSEMTSKDKFKSFEELNTKFGQVMGTAVMGGAAATAAKKADKVADDLDAFNVDDFNTKTEDDFMSSSSGSSSSADDTDLDDLLNDLHHHHHH* (SEQ ID NO:3) The nucleotide sequence of wild-type SSB protein is as follows: ATGTTTAAACGTAAATCTACTGCTGAACTCGCTGCACAAATGGCTAAACTGAATGGCAATAAAGGTTTTTCTTCTGAAGATAAAGGCGAGTGGAAACTGAAACTCGATAATGCGGGTAACGGTCAAGCAGTAATTCGTTTTCTTCCGTCTAAAAATGATGAACAAGCACCATTCGCAATTCTTGTAAATCACGGTTTCAAGAAAAATGGTAAATGGTATATTGAAACATGTTCATCTACCCATGGTGATTACGATTCTTGCCCAGTATGTCAATACATCAGTAAAAATGATCTATACAACACTGACAATAAAGAGTACAGTCTTGTTAAACGTAAAACTTCTTACTGGGCTAACATTCTTGTAGTAAAAGACCCAGCTGCTCCAGAAAACGAAGGTAAAGTATTTAAATACCGTTTCGGTAAGAAAATCTGGGATAAAATCAATGCAATGATTGCGGTTGATGTTGAAATGGGTGAAACTCCAGTTGATGTAACTTGTCCGTGGGAAGGTGCTAACTTTGTACTGAAAGTTAAACAAGTTTCTGGATTTAGTAACTACGATGAATCTAAATTCCTGAATCAATCTGCGATTCCAAACATTGACGATGAATCTTTCCAGAAAGAACTGTTCGAACAAATGGTTGACCTTTCTGAAATGACTTCTAAAGATAAATTCAAATCGTTTGAAGAACTTAATACTAAATTCGGTCAAGTTATGGGAACTGCTGTGATGGGCGGTGCTGCTGCAACTGCTGCTAAGAAAGCTGATAAAGTTGCTGATGATTTGGATGCATTCAATGTTGATGACTTCAATACAAAAACTGAAGATGATTTTATGAGCTCAAGCTCTGGTAGTTCATCTAGTGCTGATGACACGGATCTGGATGATCTGCTGAACGATCTG (SEQ ID NO:2) The nucleotide sequence of plasmid pET28a-SSB-6×His is as follows: (SEQ ID NO:4) Based on the plasmid pET28a-SSB-6×His containing the wild-type single-stranded DNA-binding protein SSB sequence, mutant plasmids of SSB protein with different point mutation sites were obtained through site-directed mutagenesis. The specific synthesized sequences included a purified 6×His tag sequence added to the 3' end and a TAA stop codon. PCR point mutation primers were designed based on the amino acid sites to be mutated. Using the pET28a-SSB-6×His plasmid as a template, relevant forward and reverse primers were designed, and the products were amplified by PCR. The mutant primers are shown in Table 1, and the PCR amplification system is shown in Table 2. Table 1 Primer sequences for SSB protein mutants

[0062] The underlined part represents the codon corresponding to the amino acid encoded by the mutant gene.

[0063] Table 2 PCR amplification system

[0064] The PCR amplification conditions for the circular plasmid were: pre-denaturation at 92℃ for 3 min, followed by 30 cycles (92℃ for 30 s, 55℃ for 30 s, 68℃ for 6 min and 30 s), and 68℃ for 15 min.

[0065] PCR products were digested with Dpn I enzyme: 1 μL of Dpn I was added to the PCR reaction system, mixed well, and treated at 37℃ for 5 min. The digested product after removing the template DNA was used for the next transformation experiment.

[0066] Acquisition, verification and preservation of mutant strains: Take 5 μL of the digestion product from the previous step, transform it into Escherichia coli BL21(DE3), spread it on a plate, and culture it until a mature single colony grows to obtain the recombinant SSB protein mutant engineered bacterial strain. Culture it overnight in a shake flask, verify it by PCR, extract the plasmid and send it for sequencing, and preserve the SSB protein mutant engineered bacterial strain that has been verified by sequencing.

[0067] Example 2 Preparation of wild-type SSB and its mutant proteins 1. Recombinant expression of wild-type SSB and mutants The mutant strain obtained in Example 1 and the wild-type original strain were activated overnight. The activated bacterial solution was transferred to LB liquid medium containing kanamycin at an inoculation rate of 1% and cultured at 37°C with a shaker at 200 rpm. When OD... 600 To induce the reaction, add isopropyl thiogalactoside (IPTG) to a final concentration of 0.2 mM until the pH reaches 0.6–1.0. The induction conditions are 37 °C for 3 h.

[0068] Collect the cultured cells by centrifugation at 6000 rpm for 30 min at 4℃, discard the supernatant, suspend the cells, homogenize under high pressure, and centrifuge again at 10000 rpm for 15 min at 4℃ to collect the supernatant. The crude enzyme solution of wild-type SSB and mutant protein can be obtained. Finally, filter through a membrane and store at low temperature for later use.

[0069] 2. Purification of wild-type SSB and mutant proteins Purification was performed using a Ni-column affinity chromatography column, with the target protein eluted in buffers containing different concentrations of imidazole. Samples were collected based on UV peak patterns, and the purity of the target protein was identified by SDS-PAGE. The samples were then combined and concentrated by ultrafiltration. Heparin column purification was then used to further remove residual DNA impurities. Finally, the mutant single-stranded binding protein was obtained after purification by molecular sieve. The same purification procedures were followed for wild-type SSB and mutant proteins.

[0070] Example 3: Determination of wild-type SSB and its mutant protein concentrations The Bradford Protein Quantitative Reagent Kit was used for the assay, and the specific method is as follows: 1. Prepare BSA Protein Standard reaction solution: Dilute BSA Protein Standard to 1 mg / ml with deionized water. Prepare reaction solutions of different concentrations of BSA Protein Standard and Bradford Protein Assay Reagent according to Table 3, and add them to a 96-well plate to create a standard curve. Table 3. Protein concentration standard curve reaction system

[0071] 2. Prepare the reaction solution for the test sample: Take an appropriate amount of the test sample and add it to a 96-well plate, add 40 μl of 5x Bradford Protein Assay Reagent, and then add deionized water to make up the total reaction volume to 200 μl and mix well.

[0072] 3. Set up three replicates. Mix the reaction solutions prepared in steps 1 and 2 in a 96-well plate and incubate at room temperature for 3-5 minutes. Measure the A595 of the sample using a microplate reader and calculate the protein concentration in the sample using a standard curve. Finally, dilute and adjust the protein concentrations of different wild-type SSBs and their mutants to be consistent, and store at low temperature for later use.

[0073] Example 4: Thermal stability test of SSB protein mutant I. Experimental Methods The SSB protein mutant obtained in Example 3 was subjected to in vitro nucleic acid thermostability experiments. The in vitro RTMA (Recombinase-transcription-mediated amplification) amplification reaction system is shown in Table 4. Table 4 In vitro RTMA amplification reaction system

[0074] RXN stands for reaction, a commonly used abbreviation in experiments, referring to the content of a substance in a single system; RNasin is synonymous with ribonuclease inhibitor, referring to a protein inhibitor of RNase; UvsX is recombinase; UvsY is recombinase auxiliary protein; RNAP is RNA polymerase.

[0075] Using canine parvovirus VP2 DNA (SEQ ID NO:17) as a template: 5'-tcagtaatatagtttgtatttcccatttgagttacaccacgtcttttatcttgttgaactcctatataaccaaagttagtacctccttcagattgaggcaaagaatttagaaatggt ggtaagcccaatgctctatttgtttgccatgtatgtgttagtctacatggtttacaatcaaaaaaaaatgttcctgtagcaaattcatcacctgttcttagtaagtgtactggcaca-3'; The primers used are: Upstream primer: 5'-TAATACGACTCACTATAGGGCACTTACTAAGAACAGGTGATGAATTTGCTACAGCSEQ-3' (SEQ ID NO: 18); Downstream primer: 5'-AGTTTGTATTTCCCATTTGAGTTACACCACGTCT-3' (SEQ ID NO: 19).

[0076] Canine parvovirus VP2 was used as the amplification target for in vitro amplification at different temperatures. The reaction conditions on the real-time PCR instrument were: constant temperature 45℃, continuous reaction for 40 min, fluorescence readings at 497 nm every 30 s, and the reaction results were monitored using the Bori FQD-96X real-time PCR system.

[0077] II. Experimental Results Table 5 shows the peak elution times at 45℃ for the SSB protein mutant with improved thermal stability. Table 5. Peak time of RTMA amplification of SSB protein mutant at a reaction temperature of 45℃

[0078] The results showed that among the SSB mutation sites E30I, K136L, T165F, K224F, E230W, and N232K, mutations in E30I and T165F led to loss of protein activity. The mutant K136L / K224F / E230W showed a significantly better peak elution time at 45℃ than other mutants, and its nucleic acid amplification performance was superior to other multi-point mutated SSB protein mutants.

[0079] Example 5: Stability test of SSB protein mutant stored at different temperatures I. Experimental Methods The mutant SSB was stored at 37°C and 45°C for 30 days, and the thermal stability of different mutants was tested by RTMA reaction. The method was the same as in Example 4, using the SSB protein mutant K136L / K224F / E230W, and RTMA amplification was performed on canine parvovirus VP2 at different temperatures.

[0080] II. Experimental Results The peak elution times of the SSB protein mutant after 30 days of storage at 37℃ and 45℃ are shown in Table 6.

[0081] Table 6. Peak elution time of SSB protein mutants after 30 days of storage at 37 and 45℃

[0082] The results showed that the stability test results of the SSB protein mutant stored at different temperatures were consistent with the thermostability test results. After 30 days of storage at 37℃ and 45℃, the peak time of K136L / K224F / E230W was significantly better than that of the wild type, indicating that it had superior nucleic acid amplification performance.

[0083] The above description of the embodiments is only for understanding the method and core ideas of the present invention. It should be noted that those skilled in the art can make various improvements and modifications to the present invention without departing from the principles of the invention, and these improvements and modifications will also fall within the protection scope of the claims of the present invention.

Claims

1. A mutant of SSB protein, characterized in that, The SSB protein mutant is obtained by mutation based on a wild-type SSB protein; wherein the amino acid sequence of the wild-type SSB protein is shown as SEQ ID NO:

1.

2. The mutant SSB protein of claim 1, wherein, The nucleotide sequence of the wild-type SSB protein is shown as SEQ ID NO: 2; Preferably, the amino acid sequence of the wild-type SSB protein is shown as SEQ ID NO: 3; Preferably, the nucleotide sequence of the wild-type SSB protein is shown as SEQ ID NO:

4.

3. The mutant SSB protein of claim 1, wherein, The mutation site of the mutant is selected from any one or more than one combination of K136L, K224F, E230W, N232K; Preferably, the mutation site of the mutant is selected from the combination of K136L / K224F / E230W.

4. A polynucleotide molecule, characterized in that, The polynucleotide molecule encodes the SSB protein mutant of any one of claims 1-3.

5. A vector, characterized in that, The vector comprises the polynucleotide molecule of claim 4.

6. A host cell, characterized in that, The host cell comprises the polynucleotide molecule of claim 4 or the vector of claim 5; Preferably, the host cell includes a prokaryotic cell or a eukaryotic cell.

7. A composite, characterized by, The complex comprises the SSB protein mutant of any one of claims 1-3 and a single-stranded target nucleic acid molecule bound to the SSB protein mutant; Preferably, the single-stranded target nucleic acid molecule is single-stranded DNA or RNA.

8. Any one of the following products: 1) a derivative comprising the SSB protein mutant of any one of claims 1-3 and a peptide tag that can be connected to the SSB protein mutant; 2) a kit comprising the SSB protein mutant of any one of claims 1-3 and one or more of a DNA polymerase, a mixture of dNTPs or dNTPs and ddNTPs, a nuclease, a buffer, a recombinase, a reducing agent, a recombinase accessory protein, a crowding agent, an ATP or ATP analog, a salt ion; 3) a reaction system comprising the SSB protein mutant of any one of claims 1-3 and one or more of a primer pair, a recombinase, a polymerase, a nuclease, a dNTP, a crowding agent, a recombinase accessory protein, an ATP or ATP analog, a salt ion; Preferably, the peptide tag includes a detection tag, a purification tag, a localization tag, a functional study tag; Preferably, the application scenarios of the kit or reaction system include nucleic acid amplification, sequencing, gene editing.

9. Any one of the following methods: 1) A method of making the host cell of claim 6, said method comprising: introducing the polynucleotide molecule of claim 4 or the vector of claim 5 into a host cell ； 2) a method for preparing the SSB protein mutant of any one of claims 1-3, the method comprising: culturing the host cell of claim 6, inducing the host cell to express, isolating and purifying the expression product, thereby obtaining the SSB protein mutant; 3) a method for stabilizing a single-stranded nucleic acid molecule, the method comprising: contacting the single-stranded nucleic acid molecule with the SSB protein mutant of any one of claims 1-3; 4) A method for improving the efficiency of nucleic acid amplification reaction, comprising: adding the SSB protein mutant of any one of claims 1-3 into the reaction system; 5) A method for enhancing the efficiency of gene editing, comprising: adding the SSB protein mutant of any one of claims 1-3 into the gene editing system before or simultaneously with the introduction of the gene editing system into the cell; 6) A method for reducing the interference of secondary structure of template in DNA sequencing, comprising: using the SSB protein mutant of any one of claims 1-3 to bind with the DNA template in the sequencing reaction.

10. Any one of the following applications: 1) The SSB protein mutant of any one of claims 1-3 for use in nucleic acid amplification reaction; 2) The SSB protein mutant of any one of claims 1-3 for use in sequencing reaction; 3) The SSB protein mutant of any one of claims 1-3 for use in gene editing technology; 4) The SSB protein mutant of any one of claims 1-3 for use in the preparation of a kit for nucleic acid amplification, sequencing or gene editing; 5) The SSB protein mutant of any one of claims 1-3 for use in the preparation of a reaction system for nucleic acid amplification, sequencing or gene editing.

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