SSB protein mutant with improved thermal stability

Abstract

The invention discloses an SSB protein mutant with improved thermal stability, the SSB protein mutant comprises a mutation site K136L / K224F / E230W / N232K, the thermal stability of the SSB protein mutant is remarkably improved, the SSB protein mutant still has stable reaction activity under the reaction condition of 45 DEG C, and can be stored for a long time under the condition of 20-45 DEG C, and the SSB protein mutant can be used as a novel SSB protein mutant. The practical application scene and the use limitation of the SSB protein nucleic acid amplification reaction are widened, and the method has wide application prospect and conversion value.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, and specifically relates to a mutant SSB protein with improved thermal stability. Background Technology

[0002] Single-stranded DNA-binding proteins (SSBs) are widely distributed in eukaryotes, bacteria, and archaea, playing crucial roles in DNA metabolism processes such as cellular DNA replication, recombination, and damage repair. They are also key to maintaining the stability of genetic information. The functions of SSB proteins include binding to single-stranded DNA (ssDNA), protecting it from degradation by nucleases; maintaining the structure of ssDNA, preventing its formation of secondary structures or reverting to double stranding; and recruiting other proteins to perform DNA replication, recombination, or repair functions.

[0003] In molecular biology, SSB protein is an important biological reagent with wide applications, primarily including improving the efficiency and yield of PCR, RT-PCR, and sequencing reactions, especially when handling complex DNA templates. SSB protein can also be used to stabilize and label single-stranded DNA, promote restriction endonuclease digestion, and enhance the elongation ability of DNA polymerases. Therefore, SSB protein has become indispensable in PCR-based techniques such as DNA sequencing, multiplex PCR, RNA reverse transcription, DNA sequencing experiments, RT-PCR, and isothermal amplification. Since SSB protein must undergo a high-temperature denaturation process during PCR amplification, it needs to be thermostable and retain its activity under high-temperature conditions. Therefore, developing SSB protein mutants with higher thermostability and better preservation of biological activity is crucial for the field of gene detection. Summary of the Invention

[0004] The purpose of this invention is to provide an SSB protein mutant with improved thermal stability to overcome the technical problems of SSB protein being temperature sensitive and difficult to store in the current field.

[0005] The above-mentioned objectives of the present invention are achieved through the following technical solutions: The first aspect of the present invention provides an SSB protein mutant, which is obtained by mutating the amino acids of wild-type SSB protein, the amino acid sequence of which 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 wild-type SSB protein is derived from Escherichia coli.

[0010] 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.

[0011] Furthermore, the mutation site of the mutant is selected from a combination of K136L / K224F / E230W / N232K.

[0012] A second 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.

[0013] Furthermore, the single-stranded target nucleic acid molecule is single-stranded DNA or RNA.

[0014] A third aspect of the present invention provides a nucleic acid molecule or a vector containing the same, said nucleic acid molecule encoding the SSB protein mutant described in the first aspect of the present invention.

[0015] A fourth aspect of the present invention provides a recombinant host cell comprising the nucleic acid molecule described in the third aspect of the present invention or the vector described in the fourth aspect of the present invention.

[0016] Furthermore, the recombinant host cell includes prokaryotic cells or eukaryotic cells.

[0017] The fifth aspect of this 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.

[0018] 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.

[0019] 3) A reaction system comprising one or more of the following: the SSB protein mutant described in the first aspect of the present invention, primer pairs, a template to be detected, a recombinase, a polymerase, a nuclease, dNTPs, a crowding agent, a recombinase helper protein, ATP or an ATP analog, and salt ions.

[0020] Furthermore, the kit also includes an instruction manual.

[0021] Furthermore, the peptide tags include detection tags, purification tags, localization tags, and functional research tags.

[0022] Furthermore, the application scenarios for the reagent kit or reaction system include nucleic acid amplification, sequencing, and gene editing.

[0023] The sixth aspect of the present invention provides any of the following methods: 1) A method for preparing the recombinant host cell according to the fourth aspect of the present invention, the method comprising the following steps: introducing the nucleic acid molecule or a vector containing the nucleic acid molecule according to the third aspect of the present invention into the host cell.

[0024] 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.

[0025] Furthermore, the method also includes gene editing of cells expressing wild-type SSB protein to induce the K136L / K224F / E230W / N232K mutation, followed by continued culturing to induce protein expression.

[0026] 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.

[0027] 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.

[0028] 5) A method for enhancing gene editing efficiency, the method comprising: adding the SSB protein mutant described in the first aspect of the present invention into a gene editing system.

[0029] 6) A method for reducing template secondary structure interference in a sequencing reaction, the method comprising: in the sequencing reaction, using the SSB protein mutant described in the first aspect of the present invention to bind to a single-stranded nucleic acid molecule template.

[0030] The seventh 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.

[0031] 2) Application of the SSB protein mutant described in the first aspect of the present invention in sequencing reactions.

[0032] 3) The application of the SSB protein mutant described in the first aspect of the present invention in gene editing technology.

[0033] 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.

[0034] 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.

[0035] Advantages and beneficial effects of the present invention: This invention discloses a thermostable SSB protein mutant containing mutation sites K136L / K224F / E230W / N232K. The thermostable SSB protein mutant exhibits significantly increased reactivity, maintaining stable activity even at 45°C, and can be stored for extended periods at temperatures ranging from 20 to 45°C. This expands the application scenarios of SSB protein in a range of nucleic acid amplification and sequencing experiments, demonstrating broad application prospects and translational value. Detailed Implementation

[0036] 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.

[0037] 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.

[0038] 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 / N232K 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), glutamic acid (E) at position 230 is replaced by tryptophan (W), and asparagine (N) at position 232 is replaced by lysine (K). All four mutations are present in the specific SSB protein mutant.

[0039] In some embodiments, the SSB protein mutant described in this invention also includes an SSB protein mutant with the same amino acid sequence as the SSB protein mutant described in this invention, and its function includes being equivalent to, slightly reduced, slightly increased or significantly increased compared to, the SSB protein mutant described in this invention. All of the above-mentioned SSB protein mutants are also included within the protection scope of this invention.

[0040] In some embodiments, "identity" is synonymous with "homology," referring to sequence similarity to a target amino acid or nucleotide sequence. Homology includes amino acid sequences having 75% or higher, 85% or higher, 90% or higher, or 95% or higher homology with the amino acid sequence of the SSB protein mutant provided by this invention. Homology can be evaluated visually or using computer software. When using computer software, the homology between two or more sequences can be expressed as a percentage (%), which can be used to evaluate the homology between related sequences. The 75% or higher homology can be 75%, 80%, 85%, 90%, or 95% or higher homology.

[0041] In this invention, the term "nucleic acid molecule" refers to any polymeric form of any length and composed of ribonucleotides or deoxyribonucleotides. In the context of this invention, it refers to a DNA sequence that, when placed under the control of a suitable regulatory sequence, is transcribed and translated into a polypeptide in a host cell. The boundaries of the coding nucleic acid sequence are determined by a start codon at the 5' (amino) end and a translation stop codon at the 3' (carboxyl) end. The coding sequence may include, but is not limited to, prokaryotic sequences, cDNA from eukaryotic mRNA, genomic DNA sequences from eukaryotic (e.g., mammalian) DNA, and even recombinant DNA sequences. The transcription stop sequence will typically be located at the 3' end of the coding sequence.

[0042] In this invention, the nucleic acid molecule may comprise natural, non-natural, or modified nucleotides; and it may comprise natural, non-natural, or modified internucleotide linkages, such as aminophosphate linkages or thiophosphate linkages, instead of phosphodiester linkages present between unmodified oligonucleotides. In some embodiments, the nucleotides do not contain any insertions, deletions, inversions, and / or substitutions. However, in some cases, it may be suitable for a nucleotide to contain one or more insertions, deletions, inversions, and / or substitutions, and therefore, nucleotides formed by these insertions, deletions, inversions, and / or substitutions are also within the scope of this invention.

[0043] In a specific embodiment of the present invention, the nucleic acid molecule refers to a nucleic acid sequence capable of encoding the SSB protein mutant described in the first aspect of the present invention. Once the coding sequence of the SSB protein mutant described in the present invention is isolated and obtained, the SSB protein mutant can be obtained in large quantities using recombination technology.

[0044] In this invention, the term "vector" refers to an artificial construct capable of delivering and preferably expressing one or more target genes or sequences in a host cell. Vectors of this invention can be plasmid vectors, viral vectors, etc. In some embodiments, a vector refers to a linear or circular nucleic acid molecule containing the nucleic acid of this invention operably linked to other segments provided for autonomous replication in a recombinant host cell, or according to an expression cassette of the nucleic acid molecule. "Operably linked" means that the nucleic acid sequence of interest is linked to a regulatory sequence in a manner that allows for the expression of the nucleotide sequence (e.g., in an in vitro transcription / translation system or when introducing the vector into a host cell).

[0045] In some embodiments, various vectors known in the art can be used, such as commercially available vectors, and then the nucleic acid encoding the above-mentioned SSB protein mutant is operatively linked to the expression regulatory sequence to form an expression vector. In some embodiments, the vector includes, but is not limited to, plasmids, phage particles, granules, artificial chromosomes, and virus-derived vectors. Specifically, the vector used in this invention is pET28a.

[0046] In some implementations, the 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. The DNA encoding the SSB protein can be effectively ligated to a multiple cloning site in the vector to guide mRNA synthesis and thus protein expression, or for homologous recombination.

[0047] In this invention, the term "recombinant host cell" refers to a host cell into which a vector is introduced, enabling the recombinant host cell to transcribe a target nucleic acid sequence and / or translate a target protein. In this invention, this genetic engineering technique can be used to enable recombinant host cells to express the SSB protein mutant described in the first aspect of this invention. In one aspect of this invention, the SSB protein mutant, as claimed and described herein, can be generated in host cells using recombinant technology. Host cells and cell lines suitable for recombinant production of various proteins are well known in the art.

[0048] In this invention, the term "host cell" refers to a cell that can be used to introduce a vector, including but not limited to prokaryotic cells such as Escherichia coli, fungal cells such as yeast cells, or cells such as fibroblasts, CHO cells, COS cells, NSO cells, HeLa cells, BHK cells, or HEK293 cells.

[0049] In some embodiments, the recombinant host cells are prepared by introducing the nucleic acid molecule described in the third aspect of the invention or a vector containing it into the host cells. The nucleic acid molecule described in the third aspect of the invention or a vector containing it can be introduced into the host cells by various suitable methods, not limited to those listed herein, such as calcium phosphate transfection, DEAE-glucan-mediated transfection, microinjection, electroporation, the TALEN method, the ZFN method, non-viral vector-mediated transfection (e.g., liposomes) or viral vector-mediated transfection (e.g., lentiviral infection, retroviral infection, adenovirus infection), and other physical, chemical, or biological means for transfer into cells, such as transposon technology, CRISPR-Cas9, etc.

[0050] 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.

[0051] 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. The dNTPs are nucleoside sources for PCR-based DNA amplification; dATP, dGTP, dCTP, and dTTP are essential. Additionally, for dNTPs, chemically modified substances for hot-start methods can be used, such as CleanAmp™ dNTPs manufactured by TriLink BioTechnologies, Inc. The recombinase is selected from T4 UvsX, RecA / Rad51, RadA, or combinations thereof. The recombinase accessory protein is selected from T4 UvsY, *E. coli* recO, *E. coli* recR, or combinations thereof. The crowding agent is selected from polyethylene glycol, polyethylene oxide, polystyrene, Ficoll, dextran, PVP, albumin, or combinations thereof. The salt ion is selected from Tris, magnesium ions, potassium ions, or combinations thereof.

[0052] In this invention, the term "primer pair" refers to a macromolecule with a specific nucleotide sequence that is stimulated to synthesize at the initiation of nucleotide polymerization and is linked to the reactant by hydrogen bonds. Such a molecule is called a primer. Primers are usually two artificially synthesized oligonucleotide sequences. One primer is complementary to one DNA template strand at one end of the target region and is called the upstream primer; the other primer is complementary to another DNA template strand at the other end of the target region and is called the downstream primer. Their function is to serve as the starting point for nucleotide polymerization, allowing nucleic acid polymerase to synthesize a new nucleic acid chain starting from its 3' end.

[0053] In this invention, the techniques 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).

[0054] In this invention, the technologies used for "sequencing" include first-generation sequencing (Sanger sequencing), second-generation sequencing (NGS, high-throughput sequencing), and third-generation sequencing.

[0055] In this invention, the technologies used for "gene editing" include CRISPR / Cas technology, transcription activator-like effector nuclease technology (TALEN), and zinc finger nuclease technology (ZFN). Those skilled in the art know that constructing nucleotides and other regulatory elements encoding gene editing tools into suitable vectors, and then transforming them into cells, can achieve editing of the intracellular genome. The types of editing include gene knockout, insertion, and base editing.

[0056] In some embodiments, the method for preparing the SSB protein mutant of the present invention is not particularly limited, as long as the SSB protein mutant of the present invention can be expressed and collected. Methods for preparing the target protein using a protein expression system are well known in the art, and exemplary methods include E. coli expression systems and yeast systems. More specifically, the method includes introducing the aforementioned vector into cells, culturing the cells, and collecting the target protein after a certain culturing time, i.e., collecting the SSB protein mutant of the present invention.

[0057] 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: MFKRKSTAELAAQMAKLNGNKGFSSEDKGEWKLKLDNAGNGQAVIRFLPSKNDEQAPFAILVNHGFKKNGKWYIETCSSTHGDYDSCPVCQYISKNDLYNTDNKEYSLVKRKTSYWANILVVKDPAAPENEGKVFKYRFGKKIWDKINAMIAVDVEMGETPVDVTCPWEGANFVLKVKQVSGFSNYDESKFLNQSAIPNIDDESFQKELFEQMVDLSEMTSKDKFKSFEELNTKFGQVMGTAVMGGAAATAAKKADKVADDLDAFNVDDFNTKTEDDFMSSSSGSSSSADDTDLDDLLNDLHHHHHH* (SEQ ID NO:3) The nucleotide sequence of the wild-type SSB protein is as follows: ATGTTTAAACGTAAATCTACTGCTGAACTCGCTGCACAAATGGCTAAACTGAATGGCAATAAAGGTTTTTCTTCTGAAGATAAAGGCGAGTGGAAACTGAAACTCGATAATGCGGGTAACGGTCAAGCAGTAATTCGTTTTCTTCCGTCTAAAAATGATGAACAAGCACCATTCGCAATTCTTGTAAATCACGGTTTCAAGAAAAATGGTAAATGGTATATTGAAACATGTTCATCTACCCATGGTGATTACGATTCTTGCCCAGTATGTCAATACATCAGTAAAAATGATCTATACAACACTGACAATAAAGAGTACAGTCTTGTTAAACGTAAAACTTCTTACTGGGCTAACATTCTTGTAGTAAAAGACCCAGCTGCTCCAGAAAACGAAGGTAAAGTATTTAAATACCGTTTCGGTAAGAAAATCTGGGATAAAATCAATGCAATGATTGCGGTTGATGTTGAAATGGGTGAAACTCCAGTTGATGTAACTTGTCCGTGGGAAGGTGCTAACTTTGTACTGAAAGTTAAACAAGTTTCTGGATTTAGTAACTACGATGAATCTAAATTCCTGAATCAATCTGCGATTCCAAACATTGACGATGAATCTTTCCAGAAAGAACTGTTCGAACAAATGGTTGACCTTTCTGAAATGACTTCTAAAGATAAATTCAAATCGTTTGAAGAACTTAATACTAAATTCGGTCAAGTTATGGGAACTGCTGTGATGGGCGGTGCTGCTGCAACTGCTGCTAAGAAAGCTGATAAAGTTGCTGATGATTTGGATGCATTCAATGTTGATGACTTCAATACAAAAACTGAAGATGATTTTATGAGCTCAAGCTCTGGTAGTTCATCTAGTGCTGATGACACGGATCTGGATGATCTGCTGAACGATCTG (SEQ ID NO:2) [[ID=2=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 synthesized sequences specifically 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

[0058] The underlined part represents the codon corresponding to the amino acid encoded by the mutant gene.

[0059] Table 2 PCR amplification system

[0060] 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.

[0061] 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.

[0062] 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.

[0063] 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.

[0064] 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.

[0065] 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.

[0066] 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

[0067] 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.

[0068] 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.

[0069] 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

[0070] 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.

[0071] 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).

[0072] 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.

[0073] 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℃

[0074] 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 / N232K 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.

[0075] 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.

[0076] 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.

[0077] Table 6. Peak elution time of SSB protein mutants after 30 days of storage at 37 and 45℃

[0078] 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 / N232K was significantly better than that of the wild type, indicating that it had advantages in nucleic acid amplification performance.

[0079] 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. An SSB protein mutant, characterized in that, The SSB protein mutant is obtained by mutating the wild-type SSB protein with the amino acid sequence shown in SEQ ID NO:1, and the SSB protein mutant has improved thermal stability.

2. The SSB protein mutant according to claim 1, characterized in that, The nucleotide sequence of the wild-type SSB protein is shown in SEQ ID NO:2; Preferably, the amino acid sequence of the wild-type SSB protein is shown in SEQ ID NO:3; Preferably, the nucleotide sequence of the wild-type SSB protein is shown in SEQ ID NO:

4.

3. The SSB protein mutant according to claim 1, characterized in that, The mutation sites of the mutants are selected from any one or a combination of one or more of K136L, K224F, E230W, and N232K; Preferably, the mutation site of the mutant is selected from a combination of K136L / K224F / E230W / N232K.

4. The SSB protein mutant according to claim 1, characterized in that, The improved thermal stability is manifested in the fact that the peak elution time of the SSB protein mutant of the present invention is better than that of the wild type under reaction conditions of 45°C, and that the SSB protein mutant of the present invention can still achieve nucleic acid amplification after being stored for 30 days under storage conditions of 45°C.

5. A complex, characterized in that, The complex comprises the SSB protein mutant as described in any one of claims 1-4 and a single-stranded target nucleic acid molecule that binds to the SSB protein mutant; Preferably, the single-stranded target nucleic acid molecule is single-stranded DNA or RNA.

6. A nucleic acid molecule or a carrier containing the same, characterized in that, The nucleic acid molecule encodes the SSB protein mutant as described in any one of claims 1-4.

7. A recombinant host cell, characterized in that, The recombinant host cell comprises the nucleic acid molecule of claim 6 or a vector comprising it; Preferably, the recombinant host cell includes a prokaryotic cell or a eukaryotic cell.

8. Any of the following products: 1) A derivative comprising the SSB protein mutant of any one of claims 1-4 and a peptide tag that can be attached to the SSB protein mutant; 2) A kit comprising the SSB protein mutant as described in any one of claims 1-4, 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. 3) A reaction system comprising the SSB protein mutant as described in any one of claims 1-4, and one or more of the following: primer pairs, recombinase, polymerase, nuclease, dNTP, congesting agent, recombinase helper protein, ATP or ATP analog, and salt ions; Preferably, the peptide tags include detection tags, purification tags, localization tags, and functional research tags; Preferably, the application scenarios of the kit or reaction system include nucleic acid amplification, sequencing, and gene editing.

9. Any of the following methods: 1) A method for preparing the recombinant host cell of claim 7, the method comprising: The nucleic acid molecule of claim 6 or a vector containing it is introduced into a host cell. ； 2) A method for preparing the SSB protein mutant according to any one of claims 1-4, the method comprising: The recombinant host cell described in claim 7 is cultured, the host cell expression is induced, and the expression product is isolated and purified to obtain 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 1-4; 4) A method for improving the efficiency of nucleic acid amplification reaction, the method comprising: adding the SSB protein mutant of any one of claims 1-4 to the reaction system; 5) A method for enhancing gene editing efficiency, the method comprising: adding the SSB protein mutant of any one of claims 1-4 into a gene editing system; 6) A method for reducing template secondary structure interference in a sequencing reaction, the method comprising: in the sequencing reaction, using an SSB protein mutant according to any one of claims 1-4 to bind to a single-stranded nucleic acid molecule template.

10. Any of the following applications: 1) The use of the SSB protein mutant according to any one of claims 1-4 in nucleic acid amplification reactions; 2) The use of the SSB protein mutant as described in any one of claims 1-4 in sequencing reactions; 3) The application of the SSB protein mutant as described in any one of claims 1-4 in gene editing technology; 4) The use of the SSB protein mutant according to any one of claims 1-4 in the preparation of kits for nucleic acid amplification, sequencing or gene editing; 5) The use of the SSB protein mutant as described in any one of claims 1-4 in the preparation of reaction systems for nucleic acid amplification, sequencing or gene editing.

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