UvsY auxiliary protein mutant with improved thermal stability

Abstract

The invention discloses a UvsY auxiliary protein mutant with improved thermal stability, the thermal stability of the UvsY auxiliary protein mutant is remarkably improved, and the core technical problem that an existing UvsY auxiliary protein is poor in thermal stability is solved. In addition, the UvsY auxiliary protein mutant can still maintain efficient in-vitro nucleic acid amplification activity after being stored for a long time, the peak appearance time is short, the amplification efficiency is stable, strict cold chain transportation and storage are not needed, and the application cost is reduced. The method can be widely applied to various scenes such as RPA and RTMA isothermal amplification technologies, adaptive gene detection and basic-level field diagnosis, and has extremely high practical application value and industrialization prospect.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to a UvsY helper protein mutant with improved thermal stability. Background Technology

[0002] In molecular biology research, the UvsY accessory protein is widely used as an important tool enzyme in fields such as isothermal DNA amplification (e.g., RPA), IPA amplification, and gene detection. The RPA reaction mainly relies on the recombinase UvsX and recombinase loading factor UvsY (accessory protein) derived from T4 phage, the single-stranded DNA-binding protein (SSB) Gp32, and the strand displacement DNA polymerase Bsu (Bacillus subtilis Pol) or Sau (Staphylococcus aureus Pol), and then the template is amplified based on the T4 phage nucleic acid replication mechanism.

[0003] The UvsY accessory protein, derived from T4 bacteriophage, belongs to the ABC family of ATPases and possesses a typical two-subunit structure containing both an ATP-binding site and an interaction site. UvsY is another key enzyme in RPA technology, functioning in conjunction with the T4 UvsX recombinase. Structural and biophysical studies have revealed that UvsY competes with single-stranded DNA-binding proteins pre-bound to single-stranded DNA for binding sites during UvsX's search for homologous sequences. UvsY promotes this competition, facilitating UvsX binding to single-stranded DNA. Through interactions with key recombinant proteins such as T4 and / or T6 UvsX recombinases, T4UvsY becomes indispensable for maintaining genome integrity and facilitating gene exchange. UvsY enhances the DNA-dependent ATPase activity of UvsX, lowering the minimum concentration required for its activity, thereby promoting strand substitution. UvsY can also form a heterotetrameric complex with UvsX recombinase, promoting the interaction between UvsX recombinase and single-stranded DNA. The UvsY protein enhances the binding of the UvsX recombinase to DNA, promoting its replacement process and assisting its entry into the cell nucleus. This enzyme has no nuclease activity.

[0004] Recombinant DNA Propagation (RPA) technology boasts advantages such as isothermal activity, speed, portability, high sensitivity, strong specificity, and ease of operation, making it a promising technology for field diagnostics at the grassroots level. Therefore, the auxiliary protein UvsY, the precursor enzyme, plays a crucial role in the RPA process. Its presence enhances the binding efficiency of UvsX to single-stranded DNA, accelerating the RPA reaction and providing strong support for related scientific research, ensuring the quality of research and experiments. However, the recombinant enzyme UvsY, currently available for industrial production, suffers from low thermostability, hindering its preservation and transportation in practical RPA applications. Summary of the Invention

[0005] In view of this, in order to overcome the above-mentioned technical problems existing in the art, the purpose of the present invention is to provide a UvsY accessory protein mutant with improved thermal stability, wherein the UvsY accessory protein mutant includes S18Y, H39W, D92F, Q128L, H39Y, H39M, H39F, S18Y / H39W, S18Y / D92F, S18Y / Q128L, S18Y / H39W / D92F, S18Y / H39W / Q128L, H39W / D92F / Q128L or S18Y / H39W / D92F / Q128L.

[0006] The present invention achieves the above-mentioned objectives by adopting the following technical solution: The first aspect of the present invention provides a UvsY accessory protein mutant with improved thermal stability, wherein the UvsY accessory protein mutant has any one of the following amino acid mutations in the amino acid sequence of the wild-type UvsY accessory protein as shown in SEQ ID NO:1: S18Y, H39W, D92F, Q128L, H39Y, H39M, H39F, S18Y / H39W, S18Y / D92F, S18Y / Q128L, S18Y / H39W / D92F, S18Y / H39W / Q128L, H39W / D92F / Q128L, S18Y / H39W / D92F / Q128L.

[0007] Furthermore, the UvsY accessory protein mutant is a combinatorial mutant, which includes any one of the following combinatorial mutation sites: S18Y / H39W, S18Y / D92F, S18Y / Q128L, S18Y / H39W / D92F, S18Y / H39W / Q128L, H39W / D92F / Q128L, and S18Y / H39W / D92F / Q128L.

[0008] Furthermore, the UvsY accessory protein mutant is a combined mutant, which includes any one of the following combined mutation sites: H39W / D92F / Q128L, S18Y / H39W / D92F / Q128L.

[0009] In this invention, the " / " between mutation sites is used to clearly indicate a combination relationship between mutation sites, that is, multiple mutation sites connected before and after the " / " exist simultaneously on the amino acid sequence of the same UvsY helper protein mutant (rather than just one). For example, "S18Y / H39W" indicates that the amino acid sequence of this mutant contains two mutation sites: serine at position 18 is mutated to tyrosine (S18Y) and histidine at position 39 is mutated to tryptophan (H39W); similarly, "S18Y / H39W / D92F / Q128L" indicates that this mutant contains four mutation sites: S18Y, H39W, D92F, and Q128L, belonging to a combined mutant. This description is a conventional notation method in the field of protein mutants, used to clearly distinguish between "single-point mutants" (containing only one mutation site, without " / ") and "combined mutants" (containing two or more mutation sites, connected by " / ").

[0010] In some embodiments, the UvsY accessory protein mutant is a single-point mutant (containing only one mutation site, with the core enhancing thermal stability), that is, any one of the following amino acid mutations occurs on the amino acid sequence of the wild-type UvsY accessory protein as shown in SEQ ID NO:1: S18Y, H39W, D92F, Q128L, H39Y, H39M, H39F. Wherein, S18Y refers to the mutation of serine (S) at position 18 of the wild-type UvsY accessory protein to tyrosine (Y); H39W refers to the mutation of histidine (H) at position 39 to tryptophan (W); H39Y refers to the mutation of histidine (H) at position 39 to tyrosine (Y); H39M refers to the mutation of histidine (H) at position 39 to methionine (M); H39F refers to the mutation of histidine (H) at position 39 to phenylalanine (F); D92F refers to the mutation of aspartic acid (D) at position 92 to phenylalanine (F); and Q128L refers to the mutation of glutamine (Q) at position 128 to leucine (L).

[0011] In some embodiments, the UvsY accessory protein mutant is a two-site combination mutant (containing two mutation sites that synergistically enhance thermal stability), that is, any one of the following amino acid mutations occurs on the amino acid sequence of the wild-type UvsY accessory protein as shown in SEQ ID NO:1: S18Y / H39W, S18Y / D92F, S18Y / Q128L. Specifically, S18Y / H39W refers to the simultaneous occurrence of the following two mutations in the amino acid sequence of the wild-type UvsY accessory protein shown in SEQ ID NO:1: serine (S) at position 18 is mutated to tyrosine (Y), and histidine (H) at position 39 is mutated to tryptophan (W); S18Y / D92F refers to the simultaneous occurrence of the following two mutations in the amino acid sequence of the wild-type UvsY accessory protein shown in SEQ ID NO:1: serine (S) at position 18 is mutated to tyrosine (Y), and aspartic acid (D) at position 92 is mutated to phenylalanine (F); and S18Y / Q128L refers to the simultaneous occurrence of the following two mutations in the amino acid sequence of the wild-type UvsY accessory protein shown in SEQ ID NO:1: serine (S) at position 18 is mutated to tyrosine (Y), and glutamine (Q) at position 128 is mutated to leucine (L).

[0012] In some embodiments, the UvsY accessory protein mutant is a three-point combination mutant (containing three mutation sites to further enhance the synergistic effect), that is, any one of the following amino acid mutations occurs on the amino acid sequence of the wild-type UvsY accessory protein as shown in SEQ ID NO:1: S18Y / H39W / D92F, S18Y / H39W / Q128L, H39W / D92F / Q128L. Specifically, S18Y / H39W / D92F refers to the simultaneous occurrence of the following three mutations in the amino acid sequence of the wild-type UvsY accessory protein shown in SEQ ID NO:1: serine at position 18 (S) mutated to tyrosine (Y), histidine at position 39 (H) mutated to tryptophan (W), and aspartic acid at position 92 (D) mutated to phenylalanine (F). S18Y / H39W / Q128L refers to the simultaneous occurrence of the following three mutations in the amino acid sequence of the wild-type UvsY accessory protein shown in SEQ ID NO:1: serine at position 18 (S) mutated to tyrosine (Y), histidine at position 39 (H) mutated to tryptophan (W), and glutamine at position 128 (Q) mutated to leucine (L). H39W / D92F / Q128L refers to the simultaneous occurrence of the following three mutations in the amino acid sequence of the wild-type UvsY accessory protein shown in SEQ ID NO:1: serine at position 18 (S) mutated to tyrosine (Y), histidine at position 39 (H) mutated to tryptophan (W), and glutamine at position 128 (Q) mutated to leucine (L). The wild-type UvsY accessory protein shown in NO:1 has the following three mutations simultaneously on its amino acid sequence: histidine (H) at position 39 is mutated to tryptophan (W), aspartic acid (D) at position 92 is mutated to phenylalanine (F), and glutamine (Q) at position 128 is mutated to leucine (L).

[0013] In some embodiments, the UvsY accessory protein mutant is a four-site combination mutant (containing four mutation sites for maximum thermostability), specifically, the following amino acid mutation occurs in the amino acid sequence of the wild-type UvsY accessory protein shown in SEQ ID NO:1: S18Y / H39W / D92F / Q128L. S18Y / H39W / D92F / Q128L refers to the simultaneous occurrence of the following four mutations in the amino acid sequence of the wild-type UvsY accessory protein shown in SEQ ID NO:1: serine (S) at position 18 is mutated to tyrosine (Y), histidine (H) at position 39 is mutated to tryptophan (W), aspartic acid (D) at position 92 is mutated to phenylalanine (F), and glutamine (Q) at position 128 is mutated to leucine (L).

[0014] A second aspect of the present invention provides a nucleic acid molecule that encodes a UvsY accessory protein mutant as described in the first aspect of the present invention; Optionally, the nucleic acid molecule is a codon-optimized sequence; Optionally, the codon-optimized sequence is adapted to the codon bias of the E. coli expression system.

[0015] In some embodiments, the nucleotide sequence of the nucleic acid molecule has at least 80% identity with the coding sequence of the wild-type UvsY accessory protein shown in SEQ ID NO:2. Its core feature is that it contains coding sequences for the corresponding mutant amino acid mutation sites. For example, the nucleotide sequence encoding S18Y is obtained by amplification with primer pairs S18Y-F and S18Y-R, and the nucleotide sequences encoding sites such as H39W, D92F, and Q128L also correspond to the corresponding specific primer pairs, ensuring that the mutation sites are accurately embedded in the coding sequence without changing the overall structural framework and functional domains of the protein.

[0016] In some implementation schemes, codon-optimized nucleic acid molecules are adaptively adjusted to the codon usage preferences of the E. coli expression system. By replacing rare codons and optimizing the codon context sequence, the expression efficiency and solubility of the UvsY helper protein mutant are significantly improved, laying the foundation for subsequent protein purification and activity verification.

[0017] A third aspect of the present invention provides a recombinant expression vector comprising the nucleic acid molecule described in the second aspect of the present invention; Optionally, the recombinant expression vector is a prokaryotic expression vector, a eukaryotic expression vector, or a shuttle vector; Optionally, the prokaryotic expression vector is a pET series vector, a pGEX series vector, a pMAL series vector, a pQE series vector, a pETBlue series vector, or a pBAD series vector; Optionally, the eukaryotic expression vector is a pPIC series vector, a pcDNA series vector, or a pYES series vector; Optionally, the shuttle vector is a pEGFP-C series vector or a pFastBac series vector.

[0018] In some embodiments, the nucleic acid molecule is inserted into the multiple cloning site of the vector backbone via restriction endonuclease ligation, forming a functional vector that can stably replicate and efficiently express the UvsY helper protein mutant. In specific embodiments, the recombinant expression vector is a prokaryotic expression vector, a eukaryotic expression vector, or a shuttle vector, and those skilled in the art can flexibly adapt it to different host cell systems according to actual expression needs. The pET-28a (+) vector is preferred, as its T7 promoter can drive efficient transcription of the target gene, and it carries a kanamycin resistance gene for easy screening of positive clones.

[0019] In some implementations, the construction process of the recombinant expression vector is as follows: using the pET-28a (+) vector as a backbone, a nucleic acid molecule encoding the UvsY accessory protein mutant is linked to an 8×histidine tag sequence at the 5' end via a linker peptide sequence, and then inserted between the NcoI and XhoI restriction sites, so that the target gene and the tag sequence form a fusion expression cassette; the recombinant expression vector also contains complete expression regulatory elements, including a promoter, a terminator, and a selection marker gene, wherein the promoter is preferably a T7 promoter, the terminator is a T7 terminator, and the selection marker gene is a kanamycin resistance gene, which can be used to rapidly screen positive recombinant vectors in a kanamycin-containing medium; after the constructed recombinant expression vector is amplified by PCR, the original template is removed by DpnI restriction enzyme digestion, and it is transformed into E. coli DH5α competent cells, and the correctness of the mutation site is verified by sequencing to ensure the sequence integrity and insertion direction accuracy of the nucleic acid molecule.

[0020] A fourth aspect of the present invention provides a recombinant host cell comprising the recombinant expression vector described in the third aspect of the present invention; Optionally, the host cell is a prokaryotic host cell or a eukaryotic host cell; Optionally, the prokaryotic host cell is Escherichia coli or Bacillus subtilis; Optionally, the eukaryotic host cell is Pichia pastoris, Saccharomyces cerevisiae, mammalian cells, or insect cells; Optionally, the Escherichia coli is E. coli DH5α, E. coli BL21 (DE3), E. coli Rosetta (DE3) or E. coli BL21 (DE3) pLysS.

[0021] In some implementations, the recombinant expression vector, after being introduced into host cells via transformation, transfection, or electroporation, can stably replicate and efficiently express the target UvsY helper protein mutant within the host cells. Optionally, the host cell can be a prokaryotic or eukaryotic host cell, which can be flexibly selected by those skilled in the art based on the type of recombinant expression vector, protein expression requirements, and application scenarios. Optionally, the prokaryotic host cell can be *Escherichia coli* or *Bacillus subtilis*, as prokaryotic host cells have advantages such as low culture cost, fast growth rate, and high expression efficiency, making them suitable for large-scale industrial production. Optionally, the eukaryotic host cell can be *Pichia pastoris*, *Saccharomyces cerevisiae*, mammalian cells, or insect cells, which can achieve correct protein folding and modification, making them suitable for applications with high requirements for protein conformation. Optionally, the *Escherichia coli* can be *E. coli* DH5α, *E. coli* BL21 (DE3), *E. coli* Rosetta (DE3), or *E. coli* BL21 (DE3) pLysS, with each strain having complementary functions to meet different experimental needs.

[0022] In some implementations, the preparation of the recombinant host cells requires selecting an appropriate introduction method based on the specific type of host cells. Specifically, for E. coli strains such as E. coli DH5α and E. coli BL21 (DE3), a heat shock transformation method is used. The specific conditions are: incubating the recombinant expression vector and competent cells on ice for 10 min, heat shocking at 42℃ for 90 s, placing on ice for 5 min, adding LB medium, activating at 37℃ and 220 rpm for 1 h, then plating on LB plates containing the corresponding screening antibiotic, culturing for 14-16 h, and picking single colonies to obtain positive transformants; for Bacillus subtilis, an electroporation transformation method is used, where a high-voltage pulse creates a transient channel in the cell membrane to promote the entry of the recombinant expression vector into the cell; for eukaryotic host cells such as Pichia pastoris and Saccharomyces cerevisiae, chemical transformation methods (such as LiAc / PEG-mediated transformation) or electroporation transformation methods can be used; for mammalian cells (such as HEK293 and CHO-K1) and insect cells (such as Sf9 and High...), the method is described below. For the five types of recombinant host cells, liposome transfection or virus-mediated transfection are used. E. coli DH5α is primarily used for the construction, amplification, and sequencing verification of recombinant expression vectors, exhibiting high transformation efficiency and supporting large-scale plasmid replication. E. coli BL21 (DE3) contains the T7 RNA polymerase gene, adaptable to T7 promoter-driven prokaryotic expression vectors, and can efficiently express UvsY accessory protein mutants. E. coli Rosetta (DE3) supplements rare tRNAs from E. coli, significantly enhancing the expression level and solubility of mutants containing rare codons such as H39W and H39F. E. coli BL21 (DE3) pLysS can express T7 lysozyme, inhibiting basal expression of the target protein, making it suitable for expressing mutants toxic to the host. The recombinant host cells obtained after screening can be verified by PCR and protein electrophoresis to confirm the integration of the recombinant expression vector and the expression efficiency of the target protein, ensuring stable and efficient production of UvsY accessory protein mutants with high thermostability.

[0023] The fifth aspect of the present invention provides a method for preparing the UvsY auxiliary protein mutant described in the first aspect of the present invention, the method comprising the following steps: based on the nucleic acid sequence of the wild-type UvsY auxiliary protein shown in SEQ ID NO.2, by designing primer pairs containing the target mutation site, introducing the mutation by PCR amplification, removing the original template by enzyme digestion, transforming into host cells to screen positive clones, and inducing protein expression to obtain the UvsY auxiliary protein mutant.

[0024] A sixth aspect of the present invention provides a method for preparing the recombinant host cell described in the fourth aspect of the present invention, the method comprising the following steps: transforming the recombinant expression vector described in the third aspect of the present invention into a host cell to obtain the recombinant host cell.

[0025] The seventh aspect of the present invention provides for application in any of the following aspects: (1) The application of the UvsY helper protein mutant described in the first aspect of the present invention in DNA isothermal amplification or IPA amplification; (2) Application of the UvsY helper protein mutant described in the first aspect of the present invention in gene detection or field diagnosis at the grassroots level.

[0026] Furthermore, the DNA isothermal amplification includes RPA amplification (recombinase polymerase amplification), RTMA amplification (recombinase-transcriptional-mediated isothermal amplification), LAMP amplification (loop-mediated isothermal amplification), RCA amplification (rolling circle amplification), HDA amplification (helicase-dependent isothermal amplification), RAA amplification (recombinase-assisted amplification), SPIA amplification (single primer isothermal amplification), or NASBA amplification (nucleic acid sequence-dependent amplification).

[0027] In some implementations, the UvsY helper protein mutant can enhance the sensitivity, specificity, and amplification speed of various isothermal amplification techniques by increasing strand substitution efficiency, promoting single-stranded DNA binding, and inhibiting single-strand renaturation, thus adapting to the nucleic acid amplification needs of different scenarios.

[0028] Compared with the prior art, the advantages and beneficial effects of the present invention are as follows: The UvsY accessory protein mutant provided by this invention exhibits significantly improved thermal stability through single-site or combined mutations at specific sites (S18Y, H39W, D92F, Q128L, H39Y, H39M, H39F, S18Y / H39W, S18Y / D92F, S18Y / Q128L, S18Y / H39W / D92F, S18Y / H39W / Q128L, H39W / D92F / Q128L, S18Y / H39W / D92F / Q128L). The Tm value reaches a maximum of 59.4℃, far exceeding the wild type's 33.4℃, thus solving the core technical problem of poor thermal stability in existing UvsY accessory proteins. Furthermore, the mutant maintains highly efficient in vitro nucleic acid amplification activity even after long-term storage at 37℃ and 45℃, with a short peak elution time and stable amplification efficiency. It eliminates the need for strict cold chain transportation and storage, reducing application costs. The UvsY helper protein mutant provided by this invention can be widely used in isothermal amplification technologies such as RPA and RTMA, and is suitable for various scenarios such as gene detection and on-site diagnosis at the grassroots level. It is especially suitable for rapid detection of low copy templates and has extremely high practical application value and industrialization prospects. Detailed Implementation

[0029] The present invention will be further illustrated below with reference to specific embodiments. These 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. The experimental consumables, reagents, and raw materials 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 specific conditions are generally performed under conventional conditions or according to the manufacturer's recommendations. In particular, the following embodiments are for illustrative purposes only and should not limit the scope of the invention in any way. It should be noted that the experimental conditions and results described in the following embodiments are for illustrative purposes only and should not, and will not, limit the invention as described in detail in the claims.

[0030] Example 1: Screening of UvsY helper protein mutants A common strategy for rationally modifying protein stability is to apply computational protein design methods (such as Rosetta), which primarily predict stabilization effects by calculating the Gibbs free energy change induced by mutations. This invention measures the change in molecular stability after mutation by calculating the change in Gibbs free energy of an enzyme molecule following a single-point mutation. DDG (or G) represents the change in free energy induced by mutation. In protein science and molecular biology, this term is often used to measure the effect of a mutation in a single amino acid residue on protein stability or binding free energy.

[0031] After site-directed mutagenesis, the interactions between amino acids in a protein change. The criteria for assessing the change in stability are: if... G is positive; mutations may reduce protein stability or binding affinity. If... G is negative, and mutations in it may increase protein stability or binding affinity. Therefore The G-value can be used to predict changes in the structural stability of a protein after site-directed mutagenesis. It is selected from a series of mutants. Mutations with high G values ​​were used to construct a UvsY helper protein mutant library. However, due to the complexity of protein structures, it is impossible to obtain mutants that meet the needs of practical applications through prediction alone. Therefore, further experimental verification and screening are needed.

[0032] The wild-type UvsY accessory protein sequence was input into the Rosetta algorithm software Cartesian_ddG to obtain... G value decreased significantly ( The information on the eight mutation sites (G value < -2) is shown in Table 1 below.

[0033] Table 1 G value decreased significantly ( Information on 8 mutation sites (G value < -2)

[0034] The amino acid sequence of the wild-type UvsY accessory protein is as follows: MRLEDLQEELKKDVFIDSTKLQYEAANNVMLYSKWLNKHSSIKKEMLRIEAQKKVALKARLDYYSGRGDGDEFSMDRYEKSEMKTVLSADKDVLKVDTSLQYWGILLDFCSGALDAIKSRGFAIKHIQDMRAFEAGK (SEQ ID NO:1); The nucleotide sequence of the codon-optimized wild-type UvsY accessory protein is shown below: ATGAGGTTGGAAGATTTACAAGAGGAACTAAAGAAGGACGTCTTCATCGATTCCACCAAACTGCAATACGAGGCCGCAAATAACGTGATGTTATATTCGAAATGGCTGAACAAACATAGCAGCATTAAGAAAGAGATGTTGCGCATTGAAGCGCAGAAAAAGGTTGCGCTGAAGGCGCGTCTCGACTATTACTCCGGCCGTGGCGACGGCGACGAATTTAGCATGGATCGCTATGAGAAATCTGAAATGAAGACCGTTCTGAGCGCTGATAAAGACGTGTTGAAGGTTGATACGTCTCTGCAATACTGGGGTATTCTGCTGGACTTCTGCAGCGGTGCATTGGATGCGATCAAGAGCCGTGGTTTCGCCATCAAGCACATCCAGGATATGCGTGCGTTTGAGGCTGGTAAATAA (SEQ ID NO:2).

[0035] Example 2 Construction of UvsY Helper Protein Mutant Recombinant Vector Based on the information of the 7 mutation sites in Example 1, the corresponding mutants were constructed. The construction method is as follows: the DNA molecule shown in SEQ ID NO:2 was linked to the 5' end of the DNA molecule encoding UvsY-wt by the linking peptide sequence (GGCAGC) and an 8× histidine tag sequence (CATCACCATCATCATCATCATCAT) was inserted between the NcoI and XhoI restriction sites of the pET-28a(+) vector to obtain the recombinant vector pET-28a(+) / UvsY-wt, which is the wild-type UvsY accessory protein.

[0036] Using the circular plasmid pET-28a(+) / UvsY-wt as a template, a PCR reaction system was prepared. Point mutations were introduced using the point mutation reaction system shown in Table 2 and the primer pairs shown in Table 3 (each point mutation is shown in Table 1), resulting in various recombinant mutant vectors. The PCR amplification program for the circular plasmid is shown in Table 4.

[0037] Table 2 Point Mutation Reaction System

[0038] Table 3 List of primers for mutants

[0039] Table 4 PCR amplification program for circular plasmids

[0040] The plasmid containing the mutant gene, obtained by PCR, was used to remove the original template plasmid using DpnI methyltransferase. The digestion reaction mixture consisted of 1 μL DpnI, 5 μL buffer, 55 μL PCR product, and water to a final volume of 50 μL. The reaction conditions were 37°C for 5 min. Incubation at 37°C could be performed on a PCR instrument or in a water bath. After DpnI digestion, the digestion product was directly transformed into *E. coli* DH5α strain, and the plasmid was extracted for sequencing.

[0041] 10 μL of correctly sequenced recombinant plasmid was added to 100 μL of BL21(DE3) competent cells (expressing host cells) and incubated on ice for 10 min; heat-shocked at 42℃ for 90 s; placed on ice for 5 min; 900 μL of LB medium was added, and the cells were activated at 37℃ and 220 rpm for 1 h on a shaker. The bacterial culture was then spread onto selection plates and cultured for 14–16 h until mature single colonies appeared. Single colonies were picked to screen transformants. These single colonies were then transferred to LB liquid medium containing kanamycin and cultured overnight at 37℃ and 200 rpm. The bacterial cells were centrifuged, and the recombinant plasmid was extracted using a plasmid extraction kit (purchased from Tiangen Biotech). The plasmid's correctness was verified.

[0042] Example 3: Expression and purification of UvsY accessory protein mutant protein (1) Seed culture: Take the UvsY helper protein mutant expression strain obtained and verified in Example 2, pick a single colony and inoculate it into 5 mL of LB medium containing kanamycin, and culture overnight.

[0043] (2) Fermentation culture: 2 mL of overnight culture medium was inoculated into LB medium containing kanamycin and cultured at 37°C with shaking until OD. 600 The value is close to 0.8~1.0.

[0044] (3) Induction culture: Isopropyl-β-D-thiogalactoside (IPTG) was added to a final concentration of 1 mM. Protein expression was induced by high temperature, with induction conditions of 37℃ for 3 h.

[0045] (4) Cell harvesting: The cell pellet was collected by centrifuging at 4°C and 6000 rpm for 20 min. The cells were resuspended by adding 5 mL of lysis buffer (20 mM Tris-HCl, 300 mM NaCl, 20 mM imidazole, pH 7.5) per gram of wet cell weight. The cells were then homogenized and lysed. The protein lysate was centrifuged at 4°C and 12000 rpm for 30 min by a high-speed refrigerated centrifuge. The supernatant was then separated and filtered to remove impurities. The filtered supernatant could be temporarily stored on ice for subsequent nickel column purification.

[0046] (5) Ni-NTA column protein purification: Equilibrate the column with two column volumes of Lysis buffer, allowing the buffer to slowly drain from the resin; slowly load the supernatant (two column volumes of sample solution); wash with Lysis buffer for 20 column volumes; then wash with Wash Buffer containing 20 mM imidazole for 10 column volumes; finally, elute the sample with Elution Buffer containing 250 mM imidazole, elution volume 3 mL. The protein obtained from Ni-NTA purification was concentrated by centrifugation using a 50 kD ultrafiltration tube and replaced with storage buffer. After uniformly quantifying the protein to 50 ng / μL using Nano Drop, the next step of activity testing was performed.

[0047] Example 4: Determination of Tm values ​​of different UvsY accessory protein mutants using differential scanning fluorometry Differential scanning fluorescence (DSF) is a cost-effective and easy-to-use label-free biophysical technique. It determines the thermal denaturation transition temperature (melting temperature, Tm) of a protein by measuring the corresponding change in fluorescence emission as temperature increases. Protein stability increases in the presence of specific compounds or ligands, manifested as an increase in melting temperature. The melting temperature Tm is calculated by assessing conformational stability. DSF utilizes the change in fluorescence intensity after environmentally sensitive dyes (such as SYPROOrange) bind to the hydrophobic regions of a protein. As temperature increases, the protein unfolds, exposing the hydrophobic regions. The dye specifically binds to these hydrophobic regions, enhancing fluorescence. The signal produced by the dye free in aqueous solution is very weak and negligible. However, once unfolded protein is present, the fluorescent dye can bind to the hydrophobic regions of the unfolded protein, producing a significant fluorescence signal.

[0048] A 20 μL reaction mixture (20 mmol / L HEPES-KOH, 150 mmol / L KCl, 1 mg enzyme, 20×SYPROOrange fluorescent dye, pH 8.0) was placed in a quantitative real-time PCR instrument. The excitation and emission wavelengths were adjusted to the corresponding wavelengths of the fluorescent dye. The heating rate was set to 1℃ / 10 s, with the temperature ranging from 25℃ to 99℃. Fluorescence intensity was measured every 1–3℃. After the reaction, the Tm value of the protein sample was calculated using data processing software. The Tm of a protein is an indicator of its thermal stability; generally, the higher the Tm, the more stable the protein.

[0049] The Tm values ​​of different UvsY accessory protein mutants are shown in Table 5. The Tm values ​​of single-point mutants S18Y, K38Y, H39W, D92F, Q128L and combined mutants are all higher than those of WT and single-point mutants N27F, S33M and N37R. The above results indicate that the UvsY accessory protein mutants provided by the present invention have higher thermal stability than WT.

[0050] Table 5. Tm values ​​of different UvsY accessory protein mutants

[0051] Example 5: In vitro nucleic acid amplification performance of UvsY helper protein mutant under different temperature storage conditions Recombinase-transcription-mediated amplification (RTMA) technology utilizes primers containing highly specific transcription promoters, corresponding RNA polymerases, single-strand binding proteins, UvsY accessory proteins, and reverse transcriptase to continuously amplify templates, providing a continuous template for recombinase polymerase amplification (RPA) and increasing amplification efficiency by more than 10 times. It is particularly effective for amplifying low-copy substrates. The components of the in vitro nucleic acid amplification reaction system (50 μL) are shown in Table 6 below.

[0052] Table 6. Composition of the in vitro nucleic acid amplification reaction system (50 μL)

[0053] 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 accessory protein; GP32 is single-strand binding protein; RNAP is RNA polymerase.

[0054] Using canine parvovirus VP2 DNA as a template (5'-tcagtaatatagtttgtatttcccatttgagttacaccacgtcttttatcttgttgaactcctatataaccaaagttagtacctccttcagattgaggcaaagaatttagaaatggtggtaagcccaatgctctatttgtttgccatgtatgtgttagtctacatggtttacaatcaaaaaaaaatgttcctgtagcaaattcatcacctgttcttagtaagtgtactggcaca-3' (SEQ ID NO:25)), the reaction was carried out at a constant temperature of 20-45℃ for 30 min on a real-time PCR instrument. Fluorescence was read every 30 s at 497 nm, and the RTMA reaction was monitored using the Bori FQD-96X real-time PCR detection system.

[0055] The primers used were: upstream primer 5'-TAATACGACTCACTATAGGGCACTTACTAAGAACAGGTGATGAATTTGCTACAGC-3' (SEQ ID NO:26); downstream primer 5'-AGTTTGTATTTCCCATTTGAGTTACACCACGTCT-3' (SEQ ID NO:27). The UvsY accessory protein mutant with significantly improved heat stability obtained in Example 3 was used instead of the wild-type UvsY accessory protein. The UvsY accessory protein mutant and wild-type UvsY accessory protein were stored at 37°C for 30 days or at 45°C for 30 days, respectively, and RTMA amplification was performed at different temperatures using canine parvovirus VP2 as the amplification target. The corresponding untreated UvsY accessory protein mutant and wild-type UvsY accessory protein were used as the untreated group. The results are shown in Table 7 below.

[0056] The results showed that the wild-type UvsY accessory protein had extremely poor thermostability. The peak time in the untreated group was 8 min 26 s, which increased to 15 min 39 s after 30 days of storage at 37℃, and completely lost its amplification activity after 30 days of storage at 45℃. In contrast, all tested combination mutants (three-point, four-point, and five-point mutant combinations) showed significantly better performance. The peak time in the untreated group was only 5 min 03 s to 7 min 02 s. After 30 days of storage at 37℃, the peak time only slightly increased to 6 min 09 s to 8 min 18 s. After 30 days of storage at 45℃, there was still a clear peak signal with a peak time of 12 min 16 s to 15 min 12 s. Among them, the five-point mutant S18Y / K38Y / H39W / D92F / Q128L performed best under all conditions. The control group had the shortest peak time and the smallest increase in peak time after high-temperature storage. These results demonstrate that the UvsY accessory protein modified by combining mutations at specific sites not only exhibits significantly higher original amplification efficiency than the wild type, but also achieves a leap in thermal stability, completely solving the application bottleneck of the wild type, which suffers from "sharp drop in efficiency during medium-temperature storage and complete inactivation during high-temperature storage." Furthermore, they prove that the combination of mutation sites has a significant synergistic effect; the more mutation sites there are, the better the thermal stability and amplification performance. This provides crucial raw material support for the application of RPA / RTMA technology in scenarios such as grassroots field diagnosis without cold chain conditions and the industrialization of in vitro diagnostic reagents, demonstrating extremely high practical application value.

[0057] Table 7. Experimental results of in vitro nucleic acid amplification performance of UvsY accessory protein mutants under different temperature storage conditions.

[0058] The Tm values ​​of different UvsY accessory protein mutants are shown in Table 5. The Tm values ​​of the combined mutants S18Y / H39W / D92F / Q128L, K38Y / H39W / D92F / Q128L, and S18Y / K38Y / H39W / D92F / Q128L are significantly higher than those of wild-type UvsY accessory protein and other combined mutants. The above results indicate that the UvsY accessory protein mutants provided by this invention have higher thermal stability and better isothermal amplification performance compared to WT.

Claims

1. A UvsY cofactor protein mutant with improved thermostability, characterized in that, The UvsY accessory protein mutant has any one of the following amino acid mutations in the amino acid sequence of the wild-type UvsY accessory protein as shown in SEQ ID NO:1: S18Y, H39W, D92F, Q128L, H39Y, H39M, H39F, S18Y / H39W, S18Y / D92F, S18Y / Q128L, S18Y / H39W / D92F, S18Y / H39W / Q128L, H39W / D92F / Q128L, S18Y / H39W / D92F / Q128L.

2. The UvsY helper protein mutant according to claim 1, characterized in that, The UvsY accessory protein mutant is a combinatorial mutant, which includes any one of the following combinatorial mutation sites: S18Y / H39W, S18Y / D92F, S18Y / Q128L, S18Y / H39W / D92F, S18Y / H39W / Q128L, H39W / D92F / Q128L, and S18Y / H39W / D92F / Q128L.

3. The UvsY helper protein mutant of claim 1, wherein, The UvsY accessory protein mutant is a combined mutant, which includes any one of the following combined mutation sites: H39W / D92F / Q128L, S18Y / H39W / D92F / Q128L.

4. A nucleic acid molecule, characterized in that, The nucleic acid molecule encodes a mutant of the UvsY accessory protein as described in any one of claims 1-3; Optionally, the nucleic acid molecule is a codon-optimized sequence; Optionally, the codon-optimized sequence is adapted to the codon bias of the E. coli expression system.

5. A recombinant expression vector, characterized in that, The recombinant expression vector comprises the nucleic acid molecule of claim 4; Optionally, the recombinant expression vector is a prokaryotic expression vector, a eukaryotic expression vector, or a shuttle vector; Optionally, the prokaryotic expression vector is a pET series vector, a pGEX series vector, a pMAL series vector, a pQE series vector, a pETBlue series vector, or a pBAD series vector; Optionally, the eukaryotic expression vector is a pPIC series vector, a pcDNA series vector, or a pYES series vector; Optionally, the shuttle vector is a pEGFP-C series vector or a pFastBac series vector.

6. A recombinant host cell, characterized in that, The recombinant host cell comprises the recombinant expression vector according to claim 5; Optionally, the host cell is a prokaryotic host cell or a eukaryotic host cell; Optionally, the prokaryotic host cell is Escherichia coli or Bacillus subtilis; Optionally, the eukaryotic host cell is Pichia pastoris, Saccharomyces cerevisiae, mammalian cells, or insect cells; Optionally, the Escherichia coli is E. coli DH5α, E. coli BL21 (DE3), E. coli Rosetta (DE3) or E. coli BL21 (DE3) pLysS.

7. A method for preparing the UvsY accessory protein mutant according to any one of claims 1-3, characterized in that, The method includes the following steps: based on the nucleic acid sequence of the wild-type UvsY accessory protein shown in SEQ ID NO.2, the UvsY accessory protein mutant is obtained by designing primer pairs containing the target mutation site, introducing mutations by PCR amplification, removing the original template by enzyme digestion, transforming into host cells to screen positive clones, and inducing protein expression.

8. A method for preparing the recombinant host cell according to claim 6, characterized in that, The method includes the following steps: transforming the recombinant expression vector of claim 5 into a host cell to obtain the recombinant host cell.

9. Applications in any of the following aspects: (1) The use of the UvsY helper protein mutant according to any one of claims 1-3 in DNA isothermal amplification or IPA amplification; (2) The application of the UvsY helper protein mutant according to any one of claims 1-3 in gene detection or field diagnosis at the grassroots level.

10. The application according to claim 9, characterized in that, The DNA isothermal amplification includes RPA amplification, RTMA amplification, LAMP amplification, RCA amplification, HDA amplification, RAA amplification, SPIA amplification, or NASBA amplification.

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