A HUH solid-binding protein and a solid-binding method mediated by the protein or nucleic acid thereof.

Abstract

This application relates to the field of biomolecular immobilization technology, specifically to a HUH immobilization protein and a method for immobilizing proteins or nucleic acids mediated by it. The HUH immobilization protein of this application comprises a HUH protein and a solid-phase material, wherein the binding force between the HUH protein and the solid-phase material is a bioaffinity force; the HUH protein is selected from at least one of A* protein, TC1 protein, and HI0217 protein. Compared to methods that can only achieve protein or nucleic acid immobilization, the immobilization peptide of this invention can immobilize two molecules in one route, and the reaction is simple, rapid, and mild, requiring no additional chemical modification. Protein immobilization is achieved in one step at room temperature, and nucleic acid immobilization is achieved in two steps. Simultaneously, the immobilized targets, such as proteins and nucleic acids, can still maintain good activity on the carrier surface.

Description

Technical Field

[0001] This application relates to the field of biomolecular immobilization technology, specifically to a HUH immobilization protein and a method for immobilizing proteins or nucleic acids mediated by it. Background Technology

[0002] Nucleic acids and proteins, as the material basis of living organisms, possess powerful functions. Functionalized materials prepared by immobilizing nucleic acid and protein molecules on solid-phase materials such as gold, polystyrene (PS), and silica have wide applications in fields such as bioimaging, biomedicine, and biosensing. However, the activity of biomolecules on material surfaces is directly related to their structure. Immobilized nucleic acids and proteins can become inactive due to unfavorable conformations, thus reducing the hybridization efficiency of surface nucleic acids or the binding efficiency with target molecules. Changes in the conformation of enzymes or antibody / antigen molecules immobilized on the surface may lead to the loss of catalytic activity or antigen-antibody recognition ability. Therefore, maintaining the function of nucleic acids and proteins while immobilizing them on a carrier is the goal of biomolecular immobilization technology development. There is currently a high demand for effective and simple biomolecular immobilization methods.

[0003] Existing nucleic acid / protein immobilization methods can be broadly categorized into physical adsorption, chemical cross-linking, and bioaffinity immobilization. Direct physical adsorption is simple but significantly impacts the activity of the immobilized molecules; chemical cross-linking methods are complex, typically requiring toxic chemical reagents and specific reaction conditions. In contrast, immobilization methods that bind proteins / peptides with high affinity to specific solid-phase materials offer better preservation of the immobilized molecules' activity. Furthermore, the immobilization process is simple, easy to operate, requires no additional chemical modification, and exhibits stability in biological environments, making it a superior alternative to traditional immobilization methods.

[0004] HUH family proteins are a class of proteins that can recognize and bind to nucleic acids. They are widely present in living organisms and play a key role in nucleic acid replication, transfer, and other processes. They are powerful tools for protein-nucleic acid linkage.

[0005] Currently, there are no reports of HUH family proteins having the ability to bind to solid materials; even fewer reports of HUH family proteins acting as a connecting bridge to immobilize nucleic acids / proteins on solid materials. Summary of the Invention

[0006] In view of this, the present invention provides a HUH solid-binding protein and a method for solid-binding proteins or nucleic acids mediated therein. The present invention discovers that the HUH protein has the ability to bind to a variety of solid-phase materials and can be used for nucleic acid / protein immobilization.

[0007] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0008] This invention is the first to discover a new function of the HUH protein: its ability to bind to a variety of solid materials, such as polymers, metals, and minerals.

[0009] In a first aspect, the present invention provides a HUH solid-phase binding protein, which includes a HUH protein and a solid-phase material, wherein the binding force between the HUH protein and the solid-phase material is a bioaffinity force.

[0010] The HUH protein is selected from at least one of the A* protein, TC1 protein, and HI0217 protein.

[0011] This HUH fixation protein can be used for the fixation of proteins and nucleic acids.

[0012] Secondly, the present invention provides a solid-phase binding method for HUH protein, the method comprising: incubating HUH protein with a solid-phase material to obtain HUH solid-phase bound protein;

[0013] The HUH protein is selected from at least one of the A* protein, TC1 protein, and HI0217 protein.

[0014] Preferably, the solid material is selected from at least one of polymers, metals, and minerals;

[0015] Preferably, the polymer is selected from at least one of polystyrene, polyurethane, polystyrene divinylbenzene, polymethyl methacrylate, polyacrylamide, polyethylene glycol terephthalate, polyvinyl acetate, polyethylene, polypropylene, polyvinyl chloride, and polyvinylpyrrolidone;

[0016] In the specific embodiments provided by this invention, the polymer is polystyrene.

[0017] Preferably, the metal is selected from at least one of gold, silver, copper, aluminum, and iron;

[0018] In the specific embodiments provided by the present invention, the metal is gold; preferably, it is gold nanoparticles.

[0019] Preferably, the mineral is selected from at least one of silicon dioxide, calcium oxide, titanium dioxide, and iron oxide.

[0020] In the specific embodiments provided by the present invention, the mineral is silicon dioxide.

[0021] Preferably, the solid material can be in the form of particles, rods, tubes, sheets, or plates.

[0022] Preferably, the incubation temperature is 30–40°C and the incubation time is 0.5–5 h.

[0023] In a specific embodiment provided by the present invention, the incubation temperature is 37°C and the incubation time is 1 hour.

[0024] This invention discovers that HUH protein has the ability to bind to various solid-phase materials—such as polymers, metals, and minerals—making HUH protein suitable for protein and nucleic acid fixation. Figure 1 ).

[0025] Thirdly, the present invention provides a method for solid-binding of proteins, comprising the following steps:

[0026] S11, the target protein is first linked to the HUH protein to obtain the target protein-HUH protein complex.

[0027] S12, the target protein-HUH protein complex and solid material are incubated;

[0028] or,

[0029] S21, HUH protein and solid material are incubated to obtain HUH solid-binding protein;

[0030] S22, the target protein and HUH-binding protein are linked in a second way;

[0031] Preferably, the first connection is performed using a method including protein fusion and / or condensation reaction;

[0032] Preferably, the second connection is achieved through a condensation reaction.

[0033] The two ligation methods (first ligation and second ligation) are two different protein immobilization methods, which ultimately yield HUH immobilized proteins for further detection of target molecules (such as antigens, antibodies, etc.).

[0034] Fourthly, the present invention provides a HUH-binding protein immobilized with a protein, the protein comprising the above-mentioned HUH-binding protein and a target protein linked to the HUH protein in the HUH-binding protein;

[0035] Preferably, the target protein includes, but is not limited to, at least one of streptavidin, enzymes, fluorescent proteins, and affinity peptides.

[0036] In a specific embodiment provided by this invention, the target protein is streptavidin. Streptavidin can bind to biotinylated molecules through affinity interactions, and can be further used for the detection of target molecules (such as antigens, antibodies, etc.).

[0037] Fifthly, the present invention provides a method for detecting an antigen, comprising the following steps:

[0038] S31, streptavidin was fused with HUH protein for expression to obtain streptavidin-HUH protein complex;

[0039] S32, the streptavidin-HUH protein complex and solid-phase material were incubated and then blocked;

[0040] S33, add biotinylated capture antibody, incubate;

[0041] S34, add the sample to be tested and incubate;

[0042] S35, add antibody containing detection marker, and incubate;

[0043] S36, Detect the detection mark.

[0044] The principle of the above antigen detection method is as follows: the streptavidin-HUH protein complex (SA-HUH) is immobilized on the surface of a solid material through affinity (SA-HUH-solid phase), and the biotinylated capture antibody is immobilized on the SA-HUH-solid phase through streptavidin-biotin affinity (capture antibody-SA-HUH-solid phase); if the sample to be tested contains antigen, the antigen binds to the capture antibody (antigen-capture antibody-SA-HUH-solid phase); finally, the labeled antibody binds to the antigen (labeled antibody-antigen-capture antibody-SA-HUH-solid phase), and the presence and / or content of the antigen can be determined by detecting the intensity of the labeled signal.

[0045] Sixthly, the present invention provides a method for solid-state binding of nucleic acids, comprising the following steps:

[0046] S41, HUH protein and solid material are incubated to obtain HUH solid-bound protein;

[0047] S42 involves incubating the target nucleic acid with the HUH-binding protein or performing a third ligation.

[0048] or,

[0049] S51, incubate or third-party ligation of HUH protein and target nucleic acid to obtain target nucleic acid-HUH protein complex;

[0050] S52, the target nucleic acid-HUH protein complex and solid material are incubated.

[0051] In step S42 or S51, since the HUH protein itself has nucleic acid binding function, the binding of the HUH protein to nucleic acid can be achieved through incubation.

[0052] Besides incubation, other methods can be used to achieve the binding of HUH protein to nucleic acids. Preferably, the third ligation method includes chemical modification and / or condensation reaction.

[0053] In a seventh aspect, the present invention provides a HUH-binding protein immobilized with DNA, comprising the above-mentioned HUH-binding protein and target DNA that is affinity-bound or linked to the HUH protein in the HUH-binding protein.

[0054] Preferably, the target DNA includes, but is not limited to, capture DNA.

[0055] Eighthly, the present invention provides a method for detecting target DNA, comprising the following steps:

[0056] S61, HUH protein and solid material are incubated to obtain HUH solid-binding protein;

[0057] S62, incubate and block the captured DNA and HUH solid-binding protein;

[0058] S63, add the sample to be tested and signal DNA containing the detection marker, and incubate;

[0059] S64, Detect the detection mark.

[0060] The principle of the above-mentioned target DNA detection method is as follows: the captured DNA binds to the HUH solid-binding protein through affinity (captured DNA-HUH-solid phase); if the sample to be tested contains target DNA, the target DNA binds to the captured DNA through complementary interaction (target DNA-captured DNA-HUH-solid phase); finally, the signal DNA binds to the target DNA (signal DNA-target DNA-captured DNA-HUH-solid phase). By detecting the intensity of the labeled signal, the presence and / or content of the target DNA can be determined.

[0061] In a specific embodiment provided by the present invention, the signal DNA is a nucleic acid fragment that can bind to the target DNA.

[0062] In specific embodiments provided by the present invention, the detection marker includes at least one of a radioactive isotope, a metal chelating agent, an enzyme, a fluorescent compound, a bioluminescent compound, or a chemiluminescent compound.

[0063] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0064] 1) Compared to methods that can only achieve protein or nucleic acid fixation, the fixation-binding peptide of the present invention can fix two molecules in one route.

[0065] 2) This invention involves a simple and rapid reaction process under mild conditions, requiring no additional chemical modification. Protein immobilization is achieved through a one-step incubation at room temperature. Nucleic acid immobilization is achieved through a two-step incubation process.

[0066] 3) In this invention, immobilized targets such as proteins and nucleic acids can still maintain good activity on the carrier surface. Attached Figure Description

[0067] Figure 1 This demonstrates the HUH protein-mediated binding and immobilization of nucleic acids or proteins with solid-phase materials.

[0068] Figure 2 Demonstrates the testing procedure for the solid-phase material binding ability of HUH protein;

[0069] Figure 3 The solid-phase material binding ability of HUH proteins is shown; where A shows the binding ability of the three HUH proteins to polystyrene (PS); B shows the binding ability of the three HUH proteins to silica; and C shows the binding ability of the three HUH proteins to gold.

[0070] Figure 4 The diagram illustrates HUH protein-mediated protein fixation; where A shows a schematic diagram of TC1 protein-mediated antigen detection; B shows that TC1 protein-mediated protein fixation can better maintain protein activity.

[0071] Figure 5 The diagram illustrates HUH protein-mediated nucleic acid immobilization. A shows a schematic diagram of TC1 protein-mediated target nucleic acid detection; B shows that TC1 protein-mediated nucleic acid immobilization better maintains nucleic acid activity. The smaller graph in B is the physical adsorption curve, i.e., a magnified version of the black line in the larger graph. The values ​​on the horizontal axis, from left to right, are 10. 5 10 10 The values ​​on the vertical axis, from bottom to top, are 200, 400, 600, 800, and 1000. Detailed Implementation

[0072] This invention discloses a HUH solid-binding protein and a method for solid-binding proteins or nucleic acids mediated therein. Those skilled in the art can refer to the content of this document and appropriately modify the process parameters to achieve the desired result. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art and are considered to be included in this invention. The methods and applications of this invention have been described through preferred embodiments. Those skilled in the art can clearly modify or appropriately change and combine the methods and applications described herein without departing from the content, spirit, and scope of this invention to realize and apply the technology of this invention.

[0073] Terminology Explanation:

[0074] HUH family proteins refer to HUH endonucleases (HUH enzymes, HUH proteins), whose main function is to catalyze the cleavage and rejoining of single-stranded DNA using tyrosine residues at their active sites, forming transient 5′-phosphotyrosine bonds with the DNA substrate. The specific types of HUH family proteins are not limited; different HUH family proteins can be freely selected based on different enzyme-specific recognition sequences and different target proteins or peptides. In specific embodiments of this invention, the HUH family protein is selected from at least one of A*, TC1, and HI0217 endonucleases.

[0075] A* endonuclease (A* protease): Derived from bacteriophage phi X174, it plays an important role in viral DNA replication, cleaving the replication initiation site and covalently ligating nucleic acids. The A* enzyme specifically recognizes the nucleic acid sequence 5'-n-CAACTTGA-n-3', where n represents one or more nucleotide bases; preferably, n is 0-30 nucleotide bases. For example, the A* enzyme specifically recognizes the nucleic acid sequence... CAACTTGA TACATACATCATCCCTCATTCA, the underlined part is the identification site.

[0076] TC1 endonuclease: A replication-associated protein from tomato yellow leaf curl virus, it cleaves the replication initiation site to initiate rolling circle replication. The TC1 enzyme specifically recognizes the nucleic acid sequence 5'-n-TATTA-n-3', where n represents one or more nucleotide bases; preferably, n is 0-30 nucleotide bases. For example, the TC1 enzyme specifically recognizes the nucleic acid sequence TATAA. TATTA CTTTTTTGTAGCACGATTGCAGCATTG. The underlined part is the identification site.

[0077] HI0217 endonuclease: A transposon-associated protein from Haemophilus influenzae, responsible for cleaving single-stranded circular DNA from the donor DNA "top strand" and then reinserting it into the DNA target. The specific recognition nucleic acid sequence for HI0217 is 5'-n-CCTAC-n-3', where n represents one or more nucleotide bases; preferably, n is 0-30 nucleotide bases. For example, the HI0217-specific recognition nucleic acid sequence is GTAGGGTGGGCTTTAGCCCACCAT. CCTAC ATTAAGTACGCATAATT, the underlined part is the identification site.

[0078] Bioaffinity: a specific interaction between biomolecules, a phenomenon in which biomolecules can recognize and attract specific substances under certain conditions (often requiring a medium).

[0079] Fusion protein: This is the expression product of two recombined genes obtained through DNA recombination technology. Two different proteins are linked into a large molecule, which can be achieved through chemical connection or gene fusion.

[0080] Condensation reaction: A reaction in which two or more organic molecules interact and combine covalently to form a large molecule, often accompanied by the loss of small molecules (such as water, hydrogen chloride, alcohol, etc.).

[0081] Streptavidin (SA): A protein with similar biological properties to avidin, it is a secretion of *Streptomyces avidinii*. Its molecular weight and biotin-binding ability are similar to avidin in egg white, with an isoelectric point of 6.0 and a much lower non-specific binding rate than avidin. The binding of biotin to streptavidin is one of the strongest known non-covalent interactions in nature, with a dissociation constant (Kd) of approximately 10. -14 mol / L. Because streptavidin-biotin complexes are well resistant to organic solvents, denaturants (such as guanidine hydrochloride), detergents (such as SDS and Triton), proteolytic enzymes, and extreme temperatures and pH, streptavidin is widely used in molecular biology and bionanotechnology.

[0082] Capture antibody: refers to an antibody immobilized on a solid phase for use in immune testing.

[0083] Capture assay: This refers to an immunoassay method that involves contacting a sample with a capture antibody immobilized on a solid phase. If the sample contains an antigen that can bind to the immobilized antibody and the reaction conditions are suitable, the antigen will form an antigen-antibody complex with the immobilized antibody, thereby being "captured" on the solid phase and subsequently detected or measured.

[0084] Capture DNA: refers to a complementary strand of a specific gene segment.

[0085] The sequences of the proteins and nucleic acids used in the following examples are as follows:

[0086] (1) A*gene nucleic acid sequence (SEQ ID NO: 1)

[0087] ATGAAATCGCGTAGACCATTTGCTATTCAGCGTTTGAATGAATGCAATGCG

[0088] ACAGGCTCATGCTGATGGTTGGTTTATCGTTTTTGACACTCTCACGTTGGC

[0089] TGACGACCGATTAGAGGCGTTTTATGATAATCCCAATGCTTTGCGTGACTA

[0090] TTTTCGTGATATTGGTCGTATGGTTCTTGCTGCCGAGGGTCGCAAGGCTAA

[0091] TGATTCACACGCCGACTGCTATCAGTATTTTTGTGTGCCTGAGTATCCAAC

[0092] AGCTAATCCACGTCTTCATTTCCATGCGGTGCACTTTATGCGGACACTTCC

[0093] TACAGGTAGCGTTGACCCTAATTTTGGTCGTCGGGTACGCAATCGCCGCC

[0094] AGTTAAATAGCGTGCAAAATACGTGGCCTTATCCCTACAGTATGCCCATC

[0095] GCAGTTCGCTACACGCAGGACGCTTTTTCACGTTCTCCCTGGTTGTGGCCT

[0096] GTTGATGCTAAAGGTGAGCCGCTTAAAGCTACCAGTTATATGGCTGTTGGT

[0097] TTCTATGTGGCTAAATTCGTTAACAAAAAGTCAGATATGGACCTTGCTGCT

[0098] AAAGGTCTAGGAGCTAAAGAATGGAACAACTCACTAAAAACCAAGCTGT

[0099] CGCTACTTCCCAAGAAGCTGTTCAGAATCAGAATGAGCCGCAACTTCGGG

[0100] ATGAAAATGCTCACAATGACAAATCTGTCCACGGAGTGCTTAATCCAACT

[0101] TACCAAGCTGGGTTACGACGCGACGCCGTTCAACCAGATATTGAAGCAGA

[0102] ACGCAAAAAGAGAGATGAGATTGAGGCTGGGAAAAGTTACTGTAGCCGA

[0103] CGTTTTGGCGGCGCAACCTGTGACGACAAATCTGCTCAAATTTATGCGCG

[0104] CTTCGATAAAAATGATTGGCGTATCCAACCTGCAGAGTTTTATCGCTTCCA

[0105] TGACGCAGAAGTTAACACTTTCGGATATTTCTGATGAGTCGAAAAATTATC

[0106] TTGATAAAGCAGGAATTACTACTGCTTGTTTACGAATTAAATCGAAGTGG

[0107] ACTGCTGGCGGAAAACACCACCACCACCACCACTGA

[0108] (2) A* protein amino acid sequence (SEQ ID NO: 2)

[0109] MKSRRPFAIQRLMNAMRQAHADGWFIVFDTLTLADDRLEAFYDNPNALRDY

[0110] FRDIGRMVLAAEGRKANDSHADCYQYFCVPEYPTANPRLHFHAVHFMRTLP

[0111] TGSVDPNFGRRVRNRRQLNSVQNTWPYPYSMPIAVRYTQDAFSRSPWLWPV

[0112] DAKGEPLKATSYMAVGFYVAKFVNKKSDMDLAAKGLGAKEWNNSLKTKLS

[0113] LLPKKLFRIRMSRNFGMKMLTMTNLSTECLIQLTKLGYDATPFNQILKQNAK

[0114] REMRLRLGKVTVADVLAAQPVTTNLLKFMRASIKMIGVSNLQSFIASMTQKL

[0115] TLSDISDESKNYLDKAGITTACLRIKSKWTAGGKHHHHHH

[0116] (3) Nucleic acid sequence of TC1 gene (SEQ ID NO: 3)

[0117] ATGCCTCGTTCAGGCCGCTTTAGCATTAAATGCAAGAACTACTTTCTGACC<000​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​(5) Nucleic acid sequence of HI0217 gene (SEQ ID NO: 5)

[0130] ATGTCTAATTATCGGCGCGATTTTACTAAACCTGGATTATATTTTTTCACA

[0131] ATTGTTTTACAAGATCCTACAAAATCTTATCTAACTGACTATATCAATGAA

[0132] TTTAGATCTTTATATAAACAAACTTGTGAACATTATCCATTCGAAACAGTA

[0133] GCAATTTGTATTTTGCCCGATCATATTCATTTACTGATGCAATTACCTGAA

[0134] AATGATGATAATTACGCAATACGCATCGCATATTTAAAAACACAATTTAC

[0135] ACGACAACTTCCAAAAGAATGCCGACAATTTAATAAAAATAGACAAAAA

[0136] TATCGAGAATCAGGTATTTGGCAACGCCGATTTTGGGAGCATTTAATTCGT

[0137] GATGATAAAGATTTAGCGAATCATTTAGATTATATTTATTACAATCCTGTG

[0138] AAACACGGCTATGTTGAGGTAGTAAAAGATTGGCCGTATTCTTCCTTCCAT

[0139] CGTGATGTGAAATGTGAGATTTATCCTGAAGATTGGGGAGGCAACCCAGA

[0140] TTTGAAAATTAAAGGTGATATACACCACCACCACCACCACTAA

[0141] (6) Amino acid sequence of HI0217 PROTEIN (SEQ ID NO: 6)

[0142] MSNYRRDFTKPGLYFFTIVLQDPTKSYLTDYINEFRSLYKQTCEHYPFETVAIC

[0143] ILPDHIHLLMQLPENDDNYAIRIAYLKTQFTRQLPKECRQFNKNRQKYRESGI

[0144] WQRRFWEHLIRDDKDLANHLDYIYYNPVKHGYVEVVKDWPYSSFHRDVKC

[0145] EIYPEDWGGNPDLKIKGDIHHHHHH

[0146] (7) Nucleic acid sequence of streptavidin SA gene (SEQ ID NO: 7)

[0147] GGAGACCCGAGCAAAGATTCTAAAGCACAAGTATCTGCTGCAGAAGCAG

[0148] GAATTACAGGCACATGGTATAATCAGCTGGGATCTACATTTATTGTTACAG

[0149] CCGGCGCAGATGGAGCTCTTACAGGAACATATGAATCTGCTGTTGGAAAT

[0150] GCAGAATCTAGATACGTGCTTACAGGAAGATATGATTCTGCACCTGCAAC

[0151] AGATGGATCCGGAACAGCACTTGGATGGACAGTTGCATGGAAAAACAATT

[0152] ATAGAAACGCACATAGCGCTACAACATGGTCTGGCCAATATGTGGGAGGT

[0153] GCAGAAGCAAGAATTAACACACAATGGCTTTTAACATCTGGAACAACAGA

[0154] AGCAAATGCATGGAAAAGTACTCTTGTTGGACATGATACATTTACAAAAG

[0155] TTAAACCTAGCGCAGCATCTATCGATGCAGCGAAAAAAGCAGGAGTTAAC

[0156] AATGGCAATCCTTTAGATGCAGTTCAACAACACCACCACCACCACCAC

[0157] (8) Amino acid sequence of streptavidin SA PROTEIN (SEQ ID NO: 8)

[0158] GDPSKDSKAQVSAAEAGITGTWYNQLGSTFIVTAGADGALTGTYESAVGNA

[0159] ESRYVLTGRYDSAPATDGSGTALGWTVAWKNNYRNAHSATTWSGQYVGG

[0160] AEARINTQWLLTSGTTEANAWKSTLVGHDTFTKVKPSAASIDAAKKAGVNN

[0161] GNPLDAVQQHHHHHH

[0162] (9) Nucleic acid sequence of SA-A* fusion protein gene (SEQ ID NO: 9)

[0163] ATGGGAGACCCGAGCAAAGATTCTAAAGCACAAGTATCTGCTGCAGAAG

[0164] CAGGAATTACAGGCACATGGTATAATCAGCTGGGATCTACATTTATTGTTA

[0165] CAGCCGGCGCAGATGGAGCTCTTACAGGAACATATGAATCTGCTGTTGGA

[0166] AATGCAGAATCTAGATACGTGCTTACAGGAAGATATGATTCTGCACCTGC

[0167] AACAGATGGATCCGGAACAGCACTTGGATGGACAGTTGCATGGAAAAAC

[0168] AATTATAGAAACGCACATAGCGCTACAACATGGTCTGGCCAATATGTGGG

[0169] AGGTGCAGAAGCAAGAATTAACACACAATGGCTTTTAACATCTGGAACAA

[0170] CAGAAGCAAATGCATGGAAAAGTACTCTTGTTGGACATGATACATTTACA

[0171] AAAGTTAAACCTAGCGCAGCATCTATCGATGCAGCGAAAAAAGCAGGAG

[0172] TTAACAATGGCAATCCTTTAGATGCAGTTCAACAAGGTGGAGGTGGATCG

[0173] AAATCGCGTAGACCATTTGCTATTCAGCGTTTGATGAATGCAATGCGACA

[0174] GGCTCATGCTGATGGTTGGTTTATCGTTTTTGACACTCTCACGTTGGCTGA

[0175] CGACCGATTAGAGGCGTTTTATGATAATCCCAATGCTTTGCGTGACTATTT

[0176] TCGTGATATTGGTCGTATGGTTCTTGCTGCCGAGGGTCGCAAGGCTAATGA

[0177] TTCACACGCCGACTGCTATCAGTATTTTTGTGTGCCTGAGTATCCAACAGC

[0178] TAATCCACGTCTTCATTTCCATGCGGTGCACTTTATGCGGACACTTCCTAC

[0179] AGGTAGCGTTGACCCTAATTTTGGTCGTCGGGTACGCAATCGCCGCCAGTT

[0180] AAATAGCGTGCAAAATACGTGGCCTTATCCCTACAGTATGCCCATCGCAG

[0181] TTCGCTACACGCAGGACGCTTTTTCACGTTCTCCCTGGTTGTGGCCTGTTG

[0182] ATGCTAAAGGTGAGCCGCTTAAAGCTACCAGTTATATGGCTGTTGGTTTCT

[0183] ATGTGGCTAAATTCGTTAACAAAAAGTCAGATATGGACCTTGCTGCTAAA

[0184] GGTCTAGGAGCTAAAGAATGGAACAACTCACTAAAAACCAAGCTGTCGCT

[0185] ACTTCCCAAGAAGCTGTTCAGAATCAGAATGAGCCGCAACTTCGGGATGA

[0186] AAATGCTCACAATGACAAATCTGTCCACGGAGTGCTTAATCCAACTTACC

[0187] AAGCTGGGTTACGACGCGACGCCGTTCAACCAGATATTGAAGCAGAACGC

[0188] AAAAAGAGAGATGAGATTGAGGCTGGGAAAAGTTACTGTAGCCGACGTTT

[0189] TGGCGGCGCAACCTGTGACGACAAATCTGCTCAAATTTATGCGCGCTTCG

[0190] ATAAAAATGATTGGCGTATCCAACCTGCAGAGTTTTATCGCTTCCATGACG

[0191] CAGAAGTTAACACTTTCGGATATTTCTGATGAGTCGAAAAATTATCTTGAT

[0192] AAAGCAGGAATTACTACTGCTTGTTTACGAATTAAATCGAAGTGGACTGC

[0193] TGGCGGAAAACACCACCACCACCACCACTGA

[0194] (10) SA-A* Fusion Protein PROTEIN Amino Acid Sequence (SEQ ID NO: 10)

[0195] MGDPSKDSKAQVSAAEAGITGTWYNQLGSTFIVTAGADGALTGTYESAVGN

[0196] AESRYVLTGRYDSAPATDGSGTALGWTVAWKNNYRNAHSATTWSGQYVG

[0197] GAEARINTQWLLTSGTTEANAWKSTLVGHDTFTKVKPSAASIDAAKKAGVN

[0198] NGNPLDAVQGGGGSKSRRPFAIQRLMNAMRQAHADGWFIVFDTLTLADDR

[0199] LEAFYDNPNALRDYFRDIGRMVLAAEGRKANDSHADCYQYFCVPEYPTANP

[0200] RLHFHAVHFMRTLPTGSVDPNFGRRVRRNRRQLNSVQNTWPYPYSMPIAVRY

[0201] TQDAFSRSPWLWPVDAKGEPLCATSYMAVGFYVAKFVNKKSDMDLAAKGL

[0202] GAKEWNNSLCTKLSLLPKKLFRIRMSRNFGMKMLTMMTNLSTECLIQLTKLGY

[0203] DATPFNQILKQNAKREMRRLGKVTVADVLAAQPVTTNLLKFMRASIKMIG

[0204] VSNLQSFIASMTQKLTLSDISDESKNYLDKAGITTACLIKSKWTAGGKHHHH

[0205] HH

[0206] (11) SA-TC1 translates to the gene (SEQ ID NO:11)

[0207] ATGGGAGACCCGAGCAAAGATTCTAAAGCACAAGTATCTGCTGCAGAAG

[0208] CAGGAATTACAGGCACATGGTATAATCAGCTGGGATCTACATTTATTGTTA

[0209] CAGCCGGCGCAGATGGAGCTCTTACAGGAACATATGAATCTGCTGTTGGA

[0210] AATGCAGAATCTAGATACGTGCTTACAGGAAGATATGATTCTGCACCTGC

[0211] AACAGATGGATCCGGAACAGCACTTGGATGGACAGTTGCATGGAAAAAC

[0212] AATTATAGAAACGCACATAGCGCTACAACATGGTCTGGCCAATATGTGGG

[0213] AGGTGCAGAAGCAAGAATTAACACACAATGGCTTTTAACATCTGGAACAA

[0214] CAGAAGCAAATGCATGGAAAAGTACTCTTGTTGGACATGATACATTTACA

[0215] AAAGTTAAACCTAGCGCAGCATCTATCGATGCAGCGAAAAAAGCAGGAG

[0216] TTAACAATGGCAATCCTTTAGATGCAGTTCAACAAGGTGGAGGTGGATCG

[0217] CCTCGTTCAGGCCGCTTTAGCATTAAATGCAAGAACTACTTTCTGACCTAT

[0218] CCGAAATGCGATCTGACCAAAGAAAACGCGCTGAGCCAGATTACCAACCT

[0219] GCAGACCCCGACCAACAAACTGTTCATCAAAATTTGCCGCGAACTGCATG

[0220] AAAACGGCCGCCTGCATCTGCATATTCTGATTCAGTTCGAGGGCAAATAT

[0221] AACTGCACCAACCAGCCATTTTTTGATCTGGTGAGCCCTACTCCCAGCGCG

[0222] CATTTTCATCCGAACATTCAGGGCGCGAAAAGCAGCAGCGATGTGAAAAG

[0223] CTACATCGATAAGGATGGCGATGTGCTGGAATGGGGCACCTTTCAGATTG

[0224] ATGGCCGTHHHHHH

[0225] (12) SA-TC1 fusion protein PROTEIN amino acid sequence (SEQ ID NO: 12)

[0226] MGDPSKDSKAQVSAAEAGITGTWYNQLGSTFIVTAGADGALTGTYESAVGN

[0227] AESRYVLTGRYDSAPATDGSGTALGWTVAWKNNYRNAHSATTWSGQYVG

[0228] GAEARINTQWLLTSGTTEANAWKSTLVGHDTFTKVKPSAASIDAAKKAGVN

[0229] NGNPLDAVQQGGGGSPRSGRFSIKCKNYFLTYPKCDLTKENALSQITNLQTPT

[0230] NKLFIKICRELHENGRLHLHILIQFEGKYNCTNQPFFDLVSPTPSAHFHPNIQGA

[0231] KSSSDVKSYIDKDGDVLEWGTFQIDGRHHHHHH

[0232] (13) SA-HI0217 fusion protein gene nucleic acid sequence (SEQ ID NO: 13)

[0233] ATGGGAGACCCGAGCAAAGATTCTAAAGCACAAGTATCTGCTGCAGAAG

[0234] CAGGAATTACAGGCACATGGTATAATCAGCTGGGATCTACATTTATTGTTA

[0235] CAGCCGGCGCAGATGGAGCTCTTACAGGAACATATGAATCTGCTGTTGGA

[0236] AATGCAGAATCTAGATACGTGCTTACAGGAAGATATGATTCTGCACCTGC

[0237] AACAGATGGATCCGGAACAGCACTTGGATGGACAGTTGCATGGAAAAAC

[0238] AATTATAGAAACGCCACATAGCGCTACAACATGGTCTGGCCAAATATGTGGG

[0239] AGGTGCAGAAGCAAGAATTAACACACAAATGGCTTTTAACATCTGGAACAA

[0240] CAGAAGCAAATGCATGGAAAAGTACTCTTGTTGGACATGATACATTTACA

[0241] AAAGTTAAACCTAGCGCAGCATCTATCGATGCAGCGAAAAAAGCAGGAG

[0242] TTAACAATGGCAATCCTTTAGATGCAGTTCAACAAGGTGGAGGTGGATCG

[0243] TCTAATTATCGGCCGATTTTACTAAACCTGGATTATATTTTTTCACAATTG

[0244] TTTTACAAGATCTCTACAAAATCTTATCTAACTGACTATATCAATGAATTTA

[0245] GATCTTTATATAAACAAACTTGTGAACATTATCCATTCGAAACAGTAGCA

[0246] ATTTGTATTTGCCCGATCATATTCATTTACTGATGCAATTACCTGAAAAT

[0247] GATGATAATTACGCAATACGCATCGCATATTTAAAAACACAATTTACACG

[0248] ACAACTTCCAAAAGAATGCCGACAATTTAATAAAAATAGACAAAAATATC

[0249] GAGAATCAGGTATTTGGCAACGCCGATTTTGGGAGCATTTAATTCGTGAT

[0250] GATAAAGATTTAGCGAATCATTTAGATTATATTTATTACAATCCTGTGAAA

[0251] CACGGCTATGTTGAGGTAGTAAAAGATTGGCCGTATTCTTCCTTCCATCGT

[0252] GATGTGAAATGTGAGATTTATCCTGAAGATTGGGGAGGCAACCCAGATTT

[0253] GAAAATTAAAGGTGATATACACCACCACCACCACCACTAA <000052**6**>(14)SA-HI0217 fusion protein PROTEIN amino acid sequence (SEQ ID NO: 14)

[0255] MGDPSKDSKAQVSAAEAGITGTWYNQLGSTFIVTAGADGALTGTYESAVGN

[0256] AESRYVLTGRYDSAPATDGSGTALGWTVAWKNNYRNAHSATTWSGQYVG

[0257] GAEARINTQWLLTSGTTEANAWKSTLVGHDTFTKVKPSAASIDAAKKAGVN

[0258] NGNPLDAVQQGGGGSSNYRRDFTKPGLYFFTIVLQDPTKSYLTDYINEFRSLY

[0259] KQTCEHYPFETVAICILPDHIHLLMQLPENDDNYAIRIAYLKTQFTRQLPKECR

[0260] QFNKNRQKYRESGIWQRRFWEHLIRDDKDLANHLDYIYYNPVKHGYVEVVK

[0261] DWPYSSFHRDVKCEIYPEDWGGNPDLKIKGDIHHHHHH

[0262] Note: There seems to be a minor error in the original text where the tag

[0254] was likely misspelled as <000052**6**> in the provided text. This has been noted but the translation has been done as per the original text.The above-mentioned nucleic acid and protein sequences are the specific sequences used in the examples. Homologous sequences of these sequences can also achieve the technical solution of the present invention and achieve similar technical effects.

[0263] For nucleic acid sequences, a homologous sequence is a nucleic acid sequence that is approximately 75% or more, 76% or more, 77% or more, 78% or more, 79% or more, 80% or more, 81% or more, 82% or more, 83% or more, 84% or more, 85% or more, 86% or more, 87% or more, 88% or more, 89% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more identical to the original nucleic acid sequence, or its corresponding cDNA molecule.

[0264] For protein sequences, homologous sequences can also be protein sequences that are approximately 75% or more, 76% or more, 77% or more, 78% or more, 79% or more, 80% or more, 81% or more, 82% or more, 83% or more, 84% or more, 85% or more, 86% or more, 87% or more, 88% or more, 89% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more identical to the original protein sequence and have the same function.

[0265] The reagents, instruments, or biological materials used in this invention can all be obtained through commercial channels.

[0266] The present invention will be further illustrated below with reference to the embodiments:

[0267] Example 1: Verification of the solid-phase material binding ability of HUH family proteins

[0268] Proteins A* (a replication-related protein from phiX174 bacteriophage), TC1 (a replication-related protein from tomato yellow leaf curl virus), and HI0217 (a transposon-related protein from Haemophilus influenzae) of the HUH family were selected as examples. Polystyrene (PS), silica, and gold were used as examples of solid-phase materials. The testing procedure is as follows: Figure 2 .

[0269] (A) Protein expression:

[0270] The genes for A*, TC1, and HI0217 proteins were cloned into expression vectors (the proteins can be well expressed in various expression vectors and hosts; here, only expression in *E. coli* and nickel affinity chromatography purification are described). The constructed functional protein expression vectors were transformed into *E. coli* expression strains, and positive clones were selected. The selected positive clones were transferred to LB medium and cultured at 37°C with shaking until the logarithmic growth phase (OD value approximately 0.5). IPTG at a working final concentration of 1 mM was added to the culture, and protein expression was induced at 25°C with shaking for 8 hours. The target proteins were purified by Ni affinity chromatography, yielding the purified HUH proteins.

[0271] (B) Solid-state combination:

[0272] (1): Verify the binding ability of HUH protein to PS.

[0273] 96-well plates containing PS material were blocked with BSA (5% w / v TBS) at 37°C for 2 h. 1 μg of HUH enzyme was added and incubated at 37°C for 1 h. The plates were washed three times with TBST, and then 100 μL of a mixture of anti-His-mouse primary antibody and HRP-goat anti-mouse secondary antibody (1 / 3000 dilution) was added and incubated at 37°C for 1 h. After washing five times with PBST, 100 μL of TMB chromogenic buffer was added and incubated at 37°C for 3 min. The reaction was terminated by adding 100 μL of 2M H₂SO₄. The absorbance was read at 450 nm using a microplate reader. The negative control was the colorimetric value obtained by adding antibody only after blocking without HUH.

[0274] The results are as follows Figure 3 A. Compared with the negative control, all three protein experimental groups had higher absorbance values, indicating that the three proteins had a high binding affinity to the PS material.

[0275] (2): Verify the binding ability of HUH protein to silica.

[0276] 96-well quartz plates (mainly composed of silica) were blocked with BSA (5% w / v TBS) at 37°C for 2 h. 1 μg of HUH enzyme was added and incubated at 37°C for 1 h. The plates were washed three times with TBST, and then 100 μL of a mixture of anti-His mouse primary antibody and HRP-goat anti-mouse secondary antibody (1 / 3000 dilution) was added and incubated at 37°C for 1 h. After washing five times with PBST, 100 μL of TMB chromogenic buffer was added and incubated at 37°C for 3 min. The reaction was terminated by adding 100 μL of 2M H₂SO₄. The absorbance was read at 450 nm using a microplate reader. The negative control was the colorimetric value obtained by adding antibody only after blocking, without the addition of HUH.

[0277] The results are as follows Figure 3B. Compared with the negative control, all three protein experimental groups showed higher absorbance values, indicating a high binding affinity between the three proteins and the silica material. Among them, TC1 and A* showed stronger binding affinity than HI0217.

[0278] (3): Verify the binding ability of HUH protein to gold.

[0279] 0.1 mg / mL, 1 mL gold nanoparticles were collected by centrifugation at 14000 rpm for 5 min. The sample was washed three times with TBS, blocked with BSA (5% w / v TBS) at 37°C for 2 h, incubated with 1 μg HUH enzyme at 37°C for 1 h, washed three times with TBST, and incubated with 100 μL of a mixture of anti-His mouse primary antibody and HRP-goat anti-mouse secondary antibody (1 / 3000 dilution) at 37°C for 1 h. After washing five times with PBST, 100 μL of TMB chromogenic solution was added and incubated at 37°C for 3 min. The reaction was terminated by adding 100 μL of 2M H2SO4. After centrifugation, the supernatant was transferred to a 96-well microplate and the absorbance was read at 450 nm. The negative control was the colorimetric value obtained by adding antibody only after blocking without HUH.

[0280] The results are as follows Figure 3 C. Compared with the negative control, all three protein experimental groups showed higher absorbance values, indicating a high binding affinity between the three proteins and gold. Among them, HI0217 showed a stronger binding affinity than the other two proteins.

[0281] The above experiments demonstrate that the HUH protein has the ability to bind to a variety of solid-phase materials.

[0282] Example 2: HUH protein-mediated protein fixation

[0283] Utilizing the solid-phase material binding ability of HUH protein, the target protein can be surface-immobilized via protein fusion. In this embodiment, the target protein—streptavidin (SA)—was immobilized on PS using TC1, and the immobilization effect was verified by detecting the antigen CRP (C-reactive protein) protein using a sandwich ELISA method, as follows: Figure 4 A. The experimental steps are as follows:

[0284] A. Target protein fusion: SA and TC1 proteins were fused by molecular cloning. The fusion protein SA-TC1 was expressed by an E. coli expression strain and purified by Ni affinity chromatography.

[0285] B. Target protein fixation: 10 μM, 100 μL of SA-TC1 protein was incubated in a 96-well PS plate at 37°C for 1 h. The control was an equal amount of SA protein directly incubated on PS. After incubation, the plate was washed three times with TBST.

[0286] C. CRP Antigen Detection: Block with blocking buffer at 37°C for 2 hours, wash 3 times, add 2 μg / mL, 100 μL of biotinylated CRP capture antibody, and incubate at 37°C for 0.5 hours. After washing, add different concentrations of CRP antigen and incubate for 1 hour to allow binding. After washing, add 2 μg / mL, 100 μL of Alexa 488-labeled fluorescent CRP antibody, incubate at 37°C for 1 hour, and wash 5 times. Read the fluorescence values ​​(Ex = 488 nm, Em = 520 nm).

[0287] The results are as follows Figure 4 B. As can be seen, the TC1 method (i.e., the HUH method in the figure) shows higher signal values ​​at each concentration, indicating that the SA immobilized by this method can bind more biotinylated antibodies, resulting in higher SA activity. The results show that TC1-mediated protein immobilization can better maintain protein activity. Example 3: HUH protein-mediated nucleic acid immobilization

[0288] Utilizing the dual function of the HUH protein—its nucleic acid binding and solid-phase binding capabilities—nucleic acids are immobilized onto the surface of a solid material. In this embodiment, the TC1 protein is used to immobilize nucleic acids onto the surface of silica particles, and the nucleic acid-immobilized silica particles are further used to detect the SARS-CoV-2 N protein gene fragment. Figure 5 A). The steps are as follows:

[0289] A. Nucleic acid fixation: Centrifuge at 8000 rpm for 1.5 min to collect 2 mg of silica particles, wash three times with TBS. Add 1 mL of 5 μM TC1 protein and incubate for 1 h, wash three times with TBS. Add 1 mL of reaction solution (TBS 2 mM Mn). 2+ 5 μM of capture nucleic acid NC-TRE with recognition sequence in ) (Bolded areas indicate nucleic acid hybridization regions), incubated at room temperature for 2 hours. Washed 3 times with TBS. Control: Nucleic acid was captured directly by physical adsorption without TC1.

[0290] B. Nucleic acid detection: Particles immobilized with nucleic acid were blocked in 5% w / v BSA, 0.1 mg / mL salmon sperm DNA, and TBS, and incubated at 37°C for 2 h. After washing three times, different concentrations of the N gene fragment (NF, 5'-CACATTGGCACCCGCAATCCTGCTAACA) were added.

[0291] ATGCTGCAATCGTGCTACA-3') and 1 μM, 1 mL of signal nucleic acid NS-FL ( (Bolded area indicates nucleic acid hybridization region), incubate at 37°C for 1 hour, wash 5 times, take the particle precipitate, resuspend in 100 μL TBS and add to a 96-well plate to read the fluorescence value (Ex = 488 nm, Em = 520 nm).

[0292] The results are as follows Figure 5 As shown in Figure B, the TC1 method exhibits higher fluorescence values ​​and linearity at each concentration. In contrast, the control group shows extremely low signal values ​​and no linearity. This indicates that the nucleic acids immobilized by the TC1 method maintain good hybridization ability and activity.

[0293] Example 4: HUH protein-mediated protein fixation

[0294] Referring to the method in Example 2, protein A* was used to mediate the fixation of the target protein.

[0295] The results showed that the fluorescence value corresponding to 500 ng / mL CRP antigen was 867 ± 21.6. This indicates that the HUH method showed a high signal value at this concentration, suggesting that the SA immobilized by this method can bind more biotinylated antibodies, resulting in higher SA activity. HUH-mediated protein immobilization can better maintain protein activity.

[0296] Example 5: HUH protein-mediated protein fixation

[0297] Referring to the method in Example 2, HI0217 was used to mediate the fixation of the target protein.

[0298] The results showed that the fluorescence value corresponding to 500 ng / mL CRP antigen was 715 ± 16.5. This indicates that the HUH method showed a high signal value at this concentration, suggesting that the SA immobilized by this method can bind more biotinylated antibodies, resulting in higher SA activity. HUH-mediated protein immobilization can better maintain protein activity.

[0299] Example 6: HUH protein-mediated protein fixation

[0300] Referring to the method in Example 2, HI0217 was used to mediate the immobilization of the target protein, and gold nanoparticles were selected as the solid phase material.

[0301] A. Fusion of target protein: SA and HI0217 proteins were fused by molecular cloning. The fusion protein SA-HI0217 was expressed by an E. coli expression strain and purified by Ni affinity chromatography.

[0302] B: Protein fixation: Centrifuge at 12000 rpm for 5 min to collect 200 μg of gold nanoparticles, and wash three times with TBS. Add 5 μM, 1 mL of SA-HI0217 protein and incubate for 1 h, then wash three times with TBS. The control is an equal amount of SA protein incubated directly on gold nanoparticles.

[0303] C. CRP Antigen Detection: Block with blocking buffer at 37℃ for 2 hours, wash 3 times, add 2 μg / mL, 100 μL of biotinylated CRP capture antibody, and incubate at 37℃ for 0.5 hours. After washing, add different concentrations of CRP antigen and incubate for 1 hour to allow binding. After washing, add 2 μg / mL, 100 μL of mouse CRP antibody and goat anti-mouse HRP-IgG and co-incubate at 37℃ for 1 hour, wash 3 times. Take the particle precipitate and add 100 μL of TMB solution, incubate for 3 minutes, then add 100 μL of 2M concentrated sulfuric acid to terminate the reaction. Take the solution and add it to a 96-well plate to read the OD450.

[0304] The results showed that the OD450 of the experimental group was 1.794±0.027, while that of the control group was 0.15±0.014. This indicates that the HUH method resulted in a higher OD value, suggesting that the SA immobilized by this method can bind more biotinylated antibodies, resulting in higher SA activity. HUH-mediated protein immobilization can better maintain protein activity.

[0305] Example 7: HUH protein-mediated nucleic acid immobilization

[0306] Referring to the method in Example 3, protein A* was used to mediate the fixation of the target nucleic acid. The fixation sequence was a capture nucleic acid NC-ARE containing an A* enzyme recognition sequence.

[0307]

[0308] The results showed that the fluorescence value corresponding to the 640pM N gene fragment was 22405.33±2367.5. It can be seen that the method of this embodiment showed a high fluorescence value in the detection of this concentration, indicating that the nucleic acid fixed by the HUH method maintained good hybridization ability and activity.

[0309] Example 8: HUH protein-mediated nucleic acid immobilization

[0310] Referring to the method in Example 3, HI0217-mediated fixation of the target nucleic acid was used. The fixation sequence was a captured nucleic acid NC-HRE containing the HI0217 enzyme recognition sequence.

[0311] The results showed that the fluorescence value corresponding to the 640pM N gene fragment was 17101±1545.3, indicating that the method in this embodiment showed a high fluorescence value at this concentration, which shows that the nucleic acid fixed by the HUH method maintained good hybridization ability and activity.

[0312] Example 9: HUH protein-mediated nucleic acid immobilization

[0313] Referring to the method in Example 3, HI0217 was used to mediate the immobilization of the target nucleic acid, and gold nanoparticles were selected as the solid phase material.

[0314] A: Nucleic acid fixation: Centrifuge at 12000 rpm for 5 min to collect 200 μg of gold nanoparticles, wash 3 times with TBS. Add 1 mL of 5 μM HI0217 protein and incubate for 1 h, wash 3 times with TBS. Add 1 mL of reaction solution (TBS 2 mM Mn). 2+ 5 μM of the capture nucleic acid NC-HRE (5'-CCTACTTTTTTAGCACGATTGCAG CATTG-3') containing the HI0217 enzyme recognition sequence was incubated at room temperature for 2 h. The sample was washed three times with TBS. The control group directly captured the nucleic acid through physical adsorption without HI0217.

[0315] B: Nucleic acid detection: Particles immobilized with nucleic acid were blocked in 5% w / v BSA, 0.1 mg / mL salmon sperm DNA, and TBS at 37°C for 2 h. After washing three times, different concentrations of the N gene fragment (NF) and 1 μM and 1 mL of signal nucleic acid NS-B (5'-CAGGATTGCGGGTGCCAATGTGTTTTT-biotin-3') were added, and the mixture was incubated at 37°C for 1 h. After washing three times, 1 μg / mL SA-HRP was added and incubated at room temperature for 30 min. After washing three times, the particle precipitate was taken and 100 μL of TMB solution was added. After incubation for 3 min, 100 μL of 2M concentrated sulfuric acid was added to terminate the reaction. The solution was then transferred to a 96-well plate and the OD450 was read.

[0316] The results showed that the OD450 of the experimental group corresponding to the 640pM N gene fragment was 1.929±0.049, while the OD450 of the control group was 0.316±0.13. This indicates that the HUH method yielded a higher OD value, suggesting that the nucleic acids fixed by the HUH method maintained good hybridization ability and activity.

[0317] Table 1

[0318] Example HUH protein solid materials Fixed proteins / nucleic acids Example 4 Protein A* PS protein Example 5 HI0217 PS protein Example 6 HI0217 gold protein Example 7 Protein A* silicon dioxide Nucleic acid Example 8 HI0217 silicon dioxide Nucleic acid Example 9 HI0217 gold Nucleic acid

[0319] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A HUH-binding protein, characterized in that, The HUH solid-binding protein includes a HUH protein and a solid-phase material, and the binding force between the HUH protein and the solid-phase material is a bioaffinity force. The HUH protein is selected from A At least one of the following: protein, TC1 protein, and HI0217 protein; The A The amino acid sequence of the protein is shown in SEQ ID NO.2; the amino acid sequence of the TC1 protein is shown in SEQ ID NO.4; the amino acid sequence of the HI0217 protein is shown in SEQ ID NO.6; The solid material is selected from one of polymers, metals, and minerals; the polymer is polystyrene; the metal is gold; and the mineral is silicon dioxide.

2. A method for solid-binding HUH protein, characterized in that, HUH protein was incubated with solid material to obtain HUH solid-bound protein; The HUH protein is selected from A At least one of the following: protein, TC1 protein, and HI0217 protein; The A The amino acid sequence of the protein is shown in SEQ ID NO.2; the amino acid sequence of the TC1 protein is shown in SEQ ID NO.4; the amino acid sequence of the HI0217 protein is shown in SEQ ID NO.6; The solid material is selected from one of polymers, metals, and minerals; the polymer is polystyrene; the metal is gold; and the mineral is silicon dioxide.

3. The solid-bonding method according to claim 2, characterized in that, The incubation temperature is 30~40℃, and the time is 0.5~5h.

4. A method for solid-binding a protein, characterized in that, Includes the following steps: S11, streptavidin SA is first linked to HUH protein to obtain streptavidin SA-HUH protein complex; S12, incubate the streptavidin SA-HUH protein complex with the solid material; The HUH protein is selected from A At least one of the following: protein, TC1 protein, and HI0217 protein; wherein A The amino acid sequence of the protein is shown in SEQ ID NO.2; the amino acid sequence of the TC1 protein is shown in SEQ ID NO.4; the amino acid sequence of the HI0217 protein is shown in SEQ ID NO.6; The solid material is selected from one of polymers, metals, and minerals; the polymer is polystyrene; the metal is gold; and the mineral is silicon dioxide.

5. The solid-bonding method according to claim 4, characterized in that, The first connection is achieved through methods including protein fusion and / or condensation reactions.

6. A HUH-binding protein immobilized with a protein, characterized in that, It includes the HUH solid-binding protein prepared by the method of claim 3, and streptavidin SA linked to the HUH protein in the HUH solid-binding protein.

7. A method for detecting antigens not intended for diagnostic purposes, characterized in that, Includes the following steps: S31, streptavidin was fused with HUH protein for expression to obtain streptavidin-HUH protein complex; S32, the streptavidin-HUH protein complex and solid-phase material were incubated and then blocked; S33, add biotinylated capture antibody, incubate; S34, add the sample to be tested and incubate; S35, add antibody containing detection marker, and incubate; S36, Detect the detection mark; The HUH protein is selected from A At least one of the following: protein, TC1 protein, and HI0217 protein; wherein A The amino acid sequence of the protein is shown in SEQ ID NO.2; the amino acid sequence of the TC1 protein is shown in SEQ ID NO.4; the amino acid sequence of the HI0217 protein is shown in SEQ ID NO.6; The solid material is selected from one of polymers, metals, and minerals; the polymer is polystyrene; the metal is gold; and the mineral is silicon dioxide.

8. A method for solid-state binding of nucleic acids, characterized in that, Includes the following steps: S41, HUH protein and solid material are incubated to obtain HUH solid-bound protein; S42, incubate the target nucleic acid and HUH solid-binding protein; or, S51, HUH protein and target nucleic acid are incubated to obtain target nucleic acid-HUH protein complex; S52, the target nucleic acid-HUH protein complex and solid material are incubated; The HUH protein is selected from A At least one of the following: protein, TC1 protein, and HI0217 protein; wherein A The amino acid sequence of the protein is shown in SEQ ID NO.2; the amino acid sequence of the TC1 protein is shown in SEQ ID NO.4; the amino acid sequence of the HI0217 protein is shown in SEQ ID NO.6; The solid material is selected from one of polymers, metals, and minerals; the polymer is polystyrene; the metal is gold; and the mineral is silicon dioxide. The target nucleic acid is selected from one of NC-HRE, NC-ARE, and NC-TRE; the nucleotide sequence of NC-HRE is 5. ’ -CCTACTTTTTTAGCACGATTGCAGCATTG-3 ’ The nucleotide sequence of NC-ARE is 5. ’ -CAACTTGATTTTTTAGCACGATTGCAGCATTG-3 ’ The nucleotide sequence of NC-TRE is 5. ’ -TATAATATTACTTTTTTGTAGCACGATTGCAGCATTG-3 ’ .

9. A HUH-binding protein immobilized with DNA, characterized in that, This includes the HUH-binding protein prepared by the method of claim 3, and capture DNA that is affinity-bound or linked to the HUH protein in the HUH-binding protein; the capture DNA is selected from one of NC-HRE, NC-ARE, and NC-TRE; the nucleotide sequence of NC-HRE is 5... ’ -CCTACTTTTTTAGCACGATTGCAGCATTG-3 ’ The nucleotide sequence of NC-ARE is 5. ’ -CAACTTGATTTTTTAGCACGATTGCAGCATTG-3 ’ The nucleotide sequence of NC-TRE is 5. ’ -TATAATATTACTTTTTTGTAGCACGATTGCAGCATTG-3 ’ .

10. A method for detecting target DNA for non-diagnostic purposes, characterized in that, Includes the following steps: S61, HUH protein and solid material are incubated to obtain HUH solid-binding protein; S62, incubate and block the captured DNA and HUH solid-binding protein; S63, add the sample to be tested and signal DNA containing the detection marker, and incubate; S64, Detect the detection mark; The HUH protein is selected from A At least one of the following: protein, TC1 protein, and HI0217 protein; wherein A The amino acid sequence of the protein is shown in SEQ ID NO.2; the amino acid sequence of the TC1 protein is shown in SEQ ID NO.4; the amino acid sequence of the HI0217 protein is shown in SEQ ID NO.6; The solid material is selected from one of polymers, metals, and minerals; the polymer is polystyrene; the metal is gold; and the mineral is silicon dioxide. The captured DNA is selected from one of NC-HRE, NC-ARE, and NC-TRE; the nucleotide sequence of NC-HRE is 5. ’ -CCTACTTTTTTAGCACGATTGCAGCATTG-3 ’ The nucleotide sequence of NC-ARE is 5. ’ -CAACTTGATTTTTTAGCACGATTGCAGCATTG-3 ’ The nucleotide sequence of NC-TRE is 5. ’ -TATAATATTACTTTTTTGTAGCACGATTGCAGCATTG-3 ’ .

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