CN116375808A - HUH solid phase binding protein and solid phase binding method of protein or nucleic acid mediated by HUH solid phase binding protein - Google Patents

HUH solid phase binding protein and solid phase binding method of protein or nucleic acid mediated by HUH solid phase binding protein Download PDF

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CN116375808A
CN116375808A CN202211743551.8A CN202211743551A CN116375808A CN 116375808 A CN116375808 A CN 116375808A CN 202211743551 A CN202211743551 A CN 202211743551A CN 116375808 A CN116375808 A CN 116375808A
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protein
huh
solid phase
phase binding
nucleic acid
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门冬
周昆
张先恩
曹姗姗
陈晨
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Wuhan Institute of Virology of CAS
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Wuhan Institute of Virology of CAS
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Abstract

The application relates to the technical field of biomolecule immobilization, in particular to a HUH solid phase binding protein and a solid phase binding method of the protein or nucleic acid mediated by the HUH solid phase binding protein. The HUH solid-phase binding protein comprises HUH protein and a solid-phase material, wherein the binding force between the HUH protein and the solid-phase material is bioaffinity force; HUH protein is selected from at least one of A protein, TC1 protein and HI0217 protein. Compared with the method only capable of realizing protein fixation or nucleic acid fixation, the solid phase binding peptide can be used for fixing two molecules in one route, has simple and rapid reaction, mild condition, no need of any additional chemical modification, realizes protein fixation by one-step incubation at normal temperature, and realizes nucleic acid fixation by two-step incubation. Meanwhile, the immobilized targets such as proteins and nucleic acids can still maintain good activity on the surface of the carrier.

Description

HUH solid phase binding protein and solid phase binding method of protein or nucleic acid mediated by HUH solid phase binding protein
Technical Field
The application relates to the technical field of biomolecule immobilization, in particular to a HUH solid phase binding protein and a solid phase binding method of the protein or nucleic acid mediated by the HUH solid phase binding protein.
Background
The nucleic acid and protein have powerful functions as the material base of living body, and the functional material prepared by fixing nucleic acid and protein molecule onto solid phase material, such as gold, polystyrene (PS) and silica, has wide application in biological imaging, biomedicine, biosensing, etc. The activity of biological molecules on the surface of the material is directly related to the structure of the biological molecules, and immobilized nucleic acids and proteins can be inactivated due to unfavorable conformations so as to reduce the hybridization efficiency of the surface nucleic acids or the binding efficiency of the immobilized nucleic acids and proteins with target molecules; the change in the immobilized conformation of the enzyme or antibody/antigen molecule on the surface may result in loss of catalytic activity or antigen-antibody recognition capability. Therefore, the immobilization of nucleic acids and proteins on a carrier while maintaining their functions is a goal of the development of biomolecular immobilization technology. Efficient and simple methods of immobilization of biomolecules are currently in high demand.
Existing nucleic acid/protein immobilization methods can be broadly divided into physical adsorption, chemical cross-linking, and bioaffinity immobilization methods. Direct physical adsorption, simple steps, but great influence on the activity of the immobilized molecules; chemical crosslinking methods, which are complex in steps, often require the participation of toxic chemical reagents and special reaction conditions. The method for fixing the biomolecules by the binding protein/peptide with high affinity to the specific solid phase material can well retain the activity of the immobilized molecules, has simple fixing process, is easy to operate, does not need additional chemical modification, has stability in biological environment, and is a good substitute for the traditional fixing method.
HUH family proteins are proteins which can recognize and bind nucleic acids, are widely used in living bodies, play a key role in the processes of nucleic acid replication, transfer and the like, and are powerful protein-nucleic acid connection tools.
At present, no report on the binding capacity between HUH family proteins and solid phase materials is found; there is no report of the immobilization of nucleic acids/proteins on solid phase materials by using HUH family proteins as a bridge.
Disclosure of Invention
In view of this, the present invention provides a HUH solid phase binding protein and a method for the solid phase binding of a protein or nucleic acid mediated thereby. The invention discovers that HUH protein has the capability of combining with various solid phase materials and can be used for fixing nucleic acid/protein.
In order to achieve the above object, the present invention provides the following technical solutions:
the invention discovers a new function of HUH protein for the first time, namely the capability of combining with various solid-phase materials such as polymers, metals, minerals and the like.
In a first aspect, the present invention provides a HUH solid phase binding protein comprising a HUH protein and a solid phase material, the binding force between the HUH protein and the solid phase material being a bioaffinity force;
HUH protein is selected from at least one of A protein, TC1 protein and HI0217 protein.
The HUH solid phase binding proteins are useful for the immobilization of proteins and nucleic acids.
In a second aspect, the present invention provides a method for solid phase binding of HUH protein, the method comprising: incubating HUH protein and a solid phase material to obtain HUH solid phase binding protein;
HUH protein is selected from at least one of A protein, TC1 protein and HI0217 protein.
Preferably, the solid phase material is selected from at least one of polymers, metals and minerals;
preferably, the polymer is at least one selected from polystyrene, polyurethane, polystyrene divinylbenzene, polymethyl methacrylate, polyacrylamide, polyethylene glycol terephthalate, polyvinyl acetate, polyethylene, polypropylene, polyvinyl chloride, polyvinylpyrrolidone;
in a specific embodiment provided by the present invention, the polymer is polystyrene.
Preferably, the metal is selected from at least one of gold, silver, copper, aluminum, iron;
in a specific embodiment provided by the invention, the metal is gold; gold nanoparticles are preferred.
Preferably, the mineral is selected from at least one of silica, calcium oxide, titanium dioxide, and ferroferric oxide.
In a specific embodiment provided herein, the mineral is silica.
Preferably, the shape of the solid phase material comprises particles, rods, tubes, sheets or plates.
Preferably, the incubation is carried out at a temperature of 30 to 40℃for a period of 0.5 to 5 hours.
In the specific example provided by the invention, the incubation temperature is 37℃and the time is 1h.
The present invention finds that HUH proteins have the ability to bind to a variety of solid phase materials, such as polymers, metals, minerals, etc., making HUH proteins useful for protein and nucleic acid immobilization (fig. 1).
In a third aspect, the present invention provides a method for solid phase binding of a protein, comprising the steps of:
s11, carrying out first connection on target protein and HUH protein to obtain target protein-HUH protein complex;
s12, incubating the target protein-HUH protein complex and a solid phase material;
or alternatively, the process may be performed,
s21, incubating HUH protein and a solid phase material to obtain HUH solid phase binding protein;
s22, carrying out second connection on the target protein and HUH solid-phase binding protein;
preferably, the first ligation comprises a protein fusion and/or condensation reaction;
preferably, the second linkage is by a condensation reaction.
The two connection methods (a first connection method and a second connection method) are protein fixing methods of two different paths, and finally the HUH solid-phase binding protein with the immobilized protein is obtained and further used for detecting target molecules (such as antigen, antibody and the like).
In a fourth aspect, the present invention provides a HUH solid phase binding protein immobilized with a protein comprising the above-described HUH solid phase binding protein, and a protein of interest linked to a HUH protein in the HUH solid phase binding protein;
preferably, the target protein includes, but is not limited to, at least one of streptavidin, an enzyme, a fluorescent protein, and an affinity peptide.
In a specific embodiment provided by the invention, the protein of interest is streptavidin. Streptavidin can bind to biotinylated molecules by affinity, and can be further used for detection of target molecules (e.g., antigens, antibodies, etc.).
In a fifth aspect, the present invention provides a method for detecting an antigen, comprising the steps of:
s31, carrying out fusion expression on streptavidin and HUH protein to obtain a streptavidin-HUH protein complex;
s32, incubating the streptavidin-HUH protein complex and a solid phase material, and sealing;
s33, adding a biotinylation capture antibody, and incubating;
s34, adding a sample to be detected, and incubating;
s35, adding an antibody containing a detection label, and incubating;
s36, detecting the detection mark.
The principle of the antigen detection method is as follows: streptavidin-HUH protein complex (SA-HUH) is immobilized on the surface of solid phase material (SA-HUH-solid phase) by affinity, and biotinylated capture antibody is immobilized on SA-HUH-solid phase (capture antibody-SA-HUH-solid phase) by streptavidin-biotin affinity; if the sample to be tested contains an antigen, the antigen is combined with a capture antibody (antigen-capture antibody-SA-HUH-solid phase); finally, the labeled antibody is combined with the antigen (labeled antibody-antigen-capture antibody-SA-HUH-solid phase), and the existence and/or the content of the antigen can be determined by detecting the signal intensity of the label.
In a sixth aspect, the present invention provides a method for solid phase binding of nucleic acids, comprising the steps of:
s41, incubating HUH protein and a solid phase material to obtain HUH solid phase binding protein;
s42, incubating or third connecting the target nucleic acid and the HUH solid-phase binding protein.
Or alternatively, the process may be performed,
s51, incubating or third connecting HUH protein and target nucleic acid to obtain target nucleic acid-HUH protein complex;
s52, incubating the target nucleic acid-HUH protein complex and the solid phase material.
In step S42 or S51, binding of the HUH protein to the nucleic acid can be achieved by incubation because the HUH protein itself has a nucleic acid binding function.
Binding of the HUH protein to the nucleic acid may be achieved by other means besides incubation. Preferably, the method used for the third ligation comprises chemical modification and/or condensation reaction.
In a seventh aspect, the present invention provides a HUH solid phase binding protein immobilized with DNA, comprising the above HUH solid phase binding protein, and target DNA affinity-bound or linked to HUH protein in the HUH solid phase binding protein;
preferably, the DNA of interest includes, but is not limited to, capture DNA.
In an eighth aspect, the present invention provides a method for detecting a target DNA, comprising the steps of:
s61, incubating HUH protein and a solid phase material to obtain HUH solid phase binding protein;
s62, incubating the capture DNA and HUH solid-phase binding protein, and sealing;
s63, adding a sample to be detected and signal DNA containing a detection mark, and incubating;
s64, detecting the detection mark.
The principle of the target DNA detection method is as follows: the capture DNA binds to the HUH solid phase binding protein by affinity (capture DNA-HUH-solid phase); if the sample to be tested contains target DNA, the target DNA is combined with the capture DNA through complementation (target DNA-capture DNA-HUH-solid phase); finally, the signal DNA is combined with the target DNA (signal DNA-target DNA-capture DNA-HUH-solid phase), and the existence and/or the content of the target DNA can be determined by detecting the signal intensity of the mark.
In a specific embodiment provided by the invention, the signal DNA is a nucleic acid fragment that binds to the target DNA.
In a specific embodiment provided herein, the detection label comprises at least one of a radioisotope, a metal chelator, an enzyme, a fluorescent compound, a bioluminescent compound, or a chemiluminescent compound.
Compared with the prior art, the invention has the following beneficial effects:
1) Compared to methods that can only achieve protein immobilization or nucleic acid immobilization, immobilization of both molecules can be performed in one route using the solid-phase binding peptides of the invention.
2) The method has the advantages of simple and rapid reaction, mild conditions and no need of any additional chemical modification. The protein fixation is realized by one-step incubation at normal temperature. Two-step incubation achieved nucleic acid immobilization.
3) In the present invention, the immobilized target such as protein, nucleic acid, etc. can still maintain a good activity on the surface of the carrier.
Drawings
FIG. 1 shows HUH protein-mediated binding immobilization of nucleic acids or proteins to a solid phase material;
FIG. 2 shows a flow chart for the solid phase material binding capacity test of HUH protein;
FIG. 3 shows the solid phase material binding capacity of HUH protein; wherein A shows the binding capacity of 3HUH proteins to Polystyrene (PS); b shows the binding capacity of 3HUH proteins to silica; c shows the binding capacity of 3HUH proteins to gold;
FIG. 4 shows HUH protein mediated protein fixation; wherein A shows a TC1 protein-mediated antigen detection schematic; b shows that TC1 protein mediated protein immobilization can better maintain protein activity;
FIG. 5 shows HUH protein mediated nucleic acid immobilization; wherein A shows a TC1 protein mediated target nucleic acid detection schematic; b shows TC1 proteinThe mediated nucleic acid immobilization can better maintain the activity of the nucleic acid, the small graph in the B is a physical adsorption curve, namely the amplification of the black line part in the large graph, and the numerical value marked by the abscissa is 10 from left to right in sequence 5 、10 10 The values marked on the ordinate are 200, 400, 600, 800 and 1000 in sequence from bottom to top.
Detailed Description
The invention discloses a HUH solid phase binding protein and a solid phase binding method of the protein or nucleic acid mediated by the same, and the technical parameters can be properly improved by the person skilled in the art by referring to the content of the text. It is expressly noted that all such similar substitutions and modifications will be apparent to those skilled in the art, and are deemed to be included in the present invention. While the methods and applications of this invention have been described in terms of preferred embodiments, it will be apparent to those skilled in the relevant art that variations and modifications can be made in the methods and applications described herein, and in the practice and application of the techniques of this invention, without departing from the spirit or scope of the invention.
Term interpretation:
HUH family proteins: refers to HUH endonuclease (HUH enzyme for short, HUH protein) whose main function is to catalyze cleavage and religation of single-stranded DNA by using active site tyrosine residues, so that a transient 5' -phosphotyrosine bond is formed between the HUH endonuclease and a DNA substrate. The specific species of the HUH family protein is not limited, and different HUH family proteins can be freely selected according to different enzyme-specific recognition sequences and different target proteins or peptides. In a specific embodiment of the invention, the HUH family protein is selected from at least one of the group consisting of a, TC1, HI0217 endonucleases.
Endonuclease (a. Protease): from phage phi X174, plays an important role in viral DNA replication, and can cleave the replication initiation site and covalently link nucleic acids. The enzyme specific recognition nucleic acid sequence is 5'-n-CAACTTGA-n-3'. Wherein n represents one or more nucleotide bases; preferably, said n is 0-30 nucleotide bases. For example, the enzyme-specific recognition nucleic acid sequence isCAACTTGATACATACATCATCCCTCATTCA, the cross-hatched portion is the recognition site.
TC1 endonuclease: replication-associated proteins from tomato yellow leaf curl virus can cleave the replication initiation site to initiate rolling circle replication. The TC1 enzyme specific recognition nucleic acid sequence is 5'-n-TATTA-n-3'. Wherein n represents one or more nucleotide bases; preferably, said n is 0-30 nucleotide bases. For example, the TC1 enzyme-specific recognition nucleic acid sequence is TATAATATTACTTTTTTGTAGCACGATTGCAGCATTG. The cross-hatched portion is the recognition site.
HI0217 endonuclease: transposition-related proteins from haemophilus influenzae are responsible for cleavage into single-stranded loops from the donor DNA "top strand" which are then reinserted into the DNA target. The specific recognition nucleic acid sequence of HI0217 enzyme is 5'-n-CCTAC-n-3'. Wherein n represents one or more nucleotide bases; preferably, said n is 0-30 nucleotide bases. For example HI0217 specific recognition nucleic acid sequence GTAGGGTGGGCTTTAGCCCACCATCCTACATTAAGTACGCATAATT, the cross-hatched portion is the recognition site.
Bioaffinity effect: the specific action between biomolecules, a phenomenon in which biomolecules can recognize and attractively bind to a specific substance under certain conditions (a medium is often required).
Fusion protein: is an expression product obtained by recombination of two genes by a DNA recombination technology. The two different proteins are linked into one macromolecule, which can be chemically linked or can be achieved by fusion of genes.
Condensation reaction: two or more organic molecules are interacted and then are combined into a macromolecule through covalent bonds, and the reaction of losing small molecules (such as water, hydrogen chloride, alcohol and the like) is frequently carried out.
Streptavidin (SA): is a protein with similar biological characteristics to avidin, is a secretion of streptomyces avidinii bacteria, has similar molecular weight and biotin binding capacity to avidin in egg white, has isoelectric point of 6.0 and has non-specific binding far lower than that of avidin. The binding of biotin to streptavidin is one of the strongest non-covalent interactions known in nature and has a dissociation constant (Kd) of about 10 -14 mol/L. Because of streptavidin-biotin complexThe substance has good tolerance to organic chemical book solvents, denaturants (such as guanidine hydrochloride), detergents (such as SDS and triton), proteolytic enzymes and extreme temperatures and pH values, so that streptavidin is widely used in molecular biology and biological nanotechnology.
Capture antibody: refers to antibodies immobilized on a solid phase for immunological testing.
Capture test: refers to an immunoassay method in which a capture antibody immobilized on a solid phase is contacted with a sample. If the sample comprises an antigen that is capable of binding 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 a solid phase and subsequently detected or assayed.
Capturing DNA: refers to a complementary strand of a fragment of a particular gene.
The sequences of the proteins and nucleic acids used in the examples below are as follows:
(1) A gene nucleic acid sequence (SEQ ID NO: 1)
ATGAAATCGCGTAGACCATTTGCTATTCAGCGTTTGATGAATGCAATGCG
ACAGGCTCATGCTGATGGTTGGTTTATCGTTTTTGACACTCTCACGTTGGC
TGACGACCGATTAGAGGCGTTTTATGATAATCCCAATGCTTTGCGTGACTA
TTTTCGTGATATTGGTCGTATGGTTCTTGCTGCCGAGGGTCGCAAGGCTAA
TGATTCACACGCCGACTGCTATCAGTATTTTTGTGTGCCTGAGTATCCAAC
AGCTAATCCACGTCTTCATTTCCATGCGGTGCACTTTATGCGGACACTTCC
TACAGGTAGCGTTGACCCTAATTTTGGTCGTCGGGTACGCAATCGCCGCC
AGTTAAATAGCGTGCAAAATACGTGGCCTTATCCCTACAGTATGCCCATC
GCAGTTCGCTACACGCAGGACGCTTTTTCACGTTCTCCCTGGTTGTGGCCT
GTTGATGCTAAAGGTGAGCCGCTTAAAGCTACCAGTTATATGGCTGTTGGT
TTCTATGTGGCTAAATTCGTTAACAAAAAGTCAGATATGGACCTTGCTGCT
AAAGGTCTAGGAGCTAAAGAATGGAACAACTCACTAAAAACCAAGCTGT
CGCTACTTCCCAAGAAGCTGTTCAGAATCAGAATGAGCCGCAACTTCGGG
ATGAAAATGCTCACAATGACAAATCTGTCCACGGAGTGCTTAATCCAACT
TACCAAGCTGGGTTACGACGCGACGCCGTTCAACCAGATATTGAAGCAGA
ACGCAAAAAGAGAGATGAGATTGAGGCTGGGAAAAGTTACTGTAGCCGA
CGTTTTGGCGGCGCAACCTGTGACGACAAATCTGCTCAAATTTATGCGCG
CTTCGATAAAAATGATTGGCGTATCCAACCTGCAGAGTTTTATCGCTTCCA
TGACGCAGAAGTTAACACTTTCGGATATTTCTGATGAGTCGAAAAATTATC
TTGATAAAGCAGGAATTACTACTGCTTGTTTACGAATTAAATCGAAGTGG
ACTGCTGGCGGAAAACACCACCACCACCACCACTGA
(2) Protein amino acid sequence (SEQ ID NO: 2)
MKSRRPFAIQRLMNAMRQAHADGWFIVFDTLTLADDRLEAFYDNPNALRDY
FRDIGRMVLAAEGRKANDSHADCYQYFCVPEYPTANPRLHFHAVHFMRTLP
TGSVDPNFGRRVRNRRQLNSVQNTWPYPYSMPIAVRYTQDAFSRSPWLWPV
DAKGEPLKATSYMAVGFYVAKFVNKKSDMDLAAKGLGAKEWNNSLKTKLS
LLPKKLFRIRMSRNFGMKMLTMTNLSTECLIQLTKLGYDATPFNQILKQNAK
REMRLRLGKVTVADVLAAQPVTTNLLKFMRASIKMIGVSNLQSFIASMTQKL
TLSDISDESKNYLDKAGITTACLRIKSKWTAGGKHHHHHH
(3) TC1 gene nucleic acid sequence (SEQ ID NO: 3)
ATGCCTCGTTCAGGCCGCTTTAGCATTAAATGCAAGAACTACTTTCTGACC
TATCCGAAATGCGATCTGACCAAAGAAAACGCGCTGAGCCAGATTACCAA
CCTGCAGACCCCGACCAACAAACTGTTCATCAAAATTTGCCGCGAACTGC
ATGAAAACGGCCGCCTGCATCTGCATATTCTGATTCAGTTCGAGGGCAAA
TATAACTGCACCAACCAGCCATTTTTTGATCTGGTGAGCCCTACTCCCAGC
GCGCATTTTCATCCGAACATTCAGGGCGCGAAAAGCAGCAGCGATGTGAA
AAGCTACATCGATAAGGATGGCGATGTGCTGGAATGGGGCACCTTTCAGA
TTGATGGCCGTCACCACCACCACCACCAC
(4) TC1 PROTEIN amino acid sequence (SEQ ID NO: 4)
MPRSGRFSIKCKNYFLTYPKCDLTKENALSQITNLQTPTNKLFIKICRELHENG
RLHLHILIQFEGKYNCTNQPFFDLVSPTPSAHFHPNIQGAKSSSDVKSYIDKDG
DVLEWGTFQIDGRHHHHHH
(5) HI0217gene nucleic acid sequence (SEQ ID NO: 5)
ATGTCTAATTATCGGCGCGATTTTACTAAACCTGGATTATATTTTTTCACA
ATTGTTTTACAAGATCCTACAAAATCTTATCTAACTGACTATATCAATGAA
TTTAGATCTTTATATAAACAAACTTGTGAACATTATCCATTCGAAACAGTA
GCAATTTGTATTTTGCCCGATCATATTCATTTACTGATGCAATTACCTGAA
AATGATGATAATTACGCAATACGCATCGCATATTTAAAAACACAATTTAC
ACGACAACTTCCAAAAGAATGCCGACAATTTAATAAAAATAGACAAAAA
TATCGAGAATCAGGTATTTGGCAACGCCGATTTTGGGAGCATTTAATTCGT
GATGATAAAGATTTAGCGAATCATTTAGATTATATTTATTACAATCCTGTG
AAACACGGCTATGTTGAGGTAGTAAAAGATTGGCCGTATTCTTCCTTCCAT
CGTGATGTGAAATGTGAGATTTATCCTGAAGATTGGGGAGGCAACCCAGA
TTTGAAAATTAAAGGTGATATACACCACCACCACCACCACTAA
(6) HI0217 PROTEIN amino acid sequence (SEQ ID NO: 6)
MSNYRRDFTKPGLYFFTIVLQDPTKSYLTDYINEFRSLYKQTCEHYPFETVAIC
ILPDHIHLLMQLPENDDNYAIRIAYLKTQFTRQLPKECRQFNKNRQKYRESGI
WQRRFWEHLIRDDKDLANHLDYIYYNPVKHGYVEVVKDWPYSSFHRDVKC
EIYPEDWGGNPDLKIKGDIHHHHHH
(7) Streptavidin SA gene nucleic acid sequence (SEQ ID NO: 7)
GGAGACCCGAGCAAAGATTCTAAAGCACAAGTATCTGCTGCAGAAGCAG
GAATTACAGGCACATGGTATAATCAGCTGGGATCTACATTTATTGTTACAG
CCGGCGCAGATGGAGCTCTTACAGGAACATATGAATCTGCTGTTGGAAAT
GCAGAATCTAGATACGTGCTTACAGGAAGATATGATTCTGCACCTGCAAC
AGATGGATCCGGAACAGCACTTGGATGGACAGTTGCATGGAAAAACAATT
ATAGAAACGCACATAGCGCTACAACATGGTCTGGCCAATATGTGGGAGGT
GCAGAAGCAAGAATTAACACACAATGGCTTTTAACATCTGGAACAACAGA
AGCAAATGCATGGAAAAGTACTCTTGTTGGACATGATACATTTACAAAAG
TTAAACCTAGCGCAGCATCTATCGATGCAGCGAAAAAAGCAGGAGTTAAC
AATGGCAATCCTTTAGATGCAGTTCAACAACACCACCACCACCACCAC
( 8) Streptavidin SA PROTEIN amino acid sequence (SEQ ID NO:8 )
GDPSKDSKAQVSAAEAGITGTWYNQLGSTFIVTAGADGALTGTYESAVGNA
ESRYVLTGRYDSAPATDGSGTALGWTVAWKNNYRNAHSATTWSGQYVGG
AEARINTQWLLTSGTTEANAWKSTLVGHDTFTKVKPSAASIDAAKKAGVNN
GNPLDAVQQHHHHHH
(9) SA-A fusion protein gene nucleic acid sequence (SEQ ID NO: 9)
ATGGGAGACCCGAGCAAAGATTCTAAAGCACAAGTATCTGCTGCAGAAG
CAGGAATTACAGGCACATGGTATAATCAGCTGGGATCTACATTTATTGTTA
CAGCCGGCGCAGATGGAGCTCTTACAGGAACATATGAATCTGCTGTTGGA
AATGCAGAATCTAGATACGTGCTTACAGGAAGATATGATTCTGCACCTGC
AACAGATGGATCCGGAACAGCACTTGGATGGACAGTTGCATGGAAAAAC
AATTATAGAAACGCACATAGCGCTACAACATGGTCTGGCCAATATGTGGG
AGGTGCAGAAGCAAGAATTAACACACAATGGCTTTTAACATCTGGAACAA
CAGAAGCAAATGCATGGAAAAGTACTCTTGTTGGACATGATACATTTACA
AAAGTTAAACCTAGCGCAGCATCTATCGATGCAGCGAAAAAAGCAGGAG
TTAACAATGGCAATCCTTTAGATGCAGTTCAACAAGGTGGAGGTGGATCG
AAATCGCGTAGACCATTTGCTATTCAGCGTTTGATGAATGCAATGCGACA
GGCTCATGCTGATGGTTGGTTTATCGTTTTTGACACTCTCACGTTGGCTGA
CGACCGATTAGAGGCGTTTTATGATAATCCCAATGCTTTGCGTGACTATTT
TCGTGATATTGGTCGTATGGTTCTTGCTGCCGAGGGTCGCAAGGCTAATGA
TTCACACGCCGACTGCTATCAGTATTTTTGTGTGCCTGAGTATCCAACAGC
TAATCCACGTCTTCATTTCCATGCGGTGCACTTTATGCGGACACTTCCTAC
AGGTAGCGTTGACCCTAATTTTGGTCGTCGGGTACGCAATCGCCGCCAGTT
AAATAGCGTGCAAAATACGTGGCCTTATCCCTACAGTATGCCCATCGCAG
TTCGCTACACGCAGGACGCTTTTTCACGTTCTCCCTGGTTGTGGCCTGTTG
ATGCTAAAGGTGAGCCGCTTAAAGCTACCAGTTATATGGCTGTTGGTTTCT
ATGTGGCTAAATTCGTTAACAAAAAGTCAGATATGGACCTTGCTGCTAAA
GGTCTAGGAGCTAAAGAATGGAACAACTCACTAAAAACCAAGCTGTCGCT
ACTTCCCAAGAAGCTGTTCAGAATCAGAATGAGCCGCAACTTCGGGATGA
AAATGCTCACAATGACAAATCTGTCCACGGAGTGCTTAATCCAACTTACC
AAGCTGGGTTACGACGCGACGCCGTTCAACCAGATATTGAAGCAGAACGC
AAAAAGAGAGATGAGATTGAGGCTGGGAAAAGTTACTGTAGCCGACGTTT
TGGCGGCGCAACCTGTGACGACAAATCTGCTCAAATTTATGCGCGCTTCG
ATAAAAATGATTGGCGTATCCAACCTGCAGAGTTTTATCGCTTCCATGACG
CAGAAGTTAACACTTTCGGATATTTCTGATGAGTCGAAAAATTATCTTGAT
AAAGCAGGAATTACTACTGCTTGTTTACGAATTAAATCGAAGTGGACTGC
TGGCGGAAAACACCACCACCACCACCACTGA
( 10 SA-A fusion PROTEIN amino acid sequence (SEQ ID NO:10 )
MGDPSKDSKAQVSAAEAGITGTWYNQLGSTFIVTAGADGALTGTYESAVGN
AESRYVLTGRYDSAPATDGSGTALGWTVAWKNNYRNAHSATTWSGQYVG
GAEARINTQWLLTSGTTEANAWKSTLVGHDTFTKVKPSAASIDAAKKAGVN
NGNPLDAVQQGGGGSKSRRPFAIQRLMNAMRQAHADGWFIVFDTLTLADDR
LEAFYDNPNALRDYFRDIGRMVLAAEGRKANDSHADCYQYFCVPEYPTANP
RLHFHAVHFMRTLPTGSVDPNFGRRVRNRRQLNSVQNTWPYPYSMPIAVRY
TQDAFSRSPWLWPVDAKGEPLKATSYMAVGFYVAKFVNKKSDMDLAAKGL
GAKEWNNSLKTKLSLLPKKLFRIRMSRNFGMKMLTMTNLSTECLIQLTKLGY
DATPFNQILKQNAKREMRLRLGKVTVADVLAAQPVTTNLLKFMRASIKMIG
VSNLQSFIASMTQKLTLSDISDESKNYLDKAGITTACLRIKSKWTAGGKHHHH
HH
( 11 SA-TC1 fusion protein gene nucleic acid sequence (SEQ ID NO:11 )
ATGGGAGACCCGAGCAAAGATTCTAAAGCACAAGTATCTGCTGCAGAAG
CAGGAATTACAGGCACATGGTATAATCAGCTGGGATCTACATTTATTGTTA
CAGCCGGCGCAGATGGAGCTCTTACAGGAACATATGAATCTGCTGTTGGA
AATGCAGAATCTAGATACGTGCTTACAGGAAGATATGATTCTGCACCTGC
AACAGATGGATCCGGAACAGCACTTGGATGGACAGTTGCATGGAAAAAC
AATTATAGAAACGCACATAGCGCTACAACATGGTCTGGCCAATATGTGGG
AGGTGCAGAAGCAAGAATTAACACACAATGGCTTTTAACATCTGGAACAA
CAGAAGCAAATGCATGGAAAAGTACTCTTGTTGGACATGATACATTTACA
AAAGTTAAACCTAGCGCAGCATCTATCGATGCAGCGAAAAAAGCAGGAG
TTAACAATGGCAATCCTTTAGATGCAGTTCAACAAGGTGGAGGTGGATCG
CCTCGTTCAGGCCGCTTTAGCATTAAATGCAAGAACTACTTTCTGACCTAT
CCGAAATGCGATCTGACCAAAGAAAACGCGCTGAGCCAGATTACCAACCT
GCAGACCCCGACCAACAAACTGTTCATCAAAATTTGCCGCGAACTGCATG
AAAACGGCCGCCTGCATCTGCATATTCTGATTCAGTTCGAGGGCAAATAT
AACTGCACCAACCAGCCATTTTTTGATCTGGTGAGCCCTACTCCCAGCGCG
CATTTTCATCCGAACATTCAGGGCGCGAAAAGCAGCAGCGATGTGAAAAG
CTACATCGATAAGGATGGCGATGTGCTGGAATGGGGCACCTTTCAGATTG
ATGGCCGTHHHHHH
( 12 SA-TC1 fusion PROTEIN amino acid sequence (SEQ ID NO:12 )
MGDPSKDSKAQVSAAEAGITGTWYNQLGSTFIVTAGADGALTGTYESAVGN
AESRYVLTGRYDSAPATDGSGTALGWTVAWKNNYRNAHSATTWSGQYVG
GAEARINTQWLLTSGTTEANAWKSTLVGHDTFTKVKPSAASIDAAKKAGVN
NGNPLDAVQQGGGGSPRSGRFSIKCKNYFLTYPKCDLTKENALSQITNLQTPT
NKLFIKICRELHENGRLHLHILIQFEGKYNCTNQPFFDLVSPTPSAHFHPNIQGA
KSSSDVKSYIDKDGDVLEWGTFQIDGRHHHHHH
( 13 SA-HI0217 fusion protein gene nucleic acid sequence (SEQ ID NO:13 )
ATGGGAGACCCGAGCAAAGATTCTAAAGCACAAGTATCTGCTGCAGAAG
CAGGAATTACAGGCACATGGTATAATCAGCTGGGATCTACATTTATTGTTA
CAGCCGGCGCAGATGGAGCTCTTACAGGAACATATGAATCTGCTGTTGGA
AATGCAGAATCTAGATACGTGCTTACAGGAAGATATGATTCTGCACCTGC
AACAGATGGATCCGGAACAGCACTTGGATGGACAGTTGCATGGAAAAAC
AATTATAGAAACGCACATAGCGCTACAACATGGTCTGGCCAATATGTGGG
AGGTGCAGAAGCAAGAATTAACACACAATGGCTTTTAACATCTGGAACAA
CAGAAGCAAATGCATGGAAAAGTACTCTTGTTGGACATGATACATTTACA
AAAGTTAAACCTAGCGCAGCATCTATCGATGCAGCGAAAAAAGCAGGAG
TTAACAATGGCAATCCTTTAGATGCAGTTCAACAAGGTGGAGGTGGATCG
TCTAATTATCGGCGCGATTTTACTAAACCTGGATTATATTTTTTCACAATTG
TTTTACAAGATCCTACAAAATCTTATCTAACTGACTATATCAATGAATTTA
GATCTTTATATAAACAAACTTGTGAACATTATCCATTCGAAACAGTAGCA
ATTTGTATTTTGCCCGATCATATTCATTTACTGATGCAATTACCTGAAAAT
GATGATAATTACGCAATACGCATCGCATATTTAAAAACACAATTTACACG
ACAACTTCCAAAAGAATGCCGACAATTTAATAAAAATAGACAAAAATATC
GAGAATCAGGTATTTGGCAACGCCGATTTTGGGAGCATTTAATTCGTGAT
GATAAAGATTTAGCGAATCATTTAGATTATATTTATTACAATCCTGTGAAA
CACGGCTATGTTGAGGTAGTAAAAGATTGGCCGTATTCTTCCTTCCATCGT
GATGTGAAATGTGAGATTTATCCTGAAGATTGGGGAGGCAACCCAGATTT
GAAAATTAAAGGTGATATACACCACCACCACCACCACTAA
( 14 SA-HI0217 fusion PROTEIN amino acid sequence (SEQ ID NO:14 )
MGDPSKDSKAQVSAAEAGITGTWYNQLGSTFIVTAGADGALTGTYESAVGN
AESRYVLTGRYDSAPATDGSGTALGWTVAWKNNYRNAHSATTWSGQYVG
GAEARINTQWLLTSGTTEANAWKSTLVGHDTFTKVKPSAASIDAAKKAGVN
NGNPLDAVQQGGGGSSNYRRDFTKPGLYFFTIVLQDPTKSYLTDYINEFRSLY
KQTCEHYPFETVAICILPDHIHLLMQLPENDDNYAIRIAYLKTQFTRQLPKECR
QFNKNRQKYRESGIWQRRFWEHLIRDDKDLANHLDYIYYNPVKHGYVEVVK
DWPYSSFHRDVKCEIYPEDWGGNPDLKIKGDIHHHHHH
The nucleic acid and protein sequences are specific sequences used in the examples, and homologous sequences of the sequences can also realize the technical scheme of the invention and can also realize similar technical effects.
For nucleic acid sequences, a homologous sequence is a nucleic acid sequence that is about 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, 99% or more identical to the original nucleic acid sequence, or a corresponding cDNA molecule thereof.
For protein sequences, homologous sequences may also be protein sequences about 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, 99% or more identical to the original protein sequence, and have the same function.
Reagents, instruments, biological materials, and the like, useful in the present invention are commercially available.
The invention is further illustrated by the following examples:
EXAMPLE 1 verification of the binding Capacity of the solid phase Material of HUH family proteins
Protein a of the HUH family (replication-associated protein from phiX174 phage), TC1 (replication-associated protein from tomato yellow leaf curl virus), and HI0217 (transposition-associated protein from haemophilus influenzae) were chosen as examples. The solid phase material is selected from Polystyrene (PS), silicon dioxide and gold as examples. The test flow is as in fig. 2.
(A) Protein expression:
genes for the a, TC1, HI0217 proteins were cloned into expression vectors (proteins can be expressed well in a variety of expression vectors and expression hosts, only expression in e.coli, nickel affinity chromatography purification are described here). And (3) transforming the constructed functional protein expression vector into an escherichia coli expression strain, and picking positive clones. The selected positive clones were transferred to LB medium, cultured at 37℃with shaking to logarithmic growth phase (OD: about 0.5). IPTG was added to the culture at a final working concentration of 1mM, and protein expression was induced by shaking culture at 25℃for 8 hours. And (3) purifying target proteins by Ni affinity chromatography to obtain purified HUH proteins.
(B) Solid phase binding:
(1): the binding capacity of HUH protein to PS was verified.
96-well plates of PS material were blocked with BSA (5% W/V TBS) at 37℃for 2h, incubated with HUH enzyme 1. Mu.g at 37℃for 1h, washed 3 times with TBST, and incubated with 100. Mu.L of a mixture of His-murine primary antibody and HRP-goat anti-murine secondary antibody (1/3000 dilution) at 37℃for 1h. After PBST was washed 5 times, 100. Mu.L of TMB developing solution was added thereto and developed at 37℃for 3 minutes, and 2M H was added thereto 2 SO 4 100. Mu.L of the reaction was terminated. The absorbance at 450nm was read by a microplate reader. Negative control (Negative) was no HUH addition, color development of antibody added after blocking only.
As a result, as shown in fig. 3A, all 3 protein experimental groups had a larger absorbance value compared to the negative control, demonstrating the high binding force of the three proteins to PS material.
(2) The binding capacity of HUH protein to silica was verified.
Quartz 96-well plates (silica as the main component) were blocked with BSA (5% W/V TBS) at 37℃for 2 hours, HUH enzyme 1. Mu.g was added and incubated at 37℃for 1 hour, and TBST was used for 3 times, 100. Mu.L of a mixture of primary anti-His murine antibody and secondary HRP-goat anti-murine antibody (1/3000 dilution) was added and incubated at 37℃for 1 hour. After PBST was washed 5 times, 100. Mu.L of TMB developing solution was added thereto and developed at 37℃for 3 minutes, and 2M H was added thereto 2 SO 4 100. Mu.L of the reaction was terminated. The absorbance at 450nm was read by a microplate reader. Negative control (Negative) was no HUH addition, color development of antibody added after blocking only.
As a result, as shown in fig. 3B, all 3 protein experimental groups had a larger absorbance value compared to the negative control, demonstrating the high binding force of the three proteins to the silica material. TC1 and a bind more strongly than HI0217.
(3) The binding capacity of HUH protein to gold was verified.
Gold nanoparticles (14000 rpm,5 min) were collected by centrifugation at 0.1mg/mL and 1 mL. TBS was washed 3 times, blocked with BSA (5% W/V TBS) at 37℃for 2 hours, HUH enzyme 1. Mu.g was added and incubated at 37℃for 1 hour, TBST was washed 3 times, and 100. Mu.L of a primary anti-His murine antibody and an HRP-goat anti-murine secondary antibody mixture (1/3000 dilution) were added and incubated at 37℃for 1 hour. PBST is washed 5 timesAdding 100 μL TMB color developing solution, developing at 37deg.C for 3min, adding 2M H 2 SO 4 100. Mu.L of the reaction was stopped, and after centrifugation, the supernatant was transferred to a 96-well ELISA plate to read the absorbance at 450 nm. Negative control (Negative) was no HUH addition, color development of antibody added after blocking only.
The results are shown in FIG. 3C, where all 3 protein experimental groups have larger absorbance values compared to the negative control, demonstrating the high binding force of the three proteins to gold. Wherein HI0217 binds more strongly than the other two proteins.
The above experiments demonstrate the ability of the HUH protein to bind to a variety of solid phase materials.
EXAMPLE 2HUH protein-mediated protein immobilization
The solid phase material binding capacity of HUH protein can be utilized to fix the target protein on the surface in a protein fusion mode. In this example, the target protein, streptavidin (SA), was immobilized on PS by TC1, and the immobilization was verified by detecting the antigen CRP (C-reactive protein) protein by sandwich ELISA, as shown in FIG. 4A. The experimental steps are as follows:
A. fusion of target protein: SA and TC1 proteins are fused through molecular cloning, the fusion protein SA-TC1 is expressed through an escherichia coli expression strain, and the fusion protein is purified through Ni affinity chromatography.
B. Fixation of target protein: 10. Mu.M, 100. Mu.L of SA-TC1 protein was incubated in 96-well PS plates at 37℃for 1h, with control being an equivalent amount of SA protein incubated directly on PS. TBST was washed 3 times after incubation was completed.
C. CRP antigen detection: blocking solution was added at 37℃for 2h, washed 3 times, 2. Mu.g/mL of biotinylated CRP capture antibody was added and incubated at 37℃for 0.5h. After washing, CRP antigen with different concentrations is added for incubation for 1h for binding. After washing, 2. Mu.g/mL of Alexa 488-labeled fluorescent CRP antibody was added and incubated at 37℃for 1h, followed by washing 5 times. Fluorescence values were read (ex=488nm, em=520 nm).
As a result, as shown in FIG. 4B, the TC1 method (i.e., HUH method in the figure) showed higher signal values at each concentration, indicating that the immobilized SA was able to bind more biotinylated antibodies and that the activity of SA was higher. The results indicate that TC 1-mediated protein immobilization can better maintain protein activity. EXAMPLE 3HUH protein-mediated nucleic acid immobilization
The dual functions of the HUH protein, i.e., the nucleic acid binding capacity as well as the solid phase binding capacity, are utilized to immobilize nucleic acids onto the surface of a solid phase material. In this example, nucleic acid was immobilized on the surface of silica particles using TC1 protein, and detection of a novel coronan protein gene fragment was further performed using the nucleic acid-immobilized silica particles (FIG. 5A). The method comprises the following steps:
A. nucleic acid immobilization: at 8000rpm, 2mg of silica particles were collected by centrifugation at 1.5min and TBS was washed 3 times. 5. Mu.M, 1mL of TC1 protein was added and incubated for 1h, and TBS was washed 3 times. 1mL of the reaction mixture (TBS 2mM Mn) 2+ ) 5 mu M of capture nucleic acid NC-TRE with recognition sequence
Figure BDA0004031517620000151
Figure BDA0004031517620000152
Coarsening into nucleic acid hybridization region), and incubating for 2h at room temperature. TBS was washed 3 times. The control directly physisorbed captured nucleic acid without TC1.
B. Nucleic acid detection: the nucleic acid-immobilized particles were blocked in 5% W/V BSA, 0.1mg/mL salmon sperm DNA, TBS at 37℃for 2 hours. After 3 times of washing, N gene fragments (NF, 5' -CACATTGGCACCCGCAATCCTGCTAACA) with different concentrations were added respectively
ATGCTGCAATCGTGCTACA-3') and 1. Mu.M, 1mL of signal nucleic acid NS-FL
Figure BDA0004031517620000153
Coarsening as nucleic acid hybridization region), incubation at 37 ℃ for 1h, washing 5 times, taking pellet, re-suspending with 100 μl TBS and adding 96-well plate to read fluorescence value (ex=48nm, em=520 nm).
As a result, as shown in fig. 5B, it can be seen that the TC1 method shows a higher fluorescence value in detection of each concentration, and has linearity. The signal value of the control group is very low and the control group is wireless. The TC1 immobilized nucleic acid maintains good hybridization ability and activity.
EXAMPLE 4HUH protein-mediated protein immobilization
Referring to the method of example 2, protein a is used to mediate protein fixation of interest.
The results showed that 500ng/mL CRP antigen corresponds to a fluorescence value of 867.+ -. 21.6. The higher signal value at this concentration indicated by the HUH method, which indicated that the immobilized SA was able to bind more biotinylated antibody and that the activity of SA was higher. HUH mediated protein fixation can better maintain protein activity.
EXAMPLE 5HUH protein-mediated protein immobilization
Referring to the method of example 2, HI0217 is used to mediate immobilization of the target protein.
The results showed that 500ng/mL CRP antigen corresponds to a fluorescence value of 715.+ -. 16.5. The higher signal value at this concentration indicated by the HUH method, which indicated that the immobilized SA was able to bind more biotinylated antibody and that the activity of SA was higher. HUH mediated protein fixation can better maintain protein activity.
EXAMPLE 6HUH protein-mediated protein immobilization
Referring to the method of example 2, HI0217 is used for mediating target protein fixation, and gold nanoparticles are used as the solid phase material.
A. Fusion of target protein: SA and HI0217 proteins are fused through molecular cloning, the fusion protein SA-HI0217 is expressed through an escherichia coli expression strain, and the fusion protein is purified through Ni affinity chromatography.
B: protein immobilization: 200 μg gold nanoparticles were collected by 5min centrifugation at 12000rpm and TBS washed 3 times. 5. Mu.M, 1mL of SA-HI0217 protein was added and incubated for 1h, and TBS was washed 3 times. The control is incubation of an equivalent amount of SA protein directly on the gold nanoparticles.
C. CRP antigen detection: blocking solution was added at 37℃for 2h, washed 3 times, 2. Mu.g/mL of biotinylated CRP capture antibody was added and incubated at 37℃for 0.5h. After washing, CRP antigen with different concentrations is added for incubation for 1h for binding. After washing, 2. Mu.g/mL of a murine CRP antibody (100. Mu.L) was added and incubated with goat anti-mouse HRP-IgG at 37℃for 1h, followed by washing 3 times. The pellet was taken and 100. Mu.L of TMB solution was added, after 3min incubation, 100. Mu.L of 2M concentrated sulfuric acid was added to stop the reaction, the solution was removed and the OD450 was read in 96 well plates.
The results showed that the experimental OD450 was 1.794 ±0.027, while the control OD450 was 0.15±0.014. The HUH method has higher OD value, which indicates that the SA immobilized by the method can bind more biotinylated antibodies, and the activity of SA is higher. HUH mediated protein fixation can better maintain protein activity.
EXAMPLE 7HUH protein-mediated nucleic acid immobilization
Referring to the method of example 3, protein a is used to mediate nucleic acid immobilization of interest. Wherein the immobilized sequence is a capture nucleic acid NC-ARE with an a-enzyme recognition sequence:
Figure BDA0004031517620000171
the result shows that the fluorescence value corresponding to the 640pM N gene fragment is 22405.33 +/-2367.5, and the method of the embodiment shows higher fluorescence value in the detection of the concentration, which proves that the nucleic acid immobilized by the HUH method maintains good hybridization capability and maintains activity.
EXAMPLE 8HUH protein-mediated nucleic acid immobilization
Referring to the method of example 3, HI0217 is used to mediate immobilization of the nucleic acid of interest. Wherein the immobilized sequence is a capture nucleic acid NC-HRE with HI0217 enzyme recognition sequence:
Figure BDA0004031517620000172
/>
Figure BDA0004031517620000173
the result shows that the fluorescence value corresponding to the 640pM N gene fragment is 17101+/-1545.3, and the method of the embodiment shows higher fluorescence value in the detection of the concentration, which proves that the nucleic acid immobilized by the HUH method maintains good hybridization capability and maintains activity.
EXAMPLE 9HUH protein-mediated nucleic acid immobilization
According to the method of example 3, HI0217 is used for mediating target nucleic acid immobilization, and gold nanoparticles are used as the solid phase material.
A: nucleic acid immobilization: 200ug of gold nanoparticles were collected by 5min centrifugation at 12000rpm and TBS washed 3 times. Adding 5 muM, 1mL HI0217 protein incubated for 1h, TBS washed 3 times. 1mL of the reaction mixture (TBS 2mM Mn) 2+ ) In (2) a 5. Mu.M capture nucleic acid NC-HRE with HI0217 enzyme recognition sequence: 5'-CCTACTTTTTTAGCACGATTGCAG CATTG-3'. Incubate at room temperature for 2h. TBS was washed 3 times. Control direct physisorption captured nucleic acid without HI0217.
B: the nucleic acid detecting portion is: the nucleic acid-immobilized particles were blocked in 5% W/V BSA, 0.1mg/mL salmon sperm DNA, TBS at 37℃for 2 hours. After 3 times of washing, N gene fragments (NF) with different concentrations and 1 mu M and 1mL of signal nucleic acid NS-B (5 '-CAGGATTGCGGGTGCCAATGTGTTTTT-biotin-3') are respectively added, the mixture is incubated for 1h at 37 ℃,1 mu g/mL SA-HRP is added after 3 times of washing, the mixture is incubated for 30min at room temperature, 100 mu L of TMB solution is added after 3 times of washing, 100 mu L of 2M concentrated sulfuric acid is added after 3min of incubation, and the reaction is stopped by taking out the solution and adding the solution into a 96-well plate to read OD450.
The results showed that the 640pM N gene fragment corresponds to an experimental OD450 of 1.929.+ -. 0.049, while the control OD450 was 0.316.+ -. 0.13. The high OD value of the HUH method shows that the nucleic acid immobilized by the HUH method maintains good hybridization capability and maintains activity.
TABLE 1
Examples HUH protein Solid phase material Immobilization of proteins/nucleic acids
Example 4 Protein A PS Proteins
Example 5 HI0217 PS Proteins
Example 6 HI0217 Gold alloy Proteins
Example 7 Protein A Silica dioxide Nucleic acid
Example 8 HI0217 Silica dioxide Nucleic acid
Example 9 HI0217 Gold alloy Nucleic acid
The foregoing is merely a preferred embodiment of the present invention and it should be noted that modifications and adaptations to those skilled in the art may be made without departing from the principles of the present invention, which are intended to be comprehended within the scope of the present invention.

Claims (12)

1. A HUH solid phase binding protein, wherein the HUH solid phase binding protein comprises 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 at least one of A-protein, TC1 protein and HI0217 protein.
2. The HUH solid phase binding protein of claim 1, wherein the solid phase material is selected from at least one of a polymer, a metal, a mineral;
preferably, the polymer is at least one selected from polystyrene, polyurethane, polystyrene divinylbenzene, polymethyl methacrylate, polyacrylamide, polyethylene glycol terephthalate, polyvinyl acetate, polyethylene, polypropylene, polyvinyl chloride, polyvinylpyrrolidone;
preferably, the metal is selected from at least one of gold, silver, copper, aluminum, iron;
preferably, the mineral is selected from at least one of silica, calcium oxide, titanium dioxide, and ferroferric oxide.
3. A solid phase binding method of HUH protein is characterized in that the HUH protein and a solid phase material are incubated to obtain the HUH solid phase binding protein;
the HUH protein is at least one of A-protein, TC1 protein and HI0217 protein.
4. The method according to claim 3, wherein the incubation is performed at a temperature of 30 to 40℃for a period of 0.5 to 5 hours.
5. A method for solid phase binding of a protein, comprising the steps of:
s11, carrying out first connection on target protein and HUH protein to obtain target protein-HUH protein complex;
s12, incubating the target protein-HUH protein complex and a solid phase material;
or alternatively, the process may be performed,
s21, incubating HUH protein and a solid phase material to obtain HUH solid phase binding protein;
s22, carrying out second connection on the target protein and HUH solid-phase binding protein.
6. The solid phase binding method according to claim 5, wherein the first ligation comprises a protein fusion and/or condensation reaction;
the second connection adopts a condensation reaction.
7. A protein immobilized HUH solid phase binding protein comprising the HUH solid phase binding protein of claim 4, and a protein of interest linked to a HUH protein in said HUH solid phase binding protein;
preferably, the target protein comprises at least one of streptavidin, enzyme, fluorescent protein and affinity peptide.
8. A method for detecting an antigen, comprising the steps of:
s31, carrying out fusion expression on streptavidin and HUH protein to obtain a streptavidin-HUH protein complex;
s32, incubating the streptavidin-HUH protein complex and a solid phase material, and sealing;
s33, adding a biotinylation capture antibody, and incubating;
s34, adding a sample to be detected, and incubating;
s35, adding an antibody containing a detection label, and incubating;
s36, detecting the detection mark.
9. A method for solid phase binding of nucleic acids, comprising the steps of:
s41, incubating HUH protein and a solid phase material to obtain HUH solid phase binding protein;
s42, incubating or third connecting the target nucleic acid and HUH solid phase binding protein;
or alternatively, the process may be performed,
s51, incubating or third connecting HUH protein and target nucleic acid to obtain target nucleic acid-HUH protein complex;
s52, incubating the target nucleic acid-HUH protein complex and the solid phase material.
10. The solid phase binding method according to claim 9, wherein the method used for the third ligation comprises chemical modification and/or condensation reaction.
11. A HUH solid phase binding protein immobilized with DNA, comprising the HUH solid phase binding protein of claim 4, and DNA of interest affinity-bound or linked to the HUH protein in the HUH solid phase binding protein;
preferably, the target DNA comprises capture DNA.
12. A method for detecting target DNA, comprising the steps of:
s61, incubating HUH protein and a solid phase material to obtain HUH solid phase binding protein;
s62, incubating the capture DNA and HUH solid-phase binding protein, and sealing;
s63, adding a sample to be detected and signal DNA containing a detection mark, and incubating;
s64, detecting the detection mark.
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