CN110452303B

CN110452303B - Method for covalently linking nucleic acid and peptide or protein and application thereof

Info

Publication number: CN110452303B
Application number: CN201910730507.5A
Authority: CN
Inventors: 门冬; 张先恩; 周昆; 曹姗姗; 周娟
Original assignee: Wuhan Institute of Virology of CAS
Current assignee: Wuhan Institute of Virology of CAS
Priority date: 2019-08-08
Filing date: 2019-08-08
Publication date: 2022-03-04
Anticipated expiration: 2039-08-08
Also published as: CN110452303A

Abstract

The invention relates to covalent connection of a protein based on TYLCV-C1 protein and nucleic acid, which utilizes the catalytic action of the TYLCV-C1 protein to covalently connect a target protein and the target nucleic acid to form a protein nucleic acid complex, has mild reaction conditions, short reaction time consumption and high efficiency of 93 percent, can react for 10min at 25 ℃, and has no negative influence on the functions and properties of the target protein and the target nucleic acid by the TYLCV-C1 protein.

Description

Method for covalently linking nucleic acid and peptide or protein and application thereof

Technical Field

The invention relates to the technical field of biological crosslinking and nanotechnology, in particular to fixed-point covalent connection of a protein and a nucleic acid based on self catalysis of TYLCV-C1 protein.

Background

Protein nucleic acid complexes have been extensively studied biotechnologically due to their functional diversity of proteins and the programmability of nucleic acids. At present, a great deal of work reports are available on the technologies of multi-enzyme systems constructed on nucleic acid nanostructures, protein immobilization on solid phase carriers such as biochips, biomolecule delivery and high-resolution imaging. However, in the above-mentioned techniques, most of the methods for linking protein and nucleic acid are chemical crosslinking, and the number and state of nucleic acid linkage cannot be determined, so that not only the target protein or nucleic acid needs to be pre-modified, but also the protein is inactivated to different extents by the reaction process, such as covalent linkage formed by reacting the nucleic acid pre-modified by a chemical crosslinking agent with amino groups or thiol groups on the surface of the protein, and covalent linkage formed by using the action of a crosslinking agent (e.g., sSMCC) to covalently bind the nucleic acid modified by alkylthio groups (Alkylthiol) with the protein having amino groups on the surface. There are also methods using enzymatic ligation, such as covalent binding between the nucleic acid modified by Z-QG (N-carbostyryl glutaminyl glycine) and the protein fused with MKHKGS short peptide under the action of transglutaminase (Transglutaminase), but such methods require modification of both nucleic acid and protein before reaction and are limited in application. There are also some non-covalent binding methods, such as protein nucleic acid linkage through the action of avidin (or streptavidin) and biotin, the binding of metal ions to protein recognition domains (such as the binding of nickel ions and polyhistidine), and the like, but these actions are relatively covalent binding, and the binding force is low, influenced by the environment, high in instability and limited in application. Therefore, the existing protein nucleic acid connection technology is yet to be developed, and a covalent fixed-point connection technology which is stable, efficient, mild in reaction condition and controllable in stoichiometric ratio is urgently needed.

Disclosure of Invention

In view of the above, the present invention provides a TYLCV-C1 protein, which is capable of recognizing a specific target nucleic acid sequence and cleaving the target nucleic acid sequence, wherein the cleaved target nucleic acid is directly covalently linked to tyrosine of TYLCV-C1 protein, and then linked to the target protein through TYLCV-C1 protein, thereby achieving site-directed and directional covalent linkage without affecting the structure or performance of the protein and the nucleic acid.

The TYLCV-C1 protein is a Replication-associated protein (Replication-associated protein) of tomato yellow leaf curl virus, is coded by a C1 gene of the virus, and plays an important role in the Replication of virus single-stranded DNA. It is cleaved at the recognition sequence of the viral gene spacer region to introduce a gap, thereby initiating Rolling Circle Replication (RCR).

The invention provides a method of covalently linking a nucleic acid and a peptide or protein, comprising covalently linking a nucleic acid of interest and a peptide or protein of interest using a TYLCV-C1 protein.

The target peptide can be a bioactive polypeptide or an artificially synthesized polypeptide and the like. For example, cytokine mimetic peptides, antibacterial active peptides, polypeptides for cardiovascular diseases, other pharmaceutically acceptable small peptides, and diagnostic polypeptides, etc. Wherein the active peptide can be immunological active peptide, nerve active peptide, cholesterol peptide, peptide for promoting mineral absorption (CPPS), enzyme regulator (such as tryptase peptide), hormone peptide such as growth hormone releasing factor (GRFS), albumin insulin synergistic peptide, antibacterial polypeptide (such as nisin, rubber extract), anticancer polypeptide (such as tumor cell necrosis factor, cyclicpeptide), and anti-AIDS peptide (such as GLQ protein).

The target protein can be monomeric protein, oligomeric protein, polymeric protein and the like. The protein can be animal protein, plant protein or artificial synthetic protein according to the source.

Wherein, the target peptide or protein can be selected according to actual needs.

The TYLCV-C1 protein comprises a complete natural TYLCV-C1 protein, an engineered TYLCV-C1 protein or a TYLCV-C1 protein domain, wherein the engineered TYLCV-C1 protein or the TYLCV-C1 protein domain has the catalytic activity of nuclease.

In a specific embodiment of the invention, the method of linking said nucleic acid and said peptide or protein further comprises, prior to linking said nucleic acid and said peptide or protein using said TYLCV-C1 protein, linking at the 5' end of said nucleic acid the sequence tatta or x-tata, wherein x represents one or more nucleotide bases.

Illustratively, x is 1-30 nucleotide bases. Preferably, x is 2-20 nucleotide bases. More preferably, x is 5-10 nucleotide bases.

In one embodiment of the invention, x is 7 nucleotide bases.

In one embodiment of the invention, the target nucleic acid is linked cgtataatatta sequences to its 5' end.

Wherein the sequence tata is a recognition sequence of the TYLCV-C1 protein, and the sequence cgtataa is helpful for the recognition of the TYLCV-C1 protein.

In a specific embodiment of the invention, wherein said TYLCV-C1 protein forms a fusion protein with said peptide or protein of interest. Of course, the linkage between the TYLCV-C1 protein and the target peptide or protein in the present invention can be performed by other methods in the prior art, such as chemical modification, non-covalent binding, etc.

In a specific embodiment of the invention, said TYLCV-C1 protein is linked to said target peptide or protein directly or via a flexible linker peptide.

In the design of fusion proteins, it is often necessary to use a linker peptide to link and fuse proteins with different functions. The design and selection of the linker peptide is critical to the expression, stability and functional activity of the fusion protein. Two common connecting peptides are flexible and rigid, wherein the flexible connecting peptide is a flexible and linear amino acid sequence easy to bend, and the rigid connecting peptide is a fixed-length amino acid sequence with a stable helical structure. The linker peptide is designed with consideration of the effects of length, amino acid composition, hydrophilicity, conformation, flexibility, and charge. The amino acid composition in the linker peptide has a greater effect on the flexible linker, while the amino acid arrangement affects the structure of the rigid linker peptide.

The length of the connecting peptide directly influences the distance between functional proteins, and the stability and activity of the fusion protein are influenced by the process or the connecting peptide with too short length. The steric hindrance effect can be caused by too short length, the proteins at two ends can not be fully folded or unfolded to form wrong configurations or block active sites, so that the fusion protein has no activity or low activity, and a large amount of dimer forms appear; the fusion protein is loose in integral structure due to overlong length, is easy to be hydrolyzed and cut by protease in host cells, and loses fusion significance.

In a specific embodiment of the invention, said TYLCV-C1 protein is linked to said peptide or protein of interest by a flexible polypeptide. The sequence of the flexible linker peptide may be selected according to the peptide or protein of interest. The sequence of the flexible linker peptide may be, for example, GGGSGGSG, (GGGGS)₆、(GGGGS)₅、(GGGGS)₄、(GGGGS)₃、(GGGGS)₂、GGGGS、GGGG、GSGGSG、GSGGSGGGSGGSGGG、GGGGSGGG、GSGGSGGG、GGGGSGGGSGG and the like.

In a specific embodiment of the invention, the TYLCV-C1 protein is linked to the peptide or protein of interest via the flexible polypeptide GGGGS.

In a first embodiment of the invention, said TYLCV-C1 protein is linked to said peptide or protein of interest by a rigid linker peptide. The sequences of the rigid linker peptides are as follows: EAAAK, (EAAAK)₂，(EAAAK)₃，(EAAAK)₄，(EAAAK)₅，(EAAAK)₆，AEAAAKA，A(EAAAK)₂A，A(EAAAK)₃A，A(EAAAK)₄A，A(EAAAK)₅A，A(EAAAK)₆A, and the like.

In a specific embodiment of the invention, the amino acid sequence of the TYLCV-C1 protein is shown in SEQ ID NO.1 or a homologous sequence thereof.

Illustratively, the homologous sequence has a homology of about 60% or more, about 70% or more, 71% or more, 72% or more, 73% or more, 74% or more, 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 with the original sequence, 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, 99.1% or more, 99.2% or more, 99.3% or more, 99.4% or more, 99.5% or more, 99.6% or more, 99.7% or more, 99.8% or more, or 99.9% or more.

In a specific embodiment of the invention, the nucleotide sequence of the TYLCV-C1 protein is shown in SEQ ID NO.2 or a degenerate sequence thereof.

Illustratively, the degenerate sequence has a homology of about 60% or more, about 70% or more, 71% or more, 72% or more, 73% or more, 74% or more, 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 to the original sequence, 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, 99.1% or more, 99.2% or more, 99.3% or more, 99.4% or more, 99.5% or more, 99.6% or more, 99.7% or more, 99.8% or more, or 99.9% or more.

In another aspect, the invention provides the use of the TYLCV-C1 protein for covalently linking a target protein to a target nucleic acid.

Illustratively, the target nucleic acid is linked at its 5' end to a sequence tata or x-tata, wherein x represents one or more nucleotide bases.

In one embodiment of the invention, x is 7 nucleotide bases.

In a specific embodiment of the invention, said TYLCV-C1 protein forms a fusion protein with said peptide or protein of interest.

Illustratively, the TYLCV-C1 protein is linked to the target peptide or protein directly or via a flexible linker peptide.

In a specific embodiment of the invention, said TYLCV-C1 protein is linked to said target peptide or protein by a flexible linker peptide. The flexible linker peptide may be selected depending on the structure, properties, etc. of the target protein. The sequence of the flexible linker peptide may be, for example, GGGSGGSG, (GGGGS)₆、(GGGGS)₅、(GGGGS)₄、(GGGGS)₃、(GGGGS)₂、GGGGS、GGGG, GSGGSG, GSGGSGGGSGGSGGG, GGGGSGGG, GSGGSGGG, GGGGSGGGSGG and the like. Preferably, the TYLCV-C1 protein is linked to the target peptide or protein via GGGGS.

In another aspect, the invention provides a fusion protein, which comprises a target protein and TYLCV-C1 protein.

Illustratively, the protein of interest and the TYLCV-C1 protein are linked directly or via a flexible linker peptide.

In a specific embodiment of the invention, said protein of interest and said TYLCV-C1 protein are linked by a flexible linker peptide.

The invention also provides a preparation method of the fusion protein, which comprises the following steps:

fusing a target protein with TYLCV-C1 protein to form a fusion protein through gene recombination, and optionally, expressing the fusion protein.

The present invention also provides a complex as described above, comprising a protein of interest and a nucleic acid of interest, said protein of interest and said nucleic acid of interest being covalently linked by catalysis by the TYLCV-C1 protein.

Illustratively, the protein of interest and the nucleic acid of interest are covalently linked by a TYLCV-C1 protein.

In a specific embodiment of the invention, said protein of interest and said nucleic acid of interest are covalently linked by a TYLCV-C1 protein.

In one embodiment of the present invention, the target protein is one of fluorescent protein Venus, pyruvate oxidase, phosphoacetyltransferase, streptococcal protein G.

In one embodiment of the invention, the sequence of the target nucleic acid is SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, or a sequence shown in figure 5.

In a specific embodiment of the invention, the complex further comprises TYLCV-C1 protein, preferably, the amino acid sequence of the TYLCV-C1 protein is shown in SEQ ID No.1 or a homologous sequence thereof.

The invention also provides a preparation method of the compound, which comprises the following steps:

mixing the TYLCV-C1 protein linked with the target protein with the target nucleic acid.

In one embodiment of the present invention, the preparation method further comprises adding metal ions, such as manganese ions, calcium ions, magnesium ions, etc., to the mixture of the TYLCV-C1 protein to which the target protein is linked and the target nucleic acid.

In one embodiment of the present invention, calcium ions and acetic acid are added to the mixture in which the TYLCV-C1 protein to which the target protein is linked is mixed with the target nucleic acid. Preferably, the calcium ion is added at a concentration of 2-4mM and the acetic acid at a concentration of 0.5-0.8%. More preferably, the concentration of calcium ions is 4 mM.

Specifically, the preparation method of the compound comprises the following steps:

(1) cloning of the fusion protein: fusing a target protein gene and a TYLCV-C1 protein gene to form a gene of a fusion protein through molecular cloning; (2) expression and purification of the fusion protein: cloning the gene of the fusion protein into an expression vector, and transforming the constructed expression vector into an escherichia coli expression strain for induced expression to obtain the fusion protein.

(3) Mixing the fusion protein with target nucleic acid, adding calcium ion with final concentration of 4mM and acetic acid with final concentration of 0.5%, and reacting at 25 deg.C for 10min to obtain protein with nucleic acid covalently linked.

The invention provides a method for connecting nucleic acid and peptide or protein, which utilizes the catalytic action of TYLCV-C1 protein to covalently connect target peptide or protein and target nucleic acid to form a protein nucleic acid complex, has mild reaction conditions, short reaction time and high efficiency, can react for 10min at 25 ℃, has almost no influence on the functions and properties of the target protein and the target nucleic acid by TYLCV-C1 protein, and can even improve the enzyme activity of enzyme with catalytic activity.

Drawings

FIG. 1 is a schematic diagram showing the connection of TYLCV-C1 protein and nucleic acid provided in the examples of the present invention. Wherein POI represents a protein of interest; TYLCV represents TYLCV-C1 protein.

FIG. 2 is a graph showing the experimental results of the complex formed by Venus fluorescent protein and nucleic acid provided in the example of the present invention.

FIG. 3 is a diagram showing the results of experiments on complexes formed between pyruvate oxidase and nucleic acids according to examples of the present invention.

FIG. 4 shows the activity of fluorescent protein Venus in Venus-TYLCV-C1-nucleic acid, a protein nucleic acid complex, determined by fluorescence intensity in an example of the present invention.

FIG. 5 is a graph showing the results of experiments on complexes formed between SPG and nucleic acids according to the present invention.

Detailed Description

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

As used herein, "comprising" is synonymous with "including," "containing," or "characterized by," and is inclusive or open-ended and does not exclude additional unrecited elements or method steps. The term "comprising" in any of the expressions herein, particularly in describing the method, use or product of the invention, is to be understood as including those products, methods and uses which consist essentially of and consist of the recited components or elements or steps. The invention illustratively described herein suitably may be practiced in the absence of any element or elements, limitation or limitations which is not specifically disclosed herein.

The terms and expressions which have been employed herein are used as terms of description and not of limitation, and there is no intention in the use of such terms and expressions of excluding any equivalents of the features shown and described or portions thereof, but it is recognized that various modifications are possible within the scope of the invention claimed. Thus, it should be understood that although the present invention has been specifically disclosed by preferred embodiments and optional features, modification and variation of the concepts herein disclosed may be resorted to by those skilled in the art, and that such modifications and variations are considered to be within the scope of this invention as defined by the appended claims.

The TYLCV-C1 protein described herein includes its related mutant or a partial domain of the TYLCV-C1 protein, which still functions as an endonuclease able to catalyze the ligation of the protein to nucleic acids.

The target protein of the present invention may be any protein, which is selected as necessary.

The target nucleic acid is any nucleic acid sequence, and only a tata sequence or an cgtataatatta sequence needs to be connected to the 5 ' end of the target nucleic acid, and if the target nucleic acid sequence contains a tata sequence or a cgtataatatta sequence at the 5 ' end, the target nucleic acid sequence does not need to be connected with a tata sequence or a cgtataatatta sequence at the 5 ' end.

For a more clear illustration of the invention, reference is now made in detail to the following examples, which are intended to be purely exemplary of the invention and are not to be interpreted as limiting the application.

Example 1 fusion and expression of fluorescent protein Venus and TYLCV-C1 protein

In the embodiment, fluorescent protein Venus is taken as target protein, and is fused and expressed with TYLCV-C1 protein.

The target protein provided in this example is linked to the TYLCV-C1 protein by a gene fusion protocol, which can also be linked by other means, such as: chemical modification or non-covalent binding, etc.

(1) Cloning of the fusion protein: nucleotide sequences of Venus and TYLCV-C1 proteins are artificially synthesized. Wherein, the nucleotide sequence of the fluorescent protein Venus is shown as SEQ ID NO: 6, the amino acid sequence of the TYLCV-C1 protein is shown as SEQ ID NO: 1, and the nucleotide sequence of the TYLCV-C1 protein is shown as SEQ ID NO: 2, respectively.

The fluorescent protein Venus is used as a target protein, and can be directly connected with TYLCV-C1 protein or connected through flexible connecting peptide. For example, the sequence of a flexible linker peptide such as GGGSGGSG, (GGGGS)₆、(GGGGS)₅、(GGGGS)₄、(GGGGS)₃、(GGGGS)₂GGGGS, GGGG, GSGGSG, GSGGSGGGSGGSGG, GGGGSGGG, GSGGSGGG, GGGGSGGGSGG and the like. In the embodiment, a fusion protein is formed between Venus and TYLCV-C1 protein through flexible connecting peptide GGGGS, wherein the sequence of the fusion protein is shown in SEQ ID NO: shown at 7.

(2) Expression and purification of the fusion protein: the gene of the fusion protein (the sequence of which is shown in SEQ ID NO: 7) was cloned into the expression vector PET32a (the protein could be well expressed in a variety of expression vectors and expression hosts, and only expression in E.coli and purification using a nickel affinity chromatography column is described here). The constructed expression vector was transformed into E.coli expression strain BL21(DE3), and positive clones were selected. The positive clones were transferred to LB medium and cultured with shaking at 37 ℃ until logarithmic phase (OD value: about 0.5). To the culture, IPTG was added at a working final concentration of 1mM, and protein expression was induced by shaking culture at 25 ℃ for 8 hours. And (3) purifying the target protein by Ni affinity chromatography to obtain a purified fusion protein Venus-TYLCV-C1.

EXAMPLE 2 preparation of a Complex of the fluorescent protein Venus with nucleic acids

The schematic diagram of protein-to-nucleic acid linkage is shown in FIG. 1. In FIG. 1, POI represents a protein of interest. In this embodiment, the target nucleic acid is any nucleic acid sequence with a tata sequence connected to the 5 'end, and preferably any nucleic acid sequence with an cgtataatatta sequence connected to the 5' end. In this example, the sequence shown in SEQ ID NO: 3, the target nucleic acid sequence 3 shown in the figure is illustrated as an example.

In SEQ ID NO: 3, a tatta sequence is inserted into the 5' end of the target nucleic acid sequence 3 shown in the specification. In this embodiment preferably the sequence shown in SEQ ID NO: 3, cgtataatatta sequence is inserted into the 5' end of the target nucleic acid sequence 3 to form a sequence shown as SEQ ID NO: 12, and (b) a nucleotide sequence shown as 12.

The fusion protein Venus-TYLCV-C1 purified in example 1 was compared with the fusion protein of SEQ ID NO: 12, adding manganese ions with the final concentration of 4mM, and reacting at 25 ℃ for 10min to obtain the Venus-TYLCV-C1-nucleic acid which is a protein-nucleic acid complex.

SDS-PAGE of the resulting Venus-TYLCV-C1-nucleic acid protein-nucleic acid complex confirmed that the molecular weight of the protein-nucleic acid complex covalently linked to the nucleic acid was large, and the band was delayed in SDS-PAGE compared with the original protein, and thus a delayed chain was present in SDS-PAGE as shown in FIG. 2. The channels from left to right in fig. 2 represent respectively: 1 is marker; 2 is a control; 3 is a complex; as can be seen from FIG. 2, channel 3 has a distinct lag band compared to control channel 2, indicating that the target protein and the target nucleic acid in this example have been covalently linked to form the protein-nucleic acid complex Venus-TYLCV-C1-nucleic acid.

Example 3 fluorescence intensity measurement of complexes formed by the fluorescent protein Venus and nucleic acids

In this example, the fluorescence intensity was used to determine the activity of the fluorescent protein Venus in the Venus-TYLCV-C1-nucleic acid protein nucleic acid complex. The Venus-TYLCV-C1-nucleic acid, the protein nucleic acid complex obtained in example 2, was subjected to fluorescence intensity measurement in a microplate reader. Taking the protein nucleic acid compound Venus-TYLCV-C1-nucleic acid 3.6ug, adding pure water to make the volume constant to 100 ul. The positive control is 3.6ug Venus-TYLCV-C1 protein; the negative control was pure water. The test conditions were: the excitation wavelength is 515nm, and the fluorescence intensity at the emission wavelength of 530nm is measured. The results are shown in FIG. 4, where the data from left to right represent: 1 is a complex; 2 is a positive control; and 3 is a negative control. It can be seen from the figure that the fluorescence intensity of the complex is not decreased compared with the positive control group, which indicates that the whole process of catalyzing the formation of the protein-nucleic acid complex between the target protein and the target nucleic acid by using the TYLCV-C1 protein provided in example 1 and example 2 has no influence on the activity of the target protein, and the target protein in the finally formed protein-nucleic acid complex can still maintain its original activity.

Example 4 fusion and expression of pyruvate oxidase with TYLCV-C1 protein

In this example, Pyruvate Oxidase (POX) was used as a target protein, and it was fused with TYLCV-C1 protein and expressed. The amino acid sequence of the TYLCV-C1 protein is shown as SEQ ID NO: 1 is shown.

The pyruvate oxidase provided in this example is linked to TYLCV-C1 protein by a gene fusion protocol, which can also be linked by other means, such as: chemical modification or non-covalent binding, etc.

(1) Cloning of the fusion protein: artificially synthesizing the nucleotide sequences of pyruvate oxidase and TYLCV-C1 protein. Wherein, the nucleotide sequence of pyruvate oxidase is shown as SEQ ID NO: 8, the nucleotide sequence of the TYLCV-C1 protein is shown as SEQ ID NO: 2, respectively.

In this embodiment, pyruvate oxidase and TYLCV-C1 protein are linked by flexible linker peptide GGGGS to form a fusion protein, abbreviated as POX-TYLCV-C1, wherein the nucleotide sequence of the fusion protein POX-TYLCV-C1 is as shown in SEQ ID NO: shown at 9.

(2) Expression and purification of the fusion protein: the gene of the fusion protein POX-TYLCV-C1 (the sequence of which is shown in SEQ ID NO: 9) was cloned into the expression vector PET32a (the protein was well expressed in a variety of expression vectors and expression hosts, and only expression in E.coli was described here and purified using a nickel affinity chromatography column). The constructed expression vector was transformed into E.coli expression strain BL21(DE3), and positive clones were selected. The positive clones were transferred to LB medium and cultured with shaking at 37 ℃ until logarithmic phase (OD value: about 0.5). To the culture, IPTG was added at a working final concentration of 1mM, and protein expression was induced by shaking culture at 25 ℃ for 8 hours. Ni affinity chromatography is used for purifying the target protein, namely the purified fusion protein POX-TYLCV-C1 is obtained.

EXAMPLE 5 preparation of a Complex of pyruvate oxidase with nucleic acid

The fusion protein POX-TYLCV-C1 purified in example 4 was compared with the amino acid sequence shown in SEQ ID NO: 4, adding magnesium ions with the final concentration of 4mM, and reacting at 37 ℃ for 10min to obtain the protein-nucleic acid complex POX-TYLCV-C1-nucleic acid.

The protein-nucleic acid complex POX-TYLCV-C1-nucleic acid was confirmed by SDS-PAGE, and the results are shown in FIG. 3. The molecular weight of the protein-nucleic acid complex covalently linked to the nucleic acid is large, and the band lags in SDS-PAGE compared with the original protein, so that a lag chain exists in SDS-PAGE electrophoresis as shown in FIG. 3. The channels from left to right in fig. 3 represent respectively: 1 is marker; 2 is a complex; and 3 is a control. As can be seen from FIG. 3, a significant lag band was observed in channel 2 compared to the control, indicating that the target protein and the target nucleic acid in this example have been covalently linked to form a protein nucleic acid complex POX-TYLCV-C1-nucleic acid.

The inventors of the present application also tested the interaction of phosphate acetyltransferase (PTA) with tyrcv-C1 protein by TYLCV-C1 protein: 4, which also forms a complex of the phosphate acetyltransferase and the nucleic acid.

Example 6 Activity assay of pyruvate oxidase in Complex

The activity of pyruvate oxidase in the fusion protein POX-TYLCV-C1 formed in example 4 and the complex POX-TYLCV-C1-nucleic acid in example 5 were tested against the activity of pyruvate oxidase expressed alone. The activity of pyruvate oxidase in this example was determined as follows:

(1) principle of measurement

Pyruvate oxidase reacts pyruvate in the sample to produce hydrogen peroxide, which under the action of catalase produces water and oxygen to consume pyruvate in the sample, which catalyzes the following reactions:

from the above reaction formula, the enzyme activity of pyruvate oxidase can be determined by measuring the absorbance value of quinonimine dye at a wavelength of 550 nm.

(2) Assay reagent

(A) Reaction mixture 1: 2.0mL of 50mmol/L disodium hydrogenphosphate-sodium dihydrogenphosphate buffer (pH7.4), 0.1mL of 2mmol/L flavin adenine dinucleotide, 0.2mL of 3mmol/L thiamine pyrophosphate, 1.0mL of 15 mmol/L4-aminoantipyrine solution, 1.0mL of 50U/mL peroxidase solution, and 1.7mL of distilled water.

(B) Reaction mixture 2: 2.0mL of 0.2% 2, 4-dichlorophenol, and 2.0mL of 0.15mol/L magnesium sulfate solution.

(C) Substrate solution: 1.0mol/L potassium pyruvate solution.

(D) Reaction termination solution: 3.72mg of disodium EDTA salt was dissolved in 100mL of water.

(E) Enzyme diluent: 50mmol/L Tris-hydrochloric acid buffer (pH 7.4).

(3) Determination of operating procedure

(A) 0.6mL of the reaction mixture 1, 0.3mL of the reaction mixture 2, and 0.1mL of the substrate solution were thoroughly mixed, and then heated to 37 ℃.

(B) Diluting a pyruvate oxidase sample by using an enzyme diluent until the unit of enzyme activity is 0.1-0.2U/mL, adding 0.02mL of the sample diluent into the mixed solution formed in the step (A), uniformly mixing, and keeping the temperature at 37 ℃ for 10 min.

(C) 2.0mL of a reaction terminator was added thereto, and the mixture was equilibrated at 37 ℃ for 5 min.

(D) 0.1mL of the above enzyme solution was taken and the absorbance value was measured at 550nm in a spectrophotometer (Shimadzu UV-2450 spectrophotometer) maintained at a constant temperature of 37 ℃ (A1).

(E) The absorbance values were determined by the procedure described above using the enzyme diluent instead of the pyruvate oxidase sample (A2).

(F) Calculating the value of Δ a: Δ a ═ a1-a 2.

(4) Definition of enzyme activity unit: under the above conditions, the amount of enzyme consuming 1. mu. mol of pyruvic acid in 1 minute was 1U.

(5) The enzyme activity calculation formula is as follows:

enzyme activity (U/mg) ═ 0.82 XDeltaA XDf ÷ C

Wherein: 10 is reaction time; vt is 3.02mL of the total volume; vs is the sample volume of 0.02 mL; df is oxygen pyruvateDilution factor of the chemoenzymatic sample; 1/2 represents 2mol of H in the reaction₂O₂To produce 1mol of quinone imine dye species; 36.88 is the molar absorption coefficient of the quinoneimine dye (cm 2/. mu.mol) under the measurement conditions; c is the concentration of enzyme in solution (mg/mL).

The raw material sources are as follows:

polyethylene glycol was purchased from Dow chemical, pyruvic acid from Shanghai spectral vibration Biotech, Inc., and peroxidase from Shanghai Jia and Biotech, Inc., respectively.

The control pyruvate oxidase was a single pyruvate oxidase prepared according to the cloning and expression method of the fusion protein of example 4 (the nucleotide sequence of which is shown in SEQ ID NO: 8); the test groups were the enzyme activities of pyruvate oxidase in the fusion protein POX-TYLCV-C1 prepared in example 4 and the enzyme activities of pyruvate oxidase in the complex POX-TYLCV-C1-nucleic acid prepared in example 5, respectively.

The results show that the enzyme activity of pyruvate oxidase of POX-TYLCV-C1 in example 4 and the enzyme activity of pyruvate oxidase of POX-TYLCV-C1-nucleic acid in example 5 are both higher than the enzyme activity of pyruvate oxidase of the control group, and the enzyme activity of pyruvate oxidase of POX-TYLCV-C1 is about 1.63 times of the enzyme activity of pyruvate oxidase of the control group; the enzyme activity of pyruvate oxidase of POX-TYLCV-C1-nucleic acid is about 1.61 times of that of the control pyruvate oxidase, and thus, in the whole process of catalyzing the pyruvate oxidase to form a protein nucleic acid complex with target nucleic acid by utilizing the TYLCV-C1 protein, the reduction of the activity of the pyruvate oxidase is not caused, but the enzyme activity of the pyruvate oxidase is enhanced, namely, the TYLCV-C1 protein can catalyze the connection of the pyruvate oxidase and the target nucleic acid, and the TYLCV-C1 protein can improve the enzyme activity of the pyruvate oxidase in the fusion protein and the complex.

The inventors of the present application also tested Phosphoacetyltransferase (PTA), a phosphoacetyltransferase, by TYLCV-C1 protein, to react with SEQ ID NO: 4 and the enzyme activity of the phosphate acetyltransferase in a complex of the phosphate acetyltransferase and the nucleic acid, wherein the phosphate acetyltransferase forms a fusion protein of the phosphate acetyltransferase and the TYLCV-C1 protein and the phosphate acetyltransferase forms a complex with the nucleic acid, it was found that the enzyme activity of the phosphate acetyltransferase of the fusion protein of the phosphate acetyltransferase and the TYLCV-C1 protein and the enzyme activity of the phosphate acetyltransferase of the complex of the phosphate acetyltransferase and the nucleic acid are both higher than the enzyme activity of the free phosphate acetyltransferase alone, and are respectively 1.54 times and 1.49 times of the enzyme activity of the free phosphate acetyltransferase alone

Example 7 formation of complexes of SPG with TYLCV-C1 protein

In this example, SPG (Streptococcus G Protein, Streptococcus Protein G) was used as a target Protein, and it was fused with TYLCV-C1 Protein and expressed. The amino acid sequence of the TYLCV-C1 protein is shown as SEQ ID NO: 1 is shown.

(1) Cloning of the fusion protein: the nucleotide sequences of the SPG and TYLCV-C1 proteins were synthesized. The nucleotide sequence of SPG is shown in SEQ ID NO: 10, the nucleotide sequence of the TYLCV-C1 protein is shown as SEQ ID NO: 2, respectively.

In this embodiment, the SPG and TYLCV-C1 proteins are linked by a flexible linker peptide ggggggs to form a fusion protein, abbreviated as SPG-TYLCV-C1, wherein the nucleotide sequence of the fusion protein SPG-TYLCV-C1 is as shown in SEQ ID NO: shown at 11.

(2) Expression and purification of the fusion protein: the gene of the fusion protein SPG-TYLCV-C1 (the sequence of which is shown in SEQ ID NO: 11) was cloned into the expression vector PET32a (the protein was well expressed in a variety of expression vectors and expression hosts, and only expression in E.coli was described here and purified using a nickel affinity chromatography column). The constructed expression vector was transformed into E.coli expression strain BL21(DE3), and positive clones were selected. The positive clones were transferred to LB medium and cultured with shaking at 37 ℃ until logarithmic phase (OD value: about 0.5). To the culture, IPTG was added at a working final concentration of 1mM, and protein expression was induced by shaking culture at 25 ℃ for 8 hours. And (4) purifying the target protein by Ni affinity chromatography to obtain the purified fusion protein SPG-TYLCV-C1.

Example 8 preparation of complexes of SPG with nucleic acids

The fusion protein SPG-TYLCV-C1 purified in example 7 was compared with the amino acid sequence of SEQ ID NO: 5, adding calcium ions with the final concentration of 4mM, and reacting at 16 ℃ for 30min to obtain the protein nucleic acid complex SPG-TYLCV-C1-nucleic acid.

The protein-nucleic acid complex SPG-TYLCV-C1-nucleic acid was confirmed by SDS-PAGE, and the results are shown in FIG. 5. The channels from left to right in fig. 5 represent: 1 is marker; 2 is a complex; and 3 is a control. As can be seen from FIG. 5, a significant hysteresis band was present in channel 2 compared to the control, indicating that the protein of interest and the nucleic acid of interest in this example have been covalently linked to form the protein-nucleic acid complex SPG-TYLCV-C1-nucleic acid.

The activity of SPG-binding antigen in the fusion protein formed in example 7 and the activity of SPG-binding antigen in the complex formed between SPG and nucleic acid in example 8 were tested against SPG alone, and the results showed that the activity of SPG in the fusion protein formed in example 7 and the activity of SPG alone in the complex formed between pyruvate oxidase and nucleic acid in example 8 were not different, and it was found that the activity of target protein was not affected throughout the formation of protein-nucleic acid complex between target protein and target nucleic acid by using TYLCV-C1 protein as provided in examples 7 and 8, and the target protein in the finally formed protein-nucleic acid complex still maintained its original activity.

Example 9 testing of the Effect of different reaction conditions on the preparation of SPG-TYLCV-C1-nucleic acid

The inventors of the present application also tested the effect of different reaction conditions on protein to nucleic acid attachment. The effect of adding different concentrations of acetic acid, calcium ions and reaction temperature on the preparation of SPG-TYLCV-C1-nucleic acid was tested against the ligation of the protein nucleic acid complex SPG-TYLCV-C1 to nucleic acid as in example 8. The specific process is as follows: the fusion protein SPG-TYLCV-C1 purified in example 7 was compared with the amino acid sequence of SEQ ID NO: 5, adding acetic acid with different concentrations and calcium ions with different concentrations, reacting at 16 ℃, 25 ℃ and 37 ℃ for 10min, performing electrophoresis, detecting a stripe gray value on an optical density scanner (Bio-Rad GS-900 optical density scanner), and calculating the connection efficiency when the reaction is finished, wherein the calculation of the connection efficiency E is as follows:

e ═ E (c)/E (c) + E (p), where E (c) is the protein-nucleic acid complex band intensity value and E (p) is the target protein band intensity value. The protocol and results are shown in Table 1.

TABLE 1 Effect of different reaction conditions on the preparation of SPG-TYLCV-C1-nucleic acids

Metal ions and addition thereof	Reaction temperature	Efficiency of reaction
			Calcium ion 4mM	37℃	67％
Acetic acid 0.2%	37℃	-
			Calcium ion 2mM	37℃	60％
Calcium ion 4mM + acetic acid 0.2%	37℃	78％
			Calcium ion 4mM	25℃	63％
Acetic acid 0.2%	25℃	-
			Calcium ion 4mM + acetic acid 0.2%	25℃	82％
Calcium ion 4mM + acetic acid 0.5%	25℃	93％
			Calcium ion 4mM + acetic acid 0.8%	25℃	87％
Calcium ion 4mM + acetic acid 1%	25℃	87％
			Calcium ion 4mM + acetic acid 2%	25℃	71％
Calcium ion 4mM	16℃	50％
			Acetic acid 0.2%	16℃	-
Calcium ion 4mM + acetic acid 0.2%	16℃	56％
			Calcium ion 4mM + acetic acid 0.5%	16℃	73％

Note: -means no reaction.

As can be seen from Table 1, different concentrations of acetic acid have an effect on the linkage of SPG-TYLCV-C1 to the nucleic acid, and in the mixture of SPG-TYLCV-C1 and the nucleic acid, SPG-TYLCV-C1 cannot covalently link to the nucleic acid to form a protein-nucleic acid complex when acetic acid alone is added; 4mM of calcium ions are more capable of promoting the connection of SPG-TYLCV-C1 to nucleic acid than 2mM of calcium ions; when calcium ions and acetic acid are simultaneously added to the mixture of SPG-TYLCV-C1 and nucleic acid, the SPG-TYLCV-C1 is more efficiently linked to the nucleic acid, especially when 4mM calcium ions and 0.5% acetic acid are added to the mixture of SPG-TYLCV-C1 and nucleic acid, the reaction efficiency is the highest, and the reaction temperature is required to be around room temperature. The inventors of the present application also tested covalent linkage of other proteins to nucleic acids, such as covalent linkage of fluorescent protein to nucleic acid in example 2, linkage of pyruvate oxidase to nucleic acid in example 5, and covalent linkage of phosphate acetyltransferase to nucleic acid, and all of the results showed that addition of 4mM calcium ion and 0.5% acetic acid during covalent linkage of target protein to target nucleic acid maximized the efficiency of the reaction for covalent linkage of target protein to target nucleic acid.

According to the embodiment, the TYLCV-C1 protein is utilized to identify the tatta sequence and catalyze the connection of the target protein and the target nucleic acid containing the tatta sequence, so that the covalent connection between the protein and the nucleic acid is realized, no chemical reagent is required to be introduced into the target protein and the target nucleic acid in the connection, only metal ions are required to be additionally added, the reaction condition is mild, the reaction time is short, the efficiency is high, the reaction can be completed within 10min at 25 ℃, and the whole preparation process and the finally formed protein nucleic acid complex have no negative influence on the target protein.

The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents and the like that are within the spirit and principle of the present invention are included in the present invention.

Sequence listing

<110> Wuhan Virus institute of Chinese academy of sciences

<120> method for covalently linking nucleic acid and peptide or protein and use thereof

<160> 12

<170> SIPOSequenceListing 1.0

<210> 1

<211> 121

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> TYLCV-C1 protein amino acid sequence

<400> 1

Met Pro Arg Ser Gly Arg Phe Ser Ile Lys Ala Lys Asn Tyr Phe Leu

1 5 10 15

Thr Tyr Pro Lys Cys Asp Leu Thr Lys Glu Asn Ala Leu Ser Gln Ile

20 25 30

Thr Asn Leu Gln Thr Pro Thr Asn Lys Leu Phe Ile Lys Ile Cys Arg

35 40 45

Glu Leu His Glu Asn Gly Glu Pro His Leu His Ile Leu Ile Gln Phe

50 55 60

Glu Gly Lys Tyr Asn Cys Thr Asn Gln Arg Phe Phe Asp Leu Val Ser

65 70 75 80

Pro Thr Arg Ser Ala His Phe His Pro Asn Ile Gln Gly Ala Lys Ser

85 90 95

Ser Ser Asp Val Lys Ser Tyr Ile Asp Lys Asp Gly Asp Val Leu Glu

100 105 110

Trp Gly Thr Phe Gln Ile Asp Gly Arg

115 120

<210> 2

<211> 363

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> TYLCV-C1 protein nucleotide sequence

<400> 2

atgcctcgtt caggccgctt tagcattaaa gcgaagaact actttctgac ctatccgaaa 60

tgcgatctga ccaaagaaaa cgcgctgagc cagattacca acctgcagac cccgaccaac 120

aaactgttca tcaaaatttg ccgcgaactg catgaaaacg gcgaaccgca tctgcatatt 180

ctgattcagt tcgagggcaa atataactgc accaaccagc gcttttttga tctggtgagc 240

cctactcgta gcgcgcattt tcatccgaac attcagggcg cgaaaagcag cagcgatgtg 300

aaaagctaca tcgataagga tggcgatgtg ctggaatggg gcacctttca gattgatggc 360

cgt 363

<210> 3

<211> 14

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> nucleotide sequence 3

<400> 3

ccggatggcc gcgc 14

<210> 4

<211> 27

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> nucleotide sequence 4

<400> 4

cgtataatat tacgggtgag gccggcg 27

<210> 5

<211> 35

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> nucleotide sequence 5

<400> 5

cgtataatat taccgaagcg accaggcgat ataat 35

<210> 6

<211> 720

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> nucleotide sequence of fluorescent protein Venus

<400> 6

atggtgagca agggcgagga gctgttcacc ggggtggtgc ccatcctggt cgagctggac 60

ggcgacgtaa acggccacaa gttcagcgtg tccggcgagg gcgagggcga tgccacctac 120

ggcaagctga ccctgaagtt gatctgcacc accggcaagc tgcccgtgcc ctggcccacc 180

ctcgtgacca ccttgggcta cggcgtgcag tgcttcgccc gctaccccga ccacatgaag 240

cagcacgact tcttcaagtc cgccatgccc gaaggctacg tccaggagcg caccatcttc 300

ttcaaggacg acggcaacta caagacccgc gccgaggtga agttcgaggg cgacaccctg 360

gtgaaccgca tcgagctgaa gggcatcgac ttcaaggagg acggcaacat cctggggcac 420

aagctggagt acaactacaa cagccacaac gtctatatca ccgccgacaa gcagaagaac 480

ggcatcaagg cgaacttcaa gatccgccac aacatcgagg acggcggcgt gcagctcgcc 540

gaccactacc agcagaacac ccccatcggc gacggccccg tgctgctgcc cgacaaccac 600

tacctgagct accagtccgc cctgagcaaa gaccccaacg agaagcgcga tcacatggtc 660

ctgctggagt tcgtgaccgc cgccgggatc actctcggca tggacgagct gtacaagtaa 720

<210> 7

<211> 1146

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> Venus-TYLCV-C1 fusion protein

<400> 7

atggtgagca agggcgagga gctgttcacc ggggtggtgc ccatcctggt cgagctggac 60

ggcgacgtaa acggccacaa gttcagcgtg tccggcgagg gcgagggcga tgccacctac 120

ggcaagctga ccctgaagtt gatctgcacc accggcaagc tgcccgtgcc ctggcccacc 180

ctcgtgacca ccttgggcta cggcgtgcag tgcttcgccc gctaccccga ccacatgaag 240

cagcacgact tcttcaagtc cgccatgccc gaaggctacg tccaggagcg caccatcttc 300

ttcaaggacg acggcaacta caagacccgc gccgaggtga agttcgaggg cgacaccctg 360

gtgaaccgca tcgagctgaa gggcatcgac ttcaaggagg acggcaacat cctggggcac 420

aagctggagt acaactacaa cagccacaac gtctatatca ccgccgacaa gcagaagaac 480

ggcatcaagg cgaacttcaa gatccgccac aacatcgagg acggcggcgt gcagctcgcc 540

gaccactacc agcagaacac ccccatcggc gacggccccg tgctgctgcc cgacaaccac 600

tacctgagct accagtccgc cctgagcaaa gaccccaacg agaagcgcga tcacatggtc 660

ctgctggagt tcgtgaccgc cgccgggatc actctcggca tggacgagct gtacaagggt 720

ggaggtggat cgggatccga aaacctttac ttccaaggcc ctcgttcagg ccgctttagc 780

attaaagcga agaactactt tctgacctat ccgaaatgcg atctgaccaa agaaaacgcg 840

ctgagccaga ttaccaacct gcagaccccg accaacaaac tgttcatcaa aatttgccgc 900

gaactgcatg aaaacggcga accgcatctg catattctga ttcagttcga gggcaaatat 960

aactgcacca accagcgctt ttttgatctg gtgagcccta ctcgtagcgc gcattttcat 1020

ccgaacattc agggcgcgaa aagcagcagc gatgtgaaaa gctacatcga taaggatggc 1080

gatgtgctgg aatggggcac ctttcagatt gatggccgtc tcgagcacca ccaccaccac 1140

cactga 1146

<210> 8

<211> 1776

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> nucleotide sequence of pyruvate oxidase

<400> 8

atgagcgaca acaagatcaa cattggtctg gcggttatga aaatcctgga aagctggggc 60

gcggacacca tttatggtat cccgagcggc accctgagca gcctgatgga tgcgatgggt 120

gaggaagaga acaacgtgaa gttcctgcag gttaaacatg aagaggtggg tgcgatggcg 180

gcggtgatgc aaagcaaatt tggtggcaac ctgggtgtga ccgttggtag cggtggcccg 240

ggtgcgagcc acctgattaa cggcctgtac gacgcggcga tggataacat tccggtggtt 300

gcgatcctgg gtagccgtcc gcagcgtgaa ctgaacatgg acgcgttcca ggagctgaac 360

caaaacccga tgtacgatca catcgcggtt tataaccgtc gtgtggcgta cgcggaacaa 420

ctgccgaagc tggttgacga ggcggcgcgt atggcgattg cgaaacgtgg tgtggcggtt 480

ctggaagttc cgggcgattt tgcgaaagtg gagatcgaca acgatcagtg gtatagcagc 540

gcgaacagcc tgcgtaaata tgcgccgatt gcgccggcgg cgcaagacat tgatgcggcg 600

gttgagctgc tgaacaacag caaacgtccg gtgatttatg cgggtatcgg cacgatgggt 660

cacggtccgg cggttcagga actggcgcgt aagattaaag cgccggtgat caccaccggt 720

aaaaacttcg aaacctttga gtgggacttc gaggcgctga ccggtagcac ctaccgtgtt 780

ggctggaagc cggcgaacga aaccatcctg gaggcggaca ccgtgctgtt tgcgggtagc 840

aacttcccgt ttagcgaagt tgagggcacc ttccgtaacg tggataactt tatccagatt 900

gacatcgatc cggcgatgct gggtaaacgt caccatgcgg atgtggcgat cctgggtgat 960

gcgggtctgg cgattgacga aatcctgaac aaggtggacg cggttgaaga gagcgcgtgg 1020

tggaccgcga acctgaaaaa cattgcgaac tggcgtgaat atatcaacat gctggagacc 1080

aaggaagagg gtgacctgca gttctaccaa gtttataacg cgattaacaa ccacgcggac 1140

gaggatgcga tttacagcat cgatgtgggc aacagcaccc agaccagcat ccgtcacctg 1200

cacatgaccc cgaaaaacat gtggcgtacc agcccgctgt tcgcgacgat gggtattgcg 1260

attccgggtg gcctgggtgc gaagaacacc tacccggatc gtcaagtttg gaacatcatt 1320

ggtgacggcg cgtttagcat gacctatccg gatgtggtta ccaacgttcg ttacaacatg 1380

ccggtgatta acgtggtttt cagcaacacc gaatatgcgt ttatcaagaa caaatacgag 1440

gacaccaaca agaacctgtt cggtgttgat tttaccgacg tggattatgc gaagattgcg 1500

gaagcgcagg gtgcgaaagg cttcaccgtg agccgtatcg aagacatgga tcgtgttatg 1560

gcggaggcgg tggcggcgaa caaggcgggc cacaccgtgg ttattgattg caaaatcacc 1620

caagaccgtc cgattccggt tgagaccctg aagctggata gcaaactgta cagcgaagac 1680

gagatcaagg cgtacaaaga acgttatgag gcggcgaacc tggtgccgtt tcgtgaatat 1740

ctggaagcgg agggtctgga gagcaagtac atcaaa 1776

<210> 9

<211> 2205

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> nucleotide sequence of fusion protein POX-TYLCV-C1

<400> 9

atgagcgaca acaagatcaa cattggtctg gcggttatga aaatcctgga aagctggggc 60

gcggacacca tttatggtat cccgagcggc accctgagca gcctgatgga tgcgatgggt 120

gaggaagaga acaacgtgaa gttcctgcag gttaaacatg aagaggtggg tgcgatggcg 180

gcggtgatgc aaagcaaatt tggtggcaac ctgggtgtga ccgttggtag cggtggcccg 240

ggtgcgagcc acctgattaa cggcctgtac gacgcggcga tggataacat tccggtggtt 300

gcgatcctgg gtagccgtcc gcagcgtgaa ctgaacatgg acgcgttcca ggagctgaac 360

caaaacccga tgtacgatca catcgcggtt tataaccgtc gtgtggcgta cgcggaacaa 420

ctgccgaagc tggttgacga ggcggcgcgt atggcgattg cgaaacgtgg tgtggcggtt 480

ctggaagttc cgggcgattt tgcgaaagtg gagatcgaca acgatcagtg gtatagcagc 540

gcgaacagcc tgcgtaaata tgcgccgatt gcgccggcgg cgcaagacat tgatgcggcg 600

gttgagctgc tgaacaacag caaacgtccg gtgatttatg cgggtatcgg cacgatgggt 660

cacggtccgg cggttcagga actggcgcgt aagattaaag cgccggtgat caccaccggt 720

aaaaacttcg aaacctttga gtgggacttc gaggcgctga ccggtagcac ctaccgtgtt 780

ggctggaagc cggcgaacga aaccatcctg gaggcggaca ccgtgctgtt tgcgggtagc 840

aacttcccgt ttagcgaagt tgagggcacc ttccgtaacg tggataactt tatccagatt 900

gacatcgatc cggcgatgct gggtaaacgt caccatgcgg atgtggcgat cctgggtgat 960

gcgggtctgg cgattgacga aatcctgaac aaggtggacg cggttgaaga gagcgcgtgg 1020

tggaccgcga acctgaaaaa cattgcgaac tggcgtgaat atatcaacat gctggagacc 1080

aaggaagagg gtgacctgca gttctaccaa gtttataacg cgattaacaa ccacgcggac 1140

gaggatgcga tttacagcat cgatgtgggc aacagcaccc agaccagcat ccgtcacctg 1200

cacatgaccc cgaaaaacat gtggcgtacc agcccgctgt tcgcgacgat gggtattgcg 1260

attccgggtg gcctgggtgc gaagaacacc tacccggatc gtcaagtttg gaacatcatt 1320

ggtgacggcg cgtttagcat gacctatccg gatgtggtta ccaacgttcg ttacaacatg 1380

ccggtgatta acgtggtttt cagcaacacc gaatatgcgt ttatcaagaa caaatacgag 1440

gacaccaaca agaacctgtt cggtgttgat tttaccgacg tggattatgc gaagattgcg 1500

gaagcgcagg gtgcgaaagg cttcaccgtg agccgtatcg aagacatgga tcgtgttatg 1560

gcggaggcgg tggcggcgaa caaggcgggc cacaccgtgg ttattgattg caaaatcacc 1620

caagaccgtc cgattccggt tgagaccctg aagctggata gcaaactgta cagcgaagac 1680

gagatcaagg cgtacaaaga acgttatgag gcggcgaacc tggtgccgtt tcgtgaatat 1740

ctggaagcgg agggtctgga gagcaagtac atcaaaggtg gaggtggatc gggatccgaa 1800

aacctttact tccaaggccc tcgttcaggc cgctttagca ttaaagcgaa gaactacttt 1860

ctgacctatc cgaaatgcga tctgaccaaa gaaaacgcgc tgagccagat taccaacctg 1920

cagaccccga ccaacaaact gttcatcaaa atttgccgcg aactgcatga aaacggcgaa 1980

ccgcatctgc atattctgat tcagttcgag ggcaaatata actgcaccaa ccagcgcttt 2040

tttgatctgg tgagccctac tcgtagcgcg cattttcatc cgaacattca gggcgcgaaa 2100

agcagcagcg atgtgaaaag ctacatcgat aaggatggcg atgtgctgga atggggcacc 2160

tttcagattg atggccgtct cgagcaccac caccaccacc actga 2205

<210> 10

<211> 171

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> nucleotide sequence of SPG

<400> 10

atgcagtaca agcttatcct gaacggtaaa accctgaaag gtgaaaccac caccgaagct 60

gttgacgctg ctaccgcgga aaaagttttc aaacagtacg ctaacgacaa cggtgttgac 120

ggtgaatgga cctacgacga cgctaccaaa accttcacgg taaccgagga t 171

<210> 11

<211> 600

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> nucleotide sequence of fusion protein SPG-TYLCV-C1

<400> 11

atgcagtaca agcttatcct gaacggtaaa accctgaaag gtgaaaccac caccgaagct 60

gttgacgctg ctaccgcgga aaaagttttc aaacagtacg ctaacgacaa cggtgttgac 120

ggtgaatgga cctacgacga cgctaccaaa accttcacgg taaccgagga tggtggaggt 180

ggatcgggat ccgaaaacct ttacttccaa ggccctcgtt caggccgctt tagcattaaa 240

gcgaagaact actttctgac ctatccgaaa tgcgatctga ccaaagaaaa cgcgctgagc 300

cagattacca acctgcagac cccgaccaac aaactgttca tcaaaatttg ccgcgaactg 360

catgaaaacg gcgaaccgca tctgcatatt ctgattcagt tcgagggcaa atataactgc 420

accaaccagc gcttttttga tctggtgagc cctactcgta gcgcgcattt tcatccgaac 480

attcagggcg cgaaaagcag cagcgatgtg aaaagctaca tcgataagga tggcgatgtg 540

ctggaatggg gcacctttca gattgatggc cgtctcgagc accaccacca ccaccactga 600

<210> 12

<211> 26

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> nucleotide sequence 12

<400> 12

cgtataatat taccggatgg ccgcgc 26

Claims

1. A method of covalently linking a nucleic acid and a peptide or protein, comprising covalently linking a nucleic acid of interest and a peptide or protein of interest using a TYLCV-C1 protein;

the TYLCV-C1 protein forms a fusion protein with the target peptide or protein;

ligating the sequence tatta or x-tata at the 5' end of the nucleic acid, x representing one or more nucleotide bases, prior to ligating the nucleic acid using the TYLCV-C1 protein;

the amino acid sequence of the TYLCV-C1 protein is shown in SEQ ID NO. 1.

2. The method of claim 1, wherein x is 1-30 nucleotide bases.

3. The method of claim 2, wherein the target nucleic acid is 5' linked to cgtataatatta sequences.

4. The method according to claim 3, wherein said TYLCV-C1 protein is linked to said peptide or protein of interest directly or via a flexible linker peptide.

5. The method according to claim 4, wherein said TYLCV-C1 protein is linked to said peptide or protein of interest via a flexible linker peptide to form a fusion protein.

Use of a TYLCV-C1 protein for the ligation of a peptide or protein of interest to a nucleic acid of interest; the amino acid sequence of the TYLCV-C1 protein is shown in SEQ ID NO. 1.

7. The use of claim 6, wherein the target nucleic acid is linked at its 5' end to a sequence tata or x-tata, wherein x represents one or more nucleotide bases.

8. The use of claim 6, wherein x is 1-30 nucleotide bases.

9. The use of claim 6, wherein the target nucleic acid is 5' linked to cgtataatatta sequences.

10. The use of claim 7, wherein said TYLCV-C1 protein forms a fusion protein with said peptide or protein of interest.

11. The use of claim 10, wherein said TYLCV-C1 protein is linked to said peptide or protein of interest directly or via a flexible linker peptide.

12. The use of claim 11, wherein the TYLCV-C1 protein is linked to the peptide or protein of interest via a flexible linker to form a fusion protein.

13. A complex comprising a protein of interest and a nucleic acid of interest, said protein of interest and said nucleic acid of interest being linked by catalysis by a TYLCV-C1 protein; the complex further comprises TYLCV-C1 protein; the target protein and the TYLCV-C1 protein form a fusion protein; the amino acid sequence of the TYLCV-C1 protein is shown in SEQ ID NO. 1.

14. A method of preparing the complex of claim 13, comprising the steps of:

the target protein was fused with TYLCV-C1 protein by gene recombination to form a fusion protein, and the fusion protein was mixed with the target nucleic acid.

15. The method of claim 14, further comprising adding a metal ion to the mixture of the fusion protein and the target nucleic acid.

16. The method of claim 15, wherein calcium ions and acetic acid are added to a mixture of the fusion protein and the target nucleic acid.