A fusion protein and its application

Abstract

The present invention provides a fusion protein and its application. The fusion protein comprises a histone binding module, a DNA methyltransferase 3α functional module, and a DNA methyltransferase 3β functional module. The fusion protein targets and binds to histone H3K27ac via the histone binding module, subsequently anchoring to cfDNA. The fusion protein then utilizes the DNA methyltransferase 3α and DNA methyltransferase 3β functional modules to catalyze the methylation of the 5' carbon position of cytosine in CpG sequences in cfDNA, thereby protecting the cfDNA through methylation.

Description

Technical Field

[0001] The present invention relates to the field of molecular biotechnology, and in particular to a fusion protein and applications thereof. Background Art

[0002] Cell-free DNA (cfDNA) refers to DNA fragments that are free from cells and range in length from 50 to 300 base pairs. It is primarily released into the extracellular environment of the human body through pathways such as apoptosis and necrosis, and is often found in physiological extracellular environments such as blood, lymph, milk, urine, and amniotic fluid. Currently, cfDNA testing is a common form of liquid biopsy on the market and has been widely used in various aspects, including tumor detection, guidance evaluation, and prognosis assessment. For example, testing tumor-derived cfDNA can reveal tumor-related mutations, heterozygous loss, gene amplification, oncoviral DNA, and hypermethylation of tumor suppressor gene promoter regions, thereby enabling non-invasive research on tumor DNA.

[0003] However, bodily fluids like plasma and serum contain large amounts of nucleases, such as deoxyribonuclease I (DNase I), which can easily cause rapid degradation of cfDNA, thus limiting the development of cfDNA testing. Therefore, developing a product that can stabilize cfDNA to prevent its inactivation by nucleases is an urgent challenge for those skilled in the art. Summary of the Invention

[0004] The present invention provides a fusion protein and a kit, which can stabilize cfDNA to prevent it from being inactivated by nucleases.

[0005] The present invention provides a recombinant nucleic acid molecule, a recombinant vector and a recombinant cell, which can produce the above fusion protein simply and quickly.

[0006] The present invention provides a use of the above-mentioned fusion protein, the above-mentioned recombinant nucleic acid molecule, the above-mentioned recombinant vector, the above-mentioned recombinant cell, and / or kit in enhancing the stability of cfDNA.

[0007] The present invention provides a fusion protein, which includes a histone binding module, a DNA methyltransferase 3α functional module and a DNA methyltransferase 3β functional module;

[0008] The amino acid sequence of the histone binding module is shown in SEQ ID NO: 1;

[0009] The amino acid sequence of the DNA methyltransferase 3α functional module is shown in SEQ ID NO: 2;

[0010] The amino acid sequence of the DNA methyltransferase 3β functional module is shown in SEQ ID NO: 3.

[0011] The fusion protein as described above, wherein the fusion protein further comprises a linker.

[0012] The fusion protein as described above, wherein the fusion protein is obtained by sequentially connecting the following modules from N-terminus to C-terminus: an N-terminal protection module, a histone binding module, a linker, a DNA methyltransferase 3α functional module, a linker, a DNA methyltransferase 3β functional module, and a histidine tag;

[0013] Wherein, the amino acid sequence of the N-terminal protection module is shown in SEQ ID NO: 4;

[0014] The amino acid sequence of the histone binding module is shown in SEQ ID NO: 1;

[0015] The amino acid sequence of the linker is shown in SEQ ID NO: 5;

[0016] The amino acid sequence of the DNA methyltransferase 3α functional module is shown in SEQ ID NO: 2;

[0017] The amino acid sequence of the DNA methyltransferase 3β functional module is shown in SEQ ID NO: 3;

[0018] The amino acid sequence of the histidine tag is shown in SEQ ID NO:6.

[0019] The fusion protein as described above, wherein the fusion protein has:

[0020] a. the amino acid sequence shown in SEQ ID NO: 7; or

[0021] b. a sequence in which one or more amino acids are substituted, deleted, added and / or replaced based on the amino acid sequence as shown in a; or

[0022] c. A sequence with 80% or more homology to the amino acid sequence shown in a or b.

[0023] The present invention provides a recombinant nucleic acid molecule, wherein the recombinant nucleic acid molecule encodes the above-mentioned fusion protein.

[0024] The present invention provides a recombinant vector, which includes the above-mentioned recombinant nucleic acid molecule.

[0025] The present invention provides a recombinant cell, comprising the above-mentioned recombinant nucleic acid molecule or the above-mentioned recombinant vector.

[0026] The present invention provides a kit comprising the above-mentioned fusion protein.

[0027] The kit as described above, wherein the kit further comprises at least one of S-adenosylmethionine and DNA methyltransferase.

[0028] The present invention provides a use of the above-mentioned fusion protein, the above-mentioned recombinant nucleic acid molecule, the above-mentioned recombinant vector, the above-mentioned recombinant cell, and / or the above-mentioned kit in enhancing the stability of cfDNA.

[0029] The present invention provides a fusion protein comprising a histone binding module, a DNA methyltransferase 3α functional module, and a DNA methyltransferase 3β functional module. This fusion protein targets and binds to histone H3K27ac via the histone binding module, subsequently anchoring to cfDNA. The DNA methyltransferase 3α and DNA methyltransferase 3β functional modules catalyze the methylation of the 5' carbon position of cytosine in CpG sequences in cfDNA, forming 5-methylcytosine. Methylation alters the spatial conformation and charge distribution of cfDNA. Nucleases (such as deoxyribonuclease I) require specific DNA structures or sequences to function. Methylation may interfere with their recognition sites, reducing the enzyme's binding affinity to DNA and thereby hindering nuclease degradation of cfDNA. Methylation also enhances the thermodynamic stability of the cfDNA duplex and raises its melting temperature, making it more resistant to nuclease cleavage. Methylation may also affect the interaction between cfDNA and histones or other binding proteins, indirectly forming a protective barrier, ultimately achieving a protective effect on cfDNA. DETAILED DESCRIPTION

[0030] In order to enable those skilled in the art to better understand the scheme of the present invention, the present invention is further described in detail below. The specific embodiments listed below are only for describing the principles and features of the present invention. The examples are only used to explain the present invention and are not intended to limit the scope of the present invention. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without making creative efforts are within the scope of protection of the present invention.

[0031] To improve the sensitivity of liquid biopsies, various technologies have been developed to enhance the stability of cell-free DNA (cfDNA). For example, DNA-binding monoclonal antibodies can specifically recognize and protect cfDNA, reducing nuclease degradation and cellular uptake. Engineering the Fc region of DNA-binding monoclonal antibodies to eliminate FcγR binding can further extend their circulation half-life and enhance cfDNA recovery. However, these approaches face sequence-dependent limitations. Another strategy involves direct inhibition of nuclease activity, but systemic inhibition may interfere with physiological processes such as DNA repair, limiting its clinical application. Therefore, the development of a biocompatible cfDNA protection technology that can fully cover the entire cfDNA population is crucial.

[0032] To solve the above problems, the present invention provides a fusion protein in a first aspect, comprising a histone binding module, a DNA methyltransferase 3α functional module and a DNA methyltransferase 3β functional module;

[0033] The amino acid sequence of the histone binding module is shown in SEQ ID NO: 1;

[0034] The amino acid sequence of the DNA methyltransferase 3α functional module is shown in SEQ ID NO: 2;

[0035] The amino acid sequence of the DNA methyltransferase 3β functional module is shown in SEQ ID NO: 3.

[0036] The histone-binding module is a functional region of the Bromo1 domain of human bromodomain-containing protein 4 (from amino acids 60 to 160 of the Bromo1 domain), and its amino acid sequence is shown in SEQ ID NO: 1. Bromodomain-containing protein 4 (Brd4) is a member of the bromodomain and extraterminal domain (Bet) family and can recognize and bind to acetylated histones through its two bromodomains (Bromo1 and Bromo2). Therefore, the histone-binding module can recognize and bind to acetylated histones.

[0037] Specifically, the Bromo1 domain of Brd4 has an "aromatic cage" structure (composed of tryptophan, phenylalanine, tyrosine, etc.) that can accommodate the acetyl group (-COCH3) of acetylated lysine, forming hydrogen bonds and hydrophobic interactions. Furthermore, its affinity for H3K27ac is significantly higher than that for non-acetylated or methylated H3K27. Furthermore, H3K27 is located at the tail of histone H3 and, after acetylation, is exposed on the nucleosome surface, facilitating the binding of the Bromo1 domain of Brd4. Therefore, the histone binding module can target the acetylation modification of lysine residue 27 on histone H3 (H3K27ac).

[0038] H3K27ac is a marker of actively transcribed regions. This modification typically occurs in transcriptionally active regions, such as gene promoters and enhancers. These regions have loose chromatin structures, making DNA more susceptible to release as cfDNA. Furthermore, following cell apoptosis or necrosis, cfDNA is more likely to originate from transcriptionally active chromatin regions. Therefore, the distribution of H3K27ac closely overlaps with the release sites of cfDNA.

[0039] After targeting and binding to histone H3K27ac, the fusion protein of the present invention anchors cfDNA, and then it can promote cfDNA methylation, thereby stabilizing cfDNA.

[0040] The function of promoting cfDNA methylation is achieved by the DNA methyltransferase 3α and DNA methyltransferase 3β functional modules in the fusion protein. The DNA methyltransferase 3α module is a functional region of DNA methyltransferase 3α (DNMT3A), including the catalytic core, and its amino acid sequence is shown in SEQ ID NO:2. The DNA methyltransferase 3β module is a functional region of DNA methyltransferase 3β (DNMT3B), including the catalytic core, and its amino acid sequence is shown in SEQ ID NO:3. DNA methyltransferases (DNMTs) are enzymes that play a key role in DNA methylation, adding a methyl group to the 5' carbon position of cytosine in DNA CpG sequences to form 5-methylcytosine. DNMT3A and DNMT3B are members of the DNA methyltransferase family and catalyze de novo DNA methylation, which involves the addition of a methyl group to the fifth carbon atom of cytosine within a DNA sequence.

[0041] On the one hand, DNMT3A or DNMT3B has a strong binding affinity for substrate DNA and a fast catalytic rate, especially in regions rich in CpG islands, and can quickly complete the methylation modification of cfDNA, while the activity of other methylases (such as DNMT1) may be limited in an in vitro cell-free environment.

[0042] On the other hand, the histone binding module of the fusion protein of the present invention can guide DNMT3A or DNMT3B to the histone binding site near cfDNA, and DNMT3A or DNMT3B itself is often bound to chromatin in cells. The spatial positioning of the two is matched, which can enhance the accuracy of methylation modification. Other methylases (such as cytoplasmic-localized methylases) lack this feature.

[0043] Furthermore, DNMT3A and DNMT3B are "de novo methyltransferases," primarily performing initial methylation modifications (i.e., "de novo methylation") on unmethylated CpG sequences during DNA replication or when a methylation template is unavailable. Other methylases, such as DNA methyltransferase 1 (DNMT1), are "maintenance methyltransferases," capable of only continuous modification based on the parent strand's methylation pattern after DNA replication and unable to initiate methylation in the absence of a template. cfDNA comes from diverse sources (e.g., apoptotic and necrotic cells), with complex methylation states and the potential lack of a complete maintenance methylation template. Therefore, the de novo methylation capabilities of DNMT3A and DNMT3B allow them to directly modify CpG sequences in various cfDNA types without relying on pre-existing methylation patterns, making them more suitable for broad-spectrum protection. Furthermore, under specific conditions, DNMT3A and DNMT3B can also methylate non-CpG sequences (e.g., CpA and CpT). Although this efficiency is lower, it may further expand the scope of cfDNA protection, a capability rarely achieved by other methylases (e.g., DNMT1).

[0044] The present invention has experimentally found that although DNMT3A and DNMT3B have high sequence similarity and domain structure, and have significant functional overlap in CpG and non-CpG DNA methylation processes, in the present invention, the fusion protein must include both the DNA methyltransferase 3α functional module and the DNA methyltransferase 3β functional module to achieve effective protection of cfDNA. The reasons may be as follows:

[0045] (1) The simultaneous use of DNMT3A and DNMT3B has functional complementarity and can cover a wider range of methylation sites and scenarios. DNMT3A prefers to methylate promoter regions rich in CpG islands, especially in embryonic stem cells and differentiated cells, where it plays a key role in maintaining gene silencing; DNMT3B is better at methylating satellite repeat sequences and DNA in the pericentromere region, and plays a significant role in chromosome stability and heterochromatin formation. cfDNA has a complex source, and its DNA sequence includes multiple regions such as promoter regions and repeat sequences. The simultaneous use of DNMT3A and DNMT3B can cover a wider range of substrate types and provide more comprehensive methylation modifications to cfDNA; the simultaneous use of DNMT3A and DNMT3B can also increase the number of methylation sites per unit cfDNA (such as modifying promoters and repeat sequences at the same time), forming a denser methylation barrier that effectively blocks the recognition and cleavage of nucleases.

[0046] (2) DNMT3A is continuously expressed in a variety of somatic cells, while DNMT3B expression is more limited to specific tissues (such as immune cells and placenta) or is upregulated in pathological conditions (such as tumors). Combined use can adapt to the methylation requirements of cfDNA from different sources. For example, when simultaneously processing cfDNA released by tumors and normal cells, the two can act on their respective preferred sequences.

[0047] (3) DNMT3A and DNMT3B can form heterodimers through the C-terminal catalytic domain, and their catalytic activity is significantly higher than that of the single homodimer.

[0048] The fusion protein provided by the present invention targets and binds to histone H3K27ac via its histone binding module, subsequently anchoring to cfDNA. It then utilizes the DNA methyltransferase 3α and DNA methyltransferase 3β functional modules to catalyze the methylation of the 5' carbon position of cytosines within CpG sequences in cfDNA, forming 5-methylcytosine. This methylation alters the spatial conformation and charge distribution of cfDNA. Nucleases (such as deoxyribonuclease I) require specific DNA structures or sequences to function. Methylation may interfere with their recognition sites, reducing the enzyme's binding affinity for DNA and thus hindering nuclease degradation of cfDNA. Methylation also enhances the thermodynamic stability of the cfDNA duplex, raising its melting temperature and making it less susceptible to nuclease degradation. Furthermore, methylation may affect the interaction of cfDNA with histones or other binding proteins, indirectly forming a protective barrier.

[0049] The present invention found through in vitro nuclease degradation experiments that the fusion protein provided by the present invention can indeed effectively inhibit the degradation of cfDNA caused by nucleases and significantly improve the stability of cfDNA.

[0050] In the above technical solution, the fusion protein also includes a linker.

[0051] A linker is an amino acid chain that connects the two building blocks of a fusion protein. It possesses a degree of flexibility to allow the proteins on either side to perform their respective functions. Linkers are an essential component of fusion proteins and play a crucial role in constructing stable, biologically active fusion proteins.

[0052] Specifically, the linker of the present invention is (Gly4Ser)3, whose amino acid sequence is shown in SEQ ID NO: 5, which can effectively enhance the degree of freedom between modules.

[0053] In the above technical solution, the fusion protein is obtained by serially connecting the following modules from N-terminus to C-terminus: N-terminal protection module, histone binding module, linker, DNA methyltransferase 3α functional module, linker, DNA methyltransferase 3β functional module, and histidine tag;

[0054] Wherein, the amino acid sequence of the N-terminal protection module is shown in SEQ ID NO: 4;

[0055] The amino acid sequence of the histone binding module is shown in SEQ ID NO: 1;

[0056] The amino acid sequence of the linker is shown in SEQ ID NO: 5;

[0057] The amino acid sequence of the DNA methyltransferase 3α functional module is shown in SEQ ID NO: 2;

[0058] The amino acid sequence of the DNA methyltransferase 3β functional module is shown in SEQ ID NO: 3;

[0059] The amino acid sequence of the histidine tag is shown in SEQ ID NO:6.

[0060] The first to 20 amino acid positions of the N-terminal protection module are a signal peptide with the amino acid sequence MVSQALRLLCLLLGLQGCLA; the signal peptide can guide the fusion protein to be secreted outside the cell through the endoplasmic reticulum-Golgi apparatus pathway, thereby improving the expression efficiency and solubility of the fusion protein. The 21st to 26th amino acid positions of the N-terminal protection module are a histidine tag, which can be used for purification of the fusion protein. The 30th amino acid position of the N-terminal protection module is leucine L, the 31st amino acid position is valine V, and the 32nd amino acid position is proline P; the peptide segment LVP can form a hydrophobic core. In addition, the N-terminal protection module resists cleavage by proteases in vivo and in vitro through steric hindrance and a specific amino acid sequence, thereby extending the half-life of the fusion protein.

[0061] The histidine tag (His-tag) is the simplest and most widely used purification tag, which has six consecutive histidine residues. Therefore, in the present invention, a histidine tag is added to the C-terminus of the fusion protein to improve the purification convenience of the fusion protein.

[0062] After long-term experimental exploration, the present invention found that by limiting the component modules and arrangement order of the fusion protein, the fusion protein obtained can ensure that the fusion protein can function, and inhibit the degradation of cfDNA caused by nucleases to the greatest extent, and significantly improve the stability of cfDNA.

[0063] Furthermore, the fusion protein may have:

[0064] a. the amino acid sequence shown in SEQ ID NO: 7; or

[0065] b. a sequence in which one or more amino acids are substituted, deleted, added and / or replaced based on the amino acid sequence as shown in a; or

[0066] c. A sequence with 80% or more homology to the amino acid sequence shown in a or b.

[0067] Based on the above research, the second aspect of the present invention provides a recombinant nucleic acid molecule, which encodes the above fusion protein.

[0068] The third aspect of the present invention provides a recombinant vector comprising the above-mentioned recombinant nucleic acid molecule.

[0069] The recombinant vector of the present invention can be a recombinant plasmid. Plasmids have the advantages of stability, reliability, and ease of operation. Therefore, a recombinant plasmid can be constructed by incorporating the aforementioned recombinant nucleic acid molecule into a plasmid. The resulting recombinant plasmid, like a plasmid, has the ability to self-replicate and maintain a constant copy number in daughter cells.

[0070] The present invention does not limit the method for recombining the recombinant nucleic acid molecule into the plasmid, and those skilled in the art can choose one according to their needs.

[0071] A fourth aspect of the present invention provides a recombinant cell, wherein the recombinant cell comprises the above-mentioned recombinant nucleic acid molecule or the above-mentioned recombinant vector.

[0072] In the present invention, recombinant cells are obtained by introducing a recombinant nucleic acid molecule or a recombinant vector carrying the recombinant nucleic acid molecule into cells to allow the cells to express a fusion protein. The methods for constructing and culturing the recombinant cells are simple and easy, and the entire process is easily controlled by humans, enabling stable production of large quantities of fusion protein.

[0073] The present invention does not limit the method for transforming the recombinant vector into the recombinant cell, and those skilled in the art can select the method as needed. For example, the recombinant vector can be introduced into the recombinant cell by heat shock transformation.

[0074] The fifth aspect of the present invention provides a kit comprising the above-mentioned fusion protein.

[0075] The above kit can be used for cfDNA extraction or for cfDNA library construction to maintain the stability of cfDNA during extraction or library construction.

[0076] Furthermore, the above-mentioned kit may also include other reagents for cfDNA extraction or for cfDNA library construction; for example, including DNA polymerase, PCR buffer, etc. for cfDNA library construction.

[0077] In a specific embodiment, the above kit further includes at least one of S-adenosylmethionine and DNA methyltransferase.

[0078] S-Adenosylmethionine (SAM), a compound produced by the enzyme adenosylmethioninase from methionine and adenosine triphosphate (ATP), is a physiologically active substance present in all human tissues and fluids. It acts as a methyl group donor (transmethylation) and participates in important biochemical reactions in the body. DNA methyltransferase 3-like (DNMT3L) is a catalytically inactive cofactor that can complex with DNMT3A and DNMT3B, enhancing their catalytic activity.

[0079] In a sixth aspect, the present invention provides a use of the above-mentioned fusion protein, the above-mentioned recombinant nucleic acid molecule, the above-mentioned recombinant vector, the above-mentioned recombinant cell, and / or the above-mentioned kit in enhancing the stability of cfDNA.

[0080] The aforementioned fusion protein, when mixed with cfDNA, can promote cfDNA methylation, thereby enhancing or improving cfDNA stability. Furthermore, the aforementioned recombinant nucleic acid molecules, recombinant vectors, or recombinant cells can be used to produce the aforementioned fusion protein, thereby enhancing cfDNA stability. It is understood that the aforementioned kit, which includes the aforementioned fusion protein, can also be used to enhance cfDNA stability.

[0081] The technical solutions of this application are further explained below with reference to specific examples. Experimental methods in the following examples, where specific conditions are not specified, were generally performed under conventional conditions or as recommended by the manufacturer. All reagents used, unless otherwise specified, were commercially available or publicly available.

[0082] Example 1:

[0083] This example provides a fusion protein comprising, from N-terminus to C-terminus, an N-terminal protection module, a histone binding module, a linker, a DNA methyltransferase 3α functional module, a linker, a DNA methyltransferase 3β functional module, and a histidine tag; its amino acid sequence is shown in Table 1. Production and purification of this fusion protein were performed by Sino Biological, resulting in a 200 µL volume and a concentration of 0.67 mg / mL.

[0084] Table 1

[0085]

[0086]

[0087]

[0088] Example 2:

[0089] To evaluate the protective effect of the above fusion protein and the methylation generated by the fusion protein on cfDNA, an in vitro nuclease degradation experiment was performed using the fusion protein in Example 1. The specific steps are as follows:

[0090] Plasma samples were collected from five healthy volunteers as biological replicates. Each volunteer collected 4 mL of plasma and aliquoted into four groups (1 mL each). In the first group, 1 μL of 0.67 mg / mL fusion protein, 1 μL of 10 mM S-adenosylmethionine (SAM), and 1 μL of 50 ng / μL DNA methyltransferase 3 like (DNMT3L) were added to each 1 mL of plasma. The mixture was then incubated at 37°C for 3 hours. In the second group, 1 μL of 10 mM SAM and 1 μL of 50 ng / μL DNMT3L were added to each 1 mL of plasma. The mixture was then incubated at 37°C for 3 hours. Groups 3 and 4 were incubated at 37°C for 3 hours without the addition of any reagents. After the incubation period, 10 μL of 0.5 mg / mL recombinant human deoxyribonuclease I (DNase I) was added to Groups 1, 2, and 3. Group 4 was incubated at 37°C for 1 hour without any reagent. Following the incubation period, cfDNA was extracted from the plasma samples of Groups 1, 2, 3, and 4 using a plasma cell-free DNA (cfDNA) extraction kit produced by Kaishuo Biotechnology. The total amount of cfDNA (100-200 bp) was calculated using a fluorescence quantification instrument and capillary electrophoresis. See Table 2 for details.

[0091] SAM is a compound produced by the enzyme adenosylmethioninase from methionine and adenosine triphosphate (ATP). It is a physiologically active substance present in all human tissues and fluids, acting as a methyl group donor (transmethylation) in important biochemical reactions. DNMT3L is a catalytically inactive cofactor that can complex with DNMT3A and DNMT3B, enhancing their catalytic activity. That is, the first group contained the fusion protein, SAM that provided methyl groups, and DNMT3L that assisted the methyltransferase, and a nuclease degradation experiment was performed, which was the experimental group; the second group did not have the fusion protein, only the SAM that provided methyl groups, and DNMT3L that assisted the methyltransferase, and a nuclease degradation experiment was performed, which was the experimental control group 1; the third group did not have the fusion protein, SAM that provided methyl groups, and DNMT3L that assisted the methyltransferase, and a nuclease degradation experiment was performed, which was the experimental control group 2; the fourth group did not have the fusion protein, SAM that provided methyl groups, and DNMT3L that assisted the methyltransferase, and no nuclease degradation experiment was performed, which was the blank control group.

[0092] Table 2

[0093]

[0094] According to Table 2, the cfDNA levels in the second and third groups were significantly lower, with average values ​​of 0.76 ng and 0.70 ng, respectively. The total cfDNA level in the first group was significantly increased, with an average value of 4.26 ng. The total cfDNA level in the fourth group was the highest, with an average value of 5.48 ng. These results demonstrate that the fusion protein provided by the present invention can target cfDNA bound to histones in plasma and, in the presence of SAM and DNMT3L, promote methylation of cfDNA, thereby protecting cfDNA from damage caused by nucleases. This fusion protein can inhibit nuclease-induced cfDNA degradation in vitro and has the potential for in vivo application.

[0095] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the above embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the above embodiments, or replace some or all of the technical features therein with equivalents. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A fusion protein, characterized in that The amino acid sequence of the fusion protein is shown in SEQ ID NO:

7.

2. A recombinant nucleic acid molecule, characterized in that The recombinant nucleic acid molecule encodes the fusion protein of claim 1.

3. A recombinant vector, characterized in that Comprising the recombinant nucleic acid molecule of claim 2.

4. A recombinant cell, characterized in that Comprising the recombinant nucleic acid molecule according to claim 2, or the recombinant vector according to claim 3.

5. A kit, characterized in that Comprising the fusion protein of claim 1.

6. The kit according to claim 5, characterized in that The kit further comprises at least one of S-adenosylmethionine and DNA methyltransferase.

7. Use of the fusion protein of claim 1, the recombinant nucleic acid molecule of claim 2, the recombinant vector of claim 3, the recombinant cell of claim 4, and / or the kit of claim 5 or 6 in enhancing cfDNA stability.

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