Efficient expression and use of human-derived enamel matrix protein

Abstract

Provided are efficient expression and a use of a human-derived enamel matrix protein. Further provided is a gene for expression of a heterologous enamel matrix protein, comprising an X-type gene and / or a Y-type gene. The gene for expression of the heterologous enamel matrix protein can heterologously express an enamel matrix protein, which has properties similar to those of a human-derived enamel matrix protein, is not easy to fold in space, and is easy to purify; the product has high uniformity and high stability; and the industrial production of enamel matrix proteins is achieved.

Description

High-efficiency expression and application of human enamel matrix protein Technical Field

[0001] The present invention belongs to the field of bioengineering technology, and specifically relates to a heterologous enamel matrix protein, and its encoding gene, expression method and application. Background Art

[0002] Tooth enamel is the hardest tissue in the human body, covering the surface of tooth crowns and composed of 96% hydroxyapatite, 3% water, and less than 1% organic matter. Enamel matrix proteins are secreted by ameloblasts during enamel development and regulate the mineralization and maturation of enamel. The accumulation of enamel matrix proteins forms enamel crystals, gradually thickening the enamel layer. Ameloblasts migrate as the enamel thickens. Enamel matrix proteins play a crucial role in enamel formation and are therefore a common research topic in periodontal therapy.

[0003] In 1997, Swedish company Biora successfully developed a porcine enamel matrix protein drug, which received FDA approval in 1999 and entered clinical use. This drug is a gel-like compound of porcine amelogenin and propylene glycol alginate. Studies have shown that it effectively promotes periodontal tissue regeneration and slows gingival atrophy. Osteogenesis and the proliferation and migration of periodontal ligament cells have also been observed.

[0004] Enamel matrix proteins are only present in enamel during embryonic development. As the enamel matures and ameloblasts gradually disappear, the enamel matrix proteins are hydrolyzed by proteases until they disappear completely. This means that enamel matrix proteins can only be extracted from tooth germs, making human sources difficult to source. Pig and bovine tooth germs are widely used because they contain a large volume of enamel. Rat teeth are also widely used as extraction materials because they contain enamel matrix proteins from different stages of development within a single tooth and are relatively inexpensive. Despite this, the low extraction yield still makes the source of enamel matrix proteins a problem. Furthermore, due to differences in experimental animals and extraction methods, the content and quality of the extracted enamel matrix proteins vary, often compromising the accuracy and reliability of research results. The lack of sufficient research materials and the uneven quality of these materials have hindered the progress of enamel matrix protein research in recent years.

[0005] The gene sequences associated with human enamel matrix proteins are genetically conserved, making the development of heterologous enamel matrix proteins possible. However, industrial-grade enamel matrix proteins are currently lacking. This is primarily because, upon expression, enamel matrix proteins accumulate to a certain level and fold, rendering the His tag unrecognizable and difficult to remove via elution. Therefore, the current method primarily involves resuspending engineered bacteria expressing enamel matrix proteins in 3% acetic acid and then purifying and isolating the enamel matrix proteins. However, this method presents certain challenges in industrialization, making it difficult to achieve industrial production of enamel matrix proteins.

[0006] Summary of the Invention

[0007] To address the shortcomings of the prior art, the present invention provides a heterologously expressed enamel matrix protein that has properties similar to those of human enamel matrix protein, is less prone to spatial folding, is easily purified, and offers high product homogeneity and stability, enabling the industrialized production of enamel matrix protein. The present invention also provides a gene for expressing the aforementioned heterologous enamel matrix protein, enabling heterologous expression of the enamel matrix protein. The present invention also provides a method for expressing the aforementioned heterologous enamel matrix protein, enabling the industrialized production of the enamel matrix protein.

[0008] To this end, one aspect of the present invention provides an enamel matrix protein, which includes an X-type enamel matrix protein and / or a Y-type enamel matrix protein; wherein the X-type enamel matrix protein has the amino acid sequence shown in SEQ ID NO: 3 or the amino acid sequence shown in SEQ ID NO: 3 after one or more amino acids are added, deleted, replaced or modified, and the Y-type enamel matrix protein has the amino acid sequence shown in SEQ ID NO: 4 or the amino acid sequence shown in SEQ ID NO: 4 after one or more amino acids are added, deleted, replaced or modified.

[0009] Another aspect of the present invention provides a nucleic acid molecule encoding the enamel matrix protein of the present invention.

[0010] In some embodiments, the nucleic acid molecule has the nucleotide sequence shown in SEQ ID NO:1 and / or SEQ ID NO:2, or has a sequence having a sequence identity of more than 80%, more than 85%, more than 90%, more than 92%, more than 95%, more than 96%, more than 97%, more than 98%, more than 99%, or more than 99.5% to the nucleotide sequence shown in SEQ ID NO:1 and / or SEQ ID NO:2.

[0011] In some embodiments, in the nucleic acid molecule, the nucleotide sequence shown in SEQ ID NO: 1 or a sequence having a sequence identity of 80% or more thereto encodes an X-type enamel matrix protein.

[0012] In some embodiments, in the nucleic acid molecule, the nucleotide sequence shown in SEQ ID NO: 2 or a sequence having a sequence identity of 80% or more thereto encodes a Y-type enamel matrix protein.

[0013] Another aspect of the present invention provides a recombinant expression vector comprising the nucleic acid molecule of the present invention.

[0014] Another aspect of the present invention provides a host cell, wherein the host cell contains the nucleic acid molecule or recombinant expression vector of the present invention.

[0015] Another aspect of the present invention provides a method for preparing enamel matrix protein, the method comprising the following steps:

[0016] 1) introducing the nucleic acid molecule of the present invention into a plasmid to obtain a recombinant plasmid;

[0017] 2) transforming the recombinant plasmid into host cells, culturing and inducing expression, and purifying to obtain enamel matrix protein.

[0018] In some embodiments, in step 1), the nucleic acid molecule further comprises a nucleotide sequence encoding a tag.

[0019] In some embodiments, the nucleic acid molecule includes a nucleotide sequence encoding at least one His tag, for example, six His tags. The insertion of multiple His tags in the present invention can significantly improve recognition rates, helping to avoid situations where partial folding of the protein structure renders the tag unrecognizable, making purification and separation difficult.

[0020] In some embodiments, the nucleic acid molecule to which the His tag is added has a nucleotide sequence as shown in SEQ ID NO: 5 and / or SEQ ID NO: 7, or a sequence having a sequence identity of more than 80%, more than 85%, more than 90%, more than 92%, more than 95%, more than 96%, more than 97%, more than 98%, more than 99%, or more than 99.5% to the nucleotide sequence as shown in SEQ ID NO: 5 and / or SEQ ID NO: 7.

[0021] In some embodiments, in the nucleic acid molecule, the nucleotide sequence shown in SEQ ID NO: 5 or a sequence having more than 80% sequence identity thereto encodes the protein shown in SEQ ID NO: 6.

[0022] In some embodiments, in the nucleic acid molecule, the nucleotide sequence shown in SEQ ID NO: 7 or a sequence having greater than 80% sequence identity thereto encodes the protein shown in SEQ ID NO: 8.

[0023] In some embodiments, in step 1), the plasmid is PET28a(+).

[0024] In some embodiments, in step 2), the host cell is Escherichia coli.

[0025] In some embodiments, the temperature for inducing expression is 15-34° C., preferably 30-34° C. In some specific embodiments, the temperature for inducing expression is 15° C., 16° C., 18° C., 20° C., 22° C., 24° C., 26° C., 28° C., 30° C., 32° C., 34° C. or any value therebetween.

[0026] In some embodiments, the time for inducing expression is 3-16 hours, preferably 3-10 hours. In some specific embodiments, the time for inducing expression is 3 hours, 4 hours, 6 hours, 8 hours, 10 hours, 12 hours, 14 hours, 16 hours or any value therebetween.

[0027] In some embodiments, the purification includes: balancing a Ni chromatography column with a binding buffer, loading a protein solution obtained by inducing expression and lysing the host cells, removing impurity proteins with a washing buffer, eluting with an elution buffer, and collecting the eluted product.

[0028] In some embodiments, the binding buffer is a PB buffer system having a working concentration of 8-12 mM and a pH of 7.0-8.0, and is supplemented with 4-6 mM imidazole and 140-160 mM sodium chloride. In some specific embodiments, in the binding buffer, the working concentration of the PB buffer system can be 8 mM, 9 mM, 10 mM, 11 mM, 12 mM, or any value therebetween, the pH can be 7.0, 7.2, 7.4, 7.6, 7.8, 8.0, or any value therebetween, the concentration of imidazole can be 4 mM, 4.5 mM, 5 mM, 5.5 mM, 6 mM, or any value therebetween, and the concentration of sodium chloride can be 140 mM, 145 mM, 150 mM, 155 mM, 160 mM, or any value therebetween.

[0029] In some embodiments, the wash buffer is a PB buffer system having a working concentration of 8-12 mM and a pH of 7.0-8.0, and is supplemented with 50-100 mM imidazole and 140-160 mM sodium chloride. In some specific embodiments, in the wash buffer, the working concentration of the PB buffer system can be 8 mM, 9 mM, 10 mM, 11 mM, 12 mM, or any value therebetween, the pH can be 7.0, 7.2, 7.4, 7.6, 7.8, 8.0, or any value therebetween, the concentration of imidazole can be 50 mM, 60 mM, 70 mM, 80 mM, 90 mM, 100 mM, or any value therebetween, and the concentration of sodium chloride can be 140 mM, 145 mM, 150 mM, 155 mM, 160 mM, or any value therebetween.

[0030] In some embodiments, the elution buffer is a PB buffer system having a working concentration of 8-12 mM and a pH of 7.0-8.0, and is supplemented with 150-250 mM imidazole and 140-160 mM sodium chloride. In some specific embodiments, in the elution buffer, the working concentration of the PB buffer system can be 8 mM, 9 mM, 10 mM, 11 mM, 12 mM, or any value therebetween, the pH can be 7.0, 7.2, 7.4, 7.6, 7.8, 8.0, or any value therebetween, the concentration of imidazole can be 150 mM, 175 mM, 200 mM, 225 mM, 250 mM, or any value therebetween, and the concentration of sodium chloride can be 140 mM, 145 mM, 150 mM, 155 mM, 160 mM, or any value therebetween.

[0031] In some specific embodiments, in the binding buffer, the working concentration of the PB buffer system is 10 mM, the pH value is 7.5, and 5 mM imidazole and 150 mM sodium chloride are added; in the wash buffer, the working concentration of the PB buffer system is 10 mM, the pH value is 7.5, and 80 mM imidazole and 150 mM sodium chloride are added; in the elution buffer, the working concentration of the PB buffer system is 10 mM, the pH value is 7.5, and 250 mM imidazole and 150 mM sodium chloride are added.

[0032] Another aspect of the present invention provides the use of the enamel matrix protein, nucleic acid molecule, recombinant expression vector, host cell or method for preparing enamel matrix protein of the present invention in the preparation of enamel matrix protein drugs.

[0033] Compared with the prior art, the present invention has the following beneficial effects:

[0034] (1) The enamel matrix protein of the present invention can be heterologously expressed, has properties similar to those of human enamel matrix protein, is not prone to spatial folding, is easy to purify, and has high product uniformity and stability.

[0035] (2) The method for expressing heterologous enamel matrix protein of the present invention is to introduce the enamel matrix protein gene into a plasmid, and then transform the plasmid into a host bacterium for expression to obtain the enamel matrix protein, which can realize the industrial production of enamel matrix protein. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] FIG1 shows the electrophoresis results of protein expression before and after induction of the fermentation bacteria in Example 2 of the present invention.

[0037] FIG2 shows the SDS-PAGE electrophoresis results of the purified heterologous enamel matrix protein obtained in Example 3 of the present invention. DETAILED DESCRIPTION

[0038] The following examples illustrate the present invention, but the present invention is not limited to these examples. Modifications to the specific embodiments of the present invention or equivalent replacements of some technical features without departing from the spirit of the present invention should be included in the scope of the technical solution claimed in the present invention.

[0039] As used herein with respect to amino acids, the term "substitution" refers to the replacement of at least one amino acid residue in an amino acid sequence with another, different, "replacement" amino acid residue. As used herein with respect to amino acids, the term "insertion" refers to the incorporation of at least one additional amino acid into an amino acid sequence. Although indels typically consist of the insertion of one or two amino acid residues, larger "peptide indels" can also be prepared, for example, insertions of about three to five, or even up to about ten, fifteen, or twenty amino acid residues. As disclosed above, the inserted residues can be naturally occurring or non-naturally occurring. As used herein with respect to amino acids, the term "deletion" refers to the removal of at least one amino acid residue from an amino acid sequence.

[0040] The enamel matrix proteins of the present invention may comprise conservative amino acid substitutions at one or more amino acid residues, for example, at essential or non-essential amino acid residues. A "conservative amino acid substitution" is a substitution of an amino acid residue with an amino acid residue having a similar side chain. Families of amino acid residues with similar side chains have been defined in the art and include basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), non-polar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), β-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Therefore, in the present invention, an essential or non-essential amino acid residue in an enamel matrix protein is preferably substituted with another amino acid residue from the same side chain family.

[0041] "Percent sequence identity" or "percent identity" between two polynucleotide or polypeptide sequences refers to the number of identical matched positions shared by the sequences over the comparison window, taking into account additions or deletions (i.e., gaps) that must be introduced for optimal alignment of the two sequences. A matched position is any position where the same nucleotide or amino acid is present in both the target sequence and the reference sequence. Since a gap is not a nucleotide or amino acid, gaps present in the target sequence are not counted. Similarly, since target sequence nucleotides or amino acids are counted and nucleotides or amino acids from the reference sequence are not counted, gaps present in the reference sequence are not counted.

[0042] Percentage sequence identity can be calculated by the following process: determine that the number of positions where identical amino acid residues or nucleic acid bases occur in both sequences, to obtain the number of matched positions, the number of matched positions divided by the total number of positions in the comparison window, and multiply the result by 100, to obtain percent sequence identity. The determination of percent sequence identity between the comparison of a sequence and two sequences can be accomplished using software that is easy to use online and download. Suitable software programs can be obtained from various sources for the comparison of protein and nucleotide sequences. A suitable program for determining percent sequence identity is bl2seq, which is a part for the BLAST suite of programs that can be obtained from the BLAST website (blast.ncbi.nlm.nih.gov) of the National Center for Biotechnology Information of the U.S. Government. Bl2seq uses BLASTN or BLASTP algorithms to compare between two sequences. BLASTN is used for comparing nucleic acid sequences, and BLASTP is used for comparing amino acid sequences. Other suitable programs are, for example, Needle, Stretcher, Water or Matcher, part of the EMBOSS suite of bioinformatics programs and also available from the European Bioinformatics Institute (EBI) at www.ebi.ac.uk / Tools / psa.

[0043] To achieve industrialized production of enamel matrix protein, the inventors found enamel matrix protein-related genes and DNA sequences from NCBI. After analyzing the DNA sequences, they found that enamel matrix protein-related genes are located on sex chromosomes and are divided into X-type and Y-type. Based on this, they further analyzed the gene sequences on the allelic chromosomes to identify conserved sequences. After codon optimization of the sequences, they obtained the gene for heterologous enamel matrix protein expression of the present invention.

[0044] In some embodiments, the gene for expressing heterologous enamel matrix proteins of the present invention comprises an X-type gene and / or a Y-type gene; the nucleotide sequence of the X-type gene is shown in SEQ ID NO: 1, and the nucleotide sequence of the Y-type gene is shown in SEQ ID NO: 2.

[0045] Furthermore, the enamel matrix protein includes X-type enamel matrix protein and / or Y-type enamel matrix protein; the amino acid sequence of the X-type enamel matrix protein is shown in SEQ ID NO: 3, and the amino acid sequence of the Y-type enamel matrix protein is shown in SEQ ID NO: 4.

[0046] In some embodiments, the method for expressing a heterologous enamel matrix protein of the present invention comprises the following steps:

[0047] 1) Tagging the gene for expressing heterologous enamel matrix protein and introducing it into the plasmid to obtain a recombinant plasmid;

[0048] 2) transforming the recombinant plasmid into host cells, culturing and inducing expression, and purifying to obtain enamel matrix protein.

[0049] Furthermore, in step 1), the tag includes a His tag.

[0050] Furthermore, in step 1), the nucleotide sequence of the tagged X-type gene is shown in SEQ ID NO: 5, and the amino acid sequence of the protein expressed thereby is shown in SEQ ID NO: 6; and / or,

[0051] The nucleotide sequence of the tagged Y-type gene is shown in SEQ ID NO: 7, and the amino acid sequence of the protein expressed by it is shown in SEQ ID NO: 8.

[0052] Furthermore, in step 1), the host bacteria is Escherichia coli, and the plasmid is PET28a(+).

[0053] Furthermore, in step 2), the specific purification operation includes: balancing the Ni chromatography column with a binding buffer, then loading the protein solution obtained by expressing and lysing the host bacteria, removing the impurity proteins adsorbed on the Ni chromatography column with a washing buffer, and then eluting with an elution buffer, collecting the eluted product, and obtaining the enamel matrix protein.

[0054] Furthermore, the binding buffer is a PB buffer system with a working concentration of 8-12 mM, a pH value of 7.0-8.0, and is added with 4-6 mM imidazole and 140-160 mM sodium chloride.

[0055] The washing buffer is a PB buffer system with a working concentration of 8-12 mM and a pH value of 7.0-8.0, and is added with 50-100 mM imidazole and 140-160 mM sodium chloride.

[0056] The elution buffer is a PB buffer system with a working concentration of 8-12 mM and a pH value of 7.0-8.0, and is added with 150-250 mM imidazole and 140-160 mM sodium chloride.

[0057] Furthermore, in step 2), the temperature for inducing expression is 15-34° C., and the induction time is 3-16 h.

[0058] In this example, no specific techniques or conditions are specified, and the operations were performed according to conventional techniques and instrument specifications in the art. All reagents or instruments used without specifying the manufacturer are conventional products that can be purchased commercially.

[0059] Example 1 Preparation of enamel matrix protein gene

[0060] We found enamel matrix protein-related genes and DNA sequences from NCBI. We analyzed the DNA sequences and found that enamel matrix protein-related genes were located on sex chromosomes. We analyzed the gene sequences on the allelic chromosomes to identify conserved sequences. After codon optimization, we obtained a gene for heterologous enamel matrix protein expression, including X-type and Y-type genes:

[0061] The nucleotide sequence of the X-type gene is shown in SEQ ID NO: 1, and the amino acid sequence of the X-type enamel matrix protein expressed by the gene is shown in SEQ ID NO: 3;

[0062] The nucleotide sequence of the Y-type gene is shown in SEQ ID NO: 2, and the amino acid sequence of the Y-type enamel matrix protein expressed by the gene is shown in SEQ ID NO: 4.

[0063] Example 2 Expression of heterologous enamel matrix proteins

[0064] A method for expressing a heterologous enamel matrix protein comprises the following steps:

[0065] 1) The gene for expressing a heterologous enamel matrix protein in Example 1 was tagged by inserting multiple His tags. The nucleotide sequence of the tagged X-type gene is shown in SEQ ID NO:5, and the amino acid sequence of the protein expressed thereby is shown in SEQ ID NO:6; the nucleotide sequence of the tagged Y-type gene is shown in SEQ ID NO:7, and the amino acid sequence of the protein expressed thereby is shown in SEQ ID NO:8.

[0066] 2) The genes obtained in step 1) were respectively introduced into the expression plasmid PET28a(+), and ligated through the Nde I restriction site and the Xho I restriction site to obtain a recombinant plasmid, which was then transformed into Escherichia coli, cultured, and induced to express by adding IPTG.

[0067] The recombinant E. coli of this example was cultured normally under a suitable environment and then induced at various temperatures and times as shown in Table 1. SDS-PAGE samples of the fermented cells before and after induction were analyzed by SDS-PAGE. After detection, the expression level of the target protein was determined by scanning to obtain the target protein expression level as a mass percentage. The SDS-PAGE electrophoresis results (Figure 2) before and after induction in Examples 2-15 show that the molecular weight of the target protein is consistent with that of a standard molecular weight marker, indicating that the induced target protein is an enamel matrix protein.

[0068] Table 1

[0069] As can be seen from Table 1, the recombinant E. coli of the present invention has a high expression at a temperature of 15-34°C and an induction time of 3-16 hours. Among them, in Example 2-15, an expression level of up to 32.7% can be achieved by induction at 34°C for 4 hours, which has the best yield.

[0070] Example 3 Purification of heterologous enamel matrix protein

[0071] The expression product obtained in Example 2 was broken into bacterial cells using a high-pressure homogenizer to achieve lysis, thereby obtaining a protein solution. The protein solution was purified according to the following steps, comprising: balancing a Ni chromatography column with a binding buffer, then loading the protein solution, removing impurity proteins adsorbed on the Ni chromatography column using a washing buffer, then eluting with an elution buffer, collecting the elution peak, and performing SDS-PAGE electrophoresis detection.

[0072] The binding buffer, washing buffer and elution buffer were all PB buffer systems, and purification was performed according to the different buffer systems shown in Table 2.

[0073] Table 2

[0074] According to the purification electrophoresis test results, the concentrations of Examples 3-5 were finally determined to be the optimal conditions, that is, in the binding buffer, the working concentration of the PB buffer system was 10 mM, the pH value was 7.5, and 5 mM imidazole and 150 mM sodium chloride were added; in the wash buffer, the working concentration of the PB buffer system was 10 mM, the pH value was 7.5, and 80 mM imidazole and 150 mM sodium chloride were added; in the elution buffer, the working concentration of the PB buffer system was 10 mM, the pH value was 7.5, and 250 mM imidazole and 150 mM sodium chloride were added, and the purification rate was the highest.

[0075] The enamel matrix protein gene of this invention has been stripped of its fragments that hinder spatial folding. Its sequence reflects the conserved sequence and core peptide of human enamel matrix protein. Double His tags are added to the C- and N-termini, making the protein more easily accessible to Ni purification media during purification. The purified protein can achieve a purity exceeding 95%, and stability testing has proven its high stability.

[0076] Obviously, the above embodiments of the present invention are merely examples for clearly illustrating the present invention, and are not limitations on the implementation methods of the present invention. For ordinary technicians in the relevant field, other different forms of changes or modifications can be made based on the above description. It is impossible to list all the implementation methods here. All obvious changes or modifications derived from the technical solution of the present invention are still within the scope of protection of the present invention.

Claims

1. An enamel matrix protein, characterized in that the enamel matrix protein comprises type X enamel matrix protein and / or type Y enamel matrix protein; wherein, the type X enamel matrix protein has the amino acid sequence shown in SEQ ID NO:3 or an amino acid sequence obtained by adding, deleting, substituting or modifying one or more amino acids in the amino acid sequence shown in SEQ ID NO:3, and the type Y enamel matrix protein has the amino acid sequence shown in SEQ ID NO:4 or an amino acid sequence obtained by adding, deleting, substituting or modifying one or more amino acids in the amino acid sequence shown in SEQ ID NO:

4.

2. A nucleic acid molecule, characterized in that the nucleic acid molecule encodes the enamel matrix protein according to claim 1.

3. The nucleic acid molecule according to claim 3, characterized in that the nucleic acid molecule has the nucleotide sequence shown in SEQ ID NO:1 and / or SEQ ID NO:2, or has a sequence with a sequence identity of more than 80% with the nucleotide sequence shown in SEQ ID NO:1 and / or SEQ ID NO:

2.

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

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

6. A method for preparing enamel matrix protein, characterized in that the method comprises the following steps: 1) Introducing the nucleic acid molecule according to claim 2 or 3 into a plasmid to obtain a recombinant plasmid; 2) Transforming the recombinant plasmid into a host cell, culturing, inducing expression, and purifying to obtain enamel matrix protein.

7. The method according to claim 6, characterized in that in step 1), the nucleic acid molecule further comprises a nucleotide sequence encoding a tag, preferably comprising a nucleotide sequence encoding at least one His tag; Preferably, the nucleic acid molecule has the nucleotide sequence shown in SEQ ID NO:5 and / or SEQ ID NO:7, or has a sequence with a sequence identity of more than 80% with the nucleotide sequence shown in SEQ ID NO:5 and / or SEQ ID NO:

7.

8. The method according to claim 6, characterized in that in step 2), the host cell is Escherichia Coli; and / or the temperature for inducing expression is 15 - 34 °C, preferably 30 - 34 °C; and / or the time for inducing expression is 3 - 16 h, preferably 3 - 10 h.

9. The method according to claim 6, characterized in that the purification comprises: equilibrating a Ni chromatography column with a binding buffer, then loading the protein solution obtained by inducing expression and lysing the host cell, removing impurities with a washing buffer, and then eluting with an elution buffer, and collecting the elution product; Preferably, the binding buffer is a PB buffer system with a working concentration of 8 - 12 mM, a pH value of 7.0 - 8.0, and 4 - 6 mM imidazole and 140 - 160 mM sodium chloride are added; Preferably, the washing buffer is a PB buffer system with a working concentration of 8 - 12 mM, a pH value of 7.0 - 8.0, and 50 - 100 mM imidazole and 140 - 160 mM sodium chloride are added; Preferably, the elution buffer is a PB buffer system with a working concentration of 8 - 12 mM, a pH value of 7.0 - 8.0, and 150 - 250 mM imidazole and 140 - 160 mM sodium chloride are added.

10. Use of the enamel matrix protein according to claim 1, the nucleic acid molecule according to claim 2 or 3, the recombinant expression vector according to claim 4, the host cell according to claim 5, or the method according to any one of claims 6 - 9 in the preparation of an enamel matrix protein drug.

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