High-efficiency expression and application of human glaze matrix protein

Abstract

The invention provides high-efficiency expression and application of the human glaze matrix protein. Also provided are genes for expression of heterologous glaze matrix proteins, including an X-type gene and / or a Y-type gene. The gene for expression of the heterologous glaze matrix protein can realize heterologous expression of the glaze matrix protein, has properties close to those of human-derived glaze matrix protein, is not easy to fold in space, is easy to purify, has strong uniformity and high stability, and realizes industrial production of the glaze matrix protein.

Description

Technical Field

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

[0002] Enamel is the hardest tissue in the human body. It covers the surface of the tooth crown and is composed of 96% hydroxyapatite, 3% water and less than 1% organic matter. Enamel matrix protein is an extracellular matrix protein secreted by ameloblasts during the enamel development period. It can regulate the mineralization and maturation of enamel. After continuous accumulation of enamel matrix protein, enamel crystals are formed to gradually thicken the enamel layer. At the same time, ameloblasts move with the thickening of the enamel. Enamel matrix protein plays an important role in the formation of enamel. Therefore, in the research of periodontal treatment, enamel matrix protein is a common research object.

[0003] In 1997, Biora Company in Sweden successfully developed a porcine-derived enamel matrix protein drug, which was certified by the FDA in 1999 and used clinically. This drug is a complex of porcine-derived amelogenin and propylene glycol alginate, in a gel form. Research shows that after administration, it can effectively promote periodontal tissue regeneration and slow down gingival recession. At the same time, osteogenesis and the proliferation and migration of periodontal ligament cells are observed.

[0004] Enamel matrix protein only exists in the enamel during embryonic development. After that, as the enamel develops and matures, ameloblasts gradually disappear, and enamel matrix protein will be hydrolyzed by proteases until it completely disappears. This means that enamel matrix protein can only be extracted from tooth germs, resulting in difficult sources of human-derived enamel matrix protein. Tooth germs of pigs and cows are widely used by researchers because they contain a relatively large volume of enamel. In addition, mouse teeth are also widely used as extraction materials because they contain enamel matrix proteins at different growth stages in the same tooth and have low costs. Nevertheless, the low extraction rate still makes the source of enamel matrix protein a problem. At the same time, due to differences in experimental animals and extraction methods, the protein contents and qualities of the enamel matrix proteins extracted by researchers are different, so it often affects the accuracy and reliability of research results. The shortage and uneven quality of research materials have led to slow progress in enamel matrix protein-related research in recent years.

[0005] The gene sequences of human-derived enamel matrix proteins are relatively conserved genetically, which makes the development of heterologous enamel matrix proteins possible. However, there is currently a lack of industrial-grade enamel matrix protein, mainly because after enamel matrix protein is expressed and accumulates to a certain amount, it will form folds, resulting in the inability to recognize the His tag and making it difficult to elute by the elution method. Therefore, currently, the engineering bacteria expressing enamel matrix protein are mainly resuspended with 3% acetic acid, and then enamel matrix protein is purified and separated. However, this method has certain industrial difficulties and is difficult to achieve the industrial production of enamel matrix protein. Summary of the Invention

[0006] In view of the deficiencies of the prior art, the present invention provides an enamel matrix protein that can be heterologously expressed, which is similar in properties to human enamel matrix protein, not easily folded in space, easy to purify, and the product has strong homogeneity and high stability, realizing the industrial production of enamel matrix protein. The present invention also provides a gene for expressing the above-mentioned heterologous enamel matrix protein, which can heterologously express enamel matrix protein. The present invention also provides a method for expressing the above-mentioned heterologous enamel matrix protein, realizing the industrial production of enamel matrix protein.

[0007] To this end, one aspect of the present invention provides an enamel matrix protein, which includes 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.

[0008] Another aspect of the present invention provides a nucleic acid molecule that encodes the enamel matrix protein of the present invention.

[0009] 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 with 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%, more than 99.5% with the nucleotide sequence shown in SEQ ID NO: 1 and / or SEQ ID NO: 2.

[0010] In some embodiments, in the nucleic acid molecule, the nucleotide sequence shown in SEQ ID NO: 1 or a sequence with a sequence identity of more than 80% thereto encodes type X enamel matrix protein.

[0011] In some embodiments, in the nucleic acid molecule, the nucleotide sequence shown in SEQ ID NO: 2 or a sequence with a sequence identity of more than 80% thereto encodes type Y enamel matrix protein.

[0012] Another aspect of the present invention provides a recombinant expression vector, which contains the nucleic acid molecule of the present invention.

[0013] Another aspect of the present invention provides a host cell, which contains the nucleic acid molecule or recombinant expression vector of the present invention.

[0014] Another aspect of the present invention provides a method for preparing enamel matrix protein, the method comprising the following steps:

[0015] 1) Introduce the nucleic acid molecule of the present invention into a plasmid to obtain a recombinant plasmid;

[0016] 2) Transform the recombinant plasmid into a host cell, culture and induce expression, and purify to obtain enamel matrix protein.

[0017] In some embodiments, in step 1), the nucleic acid molecule further comprises a nucleotide sequence encoding a tag.

[0018] In some specific embodiments, the nucleic acid molecule comprises a nucleotide sequence encoding at least one His tag, for example, comprising 6 His tags. Inserting multiple His tags in the present invention can significantly improve the recognition rate, which is beneficial to avoiding the situation that partial folding of the protein structure makes the tag unrecognizable, resulting in difficult purification and separation.

[0019] In some embodiments, the nucleic acid molecule with a His tag has the nucleotide sequence shown in SEQ ID NO: 5 and / or SEQ ID NO: 7, or has 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%, more than 99.5% with the nucleotide sequence shown in SEQ ID NO: 5 and / or SEQ ID NO: 7.

[0020] In some embodiments, in the nucleic acid molecule, the nucleotide sequence shown in SEQ ID NO: 5 or a sequence with a sequence identity of more than 80% thereto encodes the protein shown in SEQ ID NO: 6.

[0021] In some embodiments, in the nucleic acid molecule, the nucleotide sequence shown in SEQ ID NO: 7 or a sequence with a sequence identity of more than 80% thereto encodes the protein shown in SEQ ID NO: 8.

[0022] In some embodiments, in step 1), the plasmid is PET28a(+).

[0023] In some embodiments, in step 2), the host cell is Escherichia Coli.

[0024] In some embodiments, the temperature for induced expression is 15 - 34 °C, preferably 30 - 34 °C. In some specific embodiments, the temperature for induced 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.

[0025] In some embodiments, the time for induced expression is 3 - 16 h, preferably 3 - 10 h. In some specific embodiments, the time for induced expression is 3 h, 4 h, 6 h, 8 h, 10 h, 12 h, 14 h, 16 h or any value therebetween.

[0026] In some embodiments, the purification includes: equilibrating a Ni chromatography column with a binding buffer, then loading the protein solution obtained by inducing expression and lysing host cells, removing impurity proteins with a washing buffer, and then performing elution with an elution buffer, and collecting the elution product.

[0027] In some embodiments, 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 added. 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 value can be 7.0, 7.2, 7.4, 7.6, 7.8, 8.0 or any value therebetween, the concentration of imidazole is 4 mM, 4.5 mM, 5 mM, 5.5 mM, 6 mM or any value therebetween, and the concentration of sodium chloride is 140 mM, 145 mM, 150 mM, 155 mM, 160 mM or any value therebetween.

[0028] In some embodiments, 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 added. In some specific embodiments, in the washing 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 value can be 7.0, 7.2, 7.4, 7.6, 7.8, 8.0 or any value therebetween, the concentration of imidazole is 50 mM, 60 mM, 70 mM, 80 mM, 90 mM, 100 mM or any value therebetween, and the concentration of sodium chloride is 140 mM, 145 mM, 150 mM, 155 mM, 160 mM or any value therebetween.

[0029] In some embodiments, 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. 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 value can be 7.0, 7.2, 7.4, 7.6, 7.8, 8.0 or any value therebetween, the concentration of imidazole is 150 mM, 175 mM, 200 mM, 225 mM, 250 mM or any value therebetween, and the concentration of sodium chloride is 140 mM, 145 mM, 150 mM, 155 mM, 160 mM or any value therebetween.

[0030] 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 washing 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.

[0031] Another aspect of the present invention also provides the use of the enamel matrix protein, nucleic acid molecule, recombinant expression vector, host cell of the present invention or the method for preparing enamel matrix protein in the preparation of enamel matrix protein drugs.

[0032] Compared with the prior art, the present invention has the following beneficial effects:

[0033] (1) The enamel matrix protein of the present invention can be heterologously expressed, has properties similar to human enamel matrix protein, is not prone to spatial folding, is easy to purify, and the product has strong homogeneity and high stability.

[0034] (2) The expression method of the heterologous enamel matrix protein of the present invention involves introducing the enamel matrix protein gene into a plasmid, and then transforming the plasmid into a host bacterium for expression to obtain the enamel matrix protein, which can achieve the industrial production of enamel matrix protein. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1 Shows the protein expression electrophoresis results before and after induction of the fermented bacteria in Example 2 of the present invention.

[0036] Figure 2 Shows the SDS - PAGE electrophoresis results of the purified heterologous enamel matrix protein obtained in Example 3 of the present invention. DETAILED DESCRIPTION OF THE INVENTION

[0037] The following examples illustrate the present invention, but the present invention is not limited by 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 all be covered within the scope of the technical solutions claimed in the present invention.

[0038] As used herein, the term "substitution" with respect to an amino acid refers to the replacement of at least one amino acid residue in an amino acid sequence with another, different "substituted" amino acid residue. The term "insertion" as used herein with respect to an amino acid refers to the incorporation of at least one additional amino acid into an amino acid sequence. Although insertions typically consist of the insertion of 1 or 2 amino acid residues, larger "peptide insertions" can also be prepared, such as the insertion of about 3 to 5 or even up to about 10, 15 or 20 amino acid residues. As disclosed above, the inserted residues can be naturally occurring or non-naturally occurring. The term "deletion" as used herein with respect to an amino acid refers to the removal of at least one amino acid residue from an amino acid sequence.

[0039] The enamel matrix proteins of the present invention may contain conservative amino acid substitutions at one or more amino acid residues, such as at essential or non-essential amino acid residues. A "conservative amino acid substitution" is the replacement of an amino acid residue with another amino acid residue having a similar side chain. Families of amino acid residues having 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). Thus, in the present invention, an essential or non-essential amino acid residue in an enamel matrix protein is preferably replaced with another amino acid residue from the same side chain family.

[0040] The "percent sequence identity" or "identity percent" between two polynucleotide or polypeptide sequences refers to the number of identical matching positions shared by the sequences within a comparison window, taking into account additions or deletions (i.e., gaps) that must be introduced for optimal alignment of the two sequences. A matching position is any position where the same nucleotide or amino acid is present in both the target sequence and the reference sequence. Since gaps are not nucleotides or amino acids, gaps present in the target sequence are not counted. Similarly, gaps present in the reference sequence are not counted since nucleotides or amino acids from the target sequence are counted and nucleotides or amino acids from the reference sequence are not.

[0041] The percentage of sequence identity can be calculated by the following process: Determine the number of positions where the same amino acid residue or nucleic acid base appears in both of the two sequences to obtain the number of matching positions, divide the number of matching positions by the total number of positions in the comparison window, and multiply the result by 100 to obtain the percentage of sequence identity. The comparison of sequences and the determination of the percentage of sequence identity between two sequences can be accomplished using software that is readily available for online use and download. Suitable software programs are available from a variety of sources for the alignment of protein and nucleotide sequences. A suitable program for determining the percentage of sequence identity is bl2seq, which is part of the BLAST program suite available from the BLAST website (blast.ncbi.nlm.nih.gov) of the National Center for Biotechnology Information of the US government. Bl2seq uses the BLASTN or BLASTP algorithm for comparison between two sequences. BLASTN is used to compare nucleic acid sequences, while BLASTP is used to compare amino acid sequences. Other suitable programs are, for example, Needle, Stretcher, Water or Matcher, which are part of the EMBOSS suite of bioinformatics programs and are also available from the European Bioinformatics Institute (EBI) at www.ebi.ac.uk / Tools / psa.

[0042] To achieve the industrial production of enamel matrix protein, the inventors found the genes and DNA sequences related to enamel matrix protein from NCBI, analyzed the DNA sequences, and found that the genes related to enamel matrix protein are located on sex chromosomes and are divided into X-type and Y-type. In response to this, the gene sequences on the allelic sex chromosomes were further analyzed to find the conserved sequences, and after codon optimization of the sequences, the gene for heterologous expression of enamel matrix protein of the present invention was obtained.

[0043] In some embodiments, the gene for heterologous expression of enamel matrix protein of the present invention includes the X-type gene and / or the Y-type gene; the nucleotide sequence of the X-type gene is as shown in SEQ ID NO: 1, and the nucleotide sequence of the Y-type gene is as shown in SEQ ID NO: 2.

[0044] 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 as shown in SEQ ID NO: 3, and the amino acid sequence of the Y-type enamel matrix protein is as shown in SEQ ID NO: 4.

[0045] In some embodiments, the method for expressing heterologous enamel matrix protein of the present invention includes the following steps:

[0046] 1) Add a tag to the gene for heterologous expression of enamel matrix protein and introduce it into a plasmid to obtain a recombinant plasmid;

[0047] 2) Transform the recombinant plasmid into a host cell, culture it, induce expression, and purify it to obtain enamel matrix protein.

[0048] Further, in step 1), the tag includes a His tag.

[0049] Further, in step 1), the nucleotide sequence of the X-type gene after adding the tag is as shown in SEQ ID NO: 5, and the amino acid sequence of the protein expressed thereby is as shown in SEQ ID NO: 6; and / or,

[0050] The nucleotide sequence of the Y-type gene after adding the tag is as shown in SEQ ID NO: 7, and the amino acid sequence of the protein expressed thereby is as shown in SEQ ID NO: 8.

[0051] Further, in step 1), the host bacterium is Escherichia coli, and the plasmid is PET28a(+).

[0052] Further, in step 2), the specific operation of purification includes: equilibrating the Ni chromatography column with a binding buffer, then loading the protein solution obtained by expressing and lysing the host bacterium, removing the miscellaneous proteins adsorbed on the Ni chromatography column with a washing buffer, and then performing elution with an elution buffer, collecting the elution product to obtain enamel matrix protein.

[0053] Further, 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 adding 4 - 6 mM imidazole and 140 - 160 mM sodium chloride.

[0054] 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 adding 50 - 100 mM imidazole and 140 - 160 mM sodium chloride.

[0055] 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 adding 150 - 250 mM imidazole and 140 - 160 mM sodium chloride.

[0056] Further, in step 2), the temperature for induced expression is 15 - 34 °C, and the induction time is 3 - 16 h.

[0057] In this example, if no specific technology or conditions are indicated, the operations are carried out according to the conventional technical methods in the art and the content of the instrument instruction manual. For reagents or instruments without indicating the manufacturer, they are all conventional products that can be obtained through commercial purchase.

[0058] Example 1 Preparation of Enamel Matrix Protein Gene

[0059] Retrieve the genes and DNA sequences related to amelogenin from NCBI, analyze the DNA sequences, and find that the genes related to amelogenin are located on the sex chromosomes. Analyze the gene sequences on the allelic sex chromosomes, identify the conserved sequences, and after codon optimization of the sequences, obtain a gene for heterologous amelogenin expression, including X-type gene and Y-type gene:

[0060] Among them, the nucleotide sequence of the X-type gene is shown in SEQ ID NO: 1, and the amino acid sequence of the X-type amelogenin expressed by it is shown in SEQ ID NO: 3;

[0061] Among them, the nucleotide sequence of the Y-type gene is shown in SEQ ID NO: 2, and the amino acid sequence of the Y-type amelogenin expressed by it is shown in SEQ ID NO: 4.

[0062] Example 2 Expression of Heterologous Amelogenin

[0063] A method for expressing heterologous amelogenin, comprising the following steps:

[0064] 1) Insert multiple His tags into the gene for heterologous amelogenin expression in Example 1 for tagging. 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 by it 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 by it is shown in SEQ ID NO: 8.

[0065] 2) Respectively introduce the genes obtained in step 1) into the expression plasmid PET28a(+), connect through the Nde I restriction site and Xho I restriction site to obtain a recombinant plasmid, and then transform the recombinant plasmid into Escherichia coli, culture and add IPTG for induction expression.

[0066] Among them, after the recombinant Escherichia coli in this example is normally cultured in a suitable environment, induction is carried out according to different temperatures and times shown in Table 1. The cell samples before and after induction of the fermented bacteria are subjected to SDS-PAGE electrophoresis detection, and after detection, scanning is carried out to obtain the expression level of the target protein in terms of mass percentage. According to the SDS-PAGE electrophoresis results before induction and after induction in Examples 2 - 15( Figure 2 ), it shows that the molecular weight of the target protein is consistent with that of the standard molecular weight marker, indicating that the induced target protein obtained is amelogenin.

[0067] Table 1

[0068] Temperature / °C Time / h Protein expression level Example 2-1 15 16 29.8％ Example 2-2 15 10 25.4％ Example 2-3 15 4 17.8％ Example 2-4 15 3 16.2％ Example 2-5 25 16 24.6％ Example 2-6 25 10 27.9％ Example 2-7 25 4 18.3％ Example 2-8 25 3 14.6％ Example 2-9 30 16 23.9％ Example 2-10 30 10 31.8％ Example 2-11 30 4 26.7％ Example 2-12 30 3 25.8％ Example 2-13 34 16 28.2％ Example 2-14 34 10 32.9％ Example 2-15 34 4 32.7％ Example 2-16 34 3 30.6％

[0069] As can be seen from Table 1, the recombinant Escherichia coli of the present invention has a high expression at a temperature of 15 - 34°C and an induction time of 3 - 16 h. Among them, in Examples 2 - 15, an induction at 34°C for 4 h can achieve an expression level as high as 32.7%, with the best benefit.

[0070] Purification of Heterologous Enamel Matrix Protein in Example 3

[0071] The cells of the expression product obtained in Example 2 were lysed by a high-pressure homogenizer to obtain a protein solution; the protein solution was purified according to the following steps, which included: equilibrating the Ni chromatography column with a binding buffer, then loading the protein solution, removing the miscellaneous proteins adsorbed on the Ni chromatography column with a washing buffer, and then eluting with an elution buffer, collecting the elution peak, and performing SDS-PAGE electrophoresis detection.

[0072] Among them, the binding buffer, washing buffer, and elution buffer are all PB buffer systems, and purification is carried out according to different buffer systems shown in Table 2.

[0073] Table 2

[0074]

[0075] According to the purification electrophoresis detection results, the concentrations in 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 is 10 mM, the pH value is 7.5, and 5 mM imidazole and 150 mM sodium chloride are added; in the washing 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, and the purification rate is the highest.

[0076] The enamel matrix protein gene of the present invention removes the part that is not easy to fold in space. Its sequence is the conserved sequence and core peptide segment of the human enamel matrix protein sequence, and double His tags are added at the C-terminus and N-terminus. During purification, the protein can more easily bind to the Ni purification filler, facilitating purification. The purity of the purified protein can reach more than 95%, and it has been proven to have high stability through stability tests.

[0077] Obviously, the above-mentioned embodiments of the present invention are only examples for clearly explaining the present invention, rather than limitations on the implementation manners of the present invention. For those of ordinary skill in the art, other different forms of changes or variations can be made based on the above description. It is impossible to list all the implementation manners here. Any obvious changes or variations derived from the technical solutions of the present invention still fall within the protection scope of the present invention.

Claims

1. An enamel matrix protein, characterized in that, The enamel matrix protein includes 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 an enamel matrix protein, characterized in that, The method includes the following steps: 1) Introduce the nucleic acid molecule according to claim 2 or 3 into a plasmid to obtain a recombinant plasmid; 2) Transform the recombinant plasmid into a host cell, culture and induce expression, and purify to obtain the enamel matrix protein.

7. The method according to claim 6, characterized in that, In step 1), the nucleic acid molecule further includes a nucleotide sequence encoding a tag, preferably including 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 includes: equilibrating a Ni chromatography column with a binding buffer, then loading the protein solution obtained by inducing expression and lysing the host cell, removing the impurity proteins 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 adding 4-6 mM imidazole and 140-160 mM sodium chloride; 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 adding 50-100 mM imidazole and 140-160 mM sodium chloride; 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 adding 150-250 mM imidazole and 140-160 mM sodium chloride.

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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